Bioaccumulation of cadmium in marine organisms.

A general review of cadmium concentrations in marine organisms and studies of cadmium bioaccumulation is presented. Factors which influence cadmium concentrations, such as regional differences, seasonal fluctuations and salinity, are discussed and species which are likely to accumulate cadmium identified. Experimental studies designed to investigate the influence of some of these factors on cadmium bioaccumulation in a filter feeding bivalve mollusk, the American oyster (Crassostrea virginica), are presented. Field studies of seasonal dynamics of cadmium in oysters indicate patterns which may be correlated with seasonal physiological activity. The bioaccumulation of cadmium following input to estuarine systems by natural phenomena is observed. Cadmium concentrations in oysters collected from regions of different salinity suggest an inverse relationship between cadmium concentration and salinity. Laboratory experiments designed to investigate mechanisms of cadmium accumulation demonstrate that an inducible cadmium binding protein, similar to metallothiomein, is present in the American oyster.


Factors influencing cadmium accumulation and its toxicity to marine organisms.

The toxicity of dissolved cadmium to a variety of marine animals has been found to be related to salinity, with decreased toxicity observed at higher salinities. Recent data from our laboratory have demonstrated that the toxicity of cadmium to estuarine shrimp and larval fish is a function of free cadmium ion concentration, which in turn is controlled by the chloride concentration of the water. As the chloride concentration (i.e., salinity of the water) increases, the concentration of free cadmium ion decreases relative to total dissolved metal, due to its complexation with chloride ions. These observations have been given further support by measurements involving the uptake of (115m)Cd by shrimp which showed that accumulation of (115m)Cd and chloride concentration also are inversely related. Experiments also have been conducted on the physiological effects of cadmium on the respiration of excised oyster gill tissue. Although tissues from oysters exposed for 14 days to 0.1 ppm total dissolved cadmium accumulated significant quantities of metal, no measurable effects on respiration rates were detected. Higher doses (0.3 and 0.6 ppm) caused both mortalities of oysters and accelerated respiration of excised oyster gill. Exposure to 0.1 ppm cadmium also caused the induction of and/or increased binding of cadmium to a specific low molecular weight protein in oysters. This protein appeared to have a detoxification function at low cadmium exposure levels, but in animals exposed to 0.6 ppm cadmium the induction mechanism apparently became saturated, allowing the excess cadmium to bind critical sites with resultant damage.


Influence of some factors on cadmium pharmacokinetics and toxicity.

Cadmium metabolism in the young and in conditions of dietary contamination with ash from coal gasification were investigated. The experiments were performed in adult rats which received ash in the diet (5%) and/or cadmium in drinking water (100 ppm) over a period of five weeks and in sucklings whose mothers were given the same treatment throughout pregnancy and lactation. In pharmacokinetic studies, (115m)Cd was administered orally or intraperitoneally to determine the intestinal absorption, retention, and distribution. Cadmium toxicity (LD(50)) was determined in different age groups of animals treated with ash for five weeks before a single oral or intraperitoneal administration of cadmium chloride. After intraperitoneal administration, (115m)Cd body retention decreased with age and was independent of the dietary treatment. Sucklings had a higher retention in the blood, carcass, and gut than adults. After oral administration, sucklings had a much higher body retention than adults regardless of the dietary treatment of their mothers. Cadmium toxicity was also independent of the dietary treatment. Most striking was a very high oral toxicity of cadmium in sucklings. It is concluded that the young might be at a special risk at the same level of environmental cadmium exposure because of the high oral cadmium toxicity at this age which is most probably due to a high cadmium retention in the gut. It is also concluded that the mixture of elements contained in ash is not likely to influence cadmium metabolism and toxicity in conditions of dietary exposure.


Some effects of oral ingestion of cadmium on zinc, copper, and iron metabolism.

Data are presented to show that ingestion of cadmium chloride by rats at low levels leads to alteration of zinc metabolism in the liver, even though the formation of metallothionein is not evident. A dose-response relationship between amount of cadmium ingested and degree of perturbation of zinc metabolism in liver was found. Oral cadmium was shown to cause emphysema and reduce pulmonary function in male rats; the effect was less severe or delayed in onset if dietary zinc concentration was high. Interference with copper and iron metabolism was shown to occur in rats given low levels of cadmium orally. Depression of copper and iron metabolism of the rat fetus was found to occur when dams received very low doses of cadmium during gestation, even though very little cadmium passed the placental barrier.


Protease deficiency in plasma of patients with cystic fibrosis. Reduced reaction of 4-methylumbelliferylguanidinobenzoate with plasma of patients with cystic fibrosis.

Protease activity in plasma is assayed using 4-methylumbelliferylguanidinobenzoate. The assay is modified by carrying out the reaction in the presence and absence of benzamidine, a competitive inhibitor of trypsin-like proteases. The parameters of the assay are described in detail. Using this assay, our earlier demonstration of a deficiency of protease activity in plasma of patients with cystic fibrosis is confirmed. The activity, corrected for the nonspecific hydrolysis of 4-methylumbelliferylguanidinobenzoate by benzamidine, is expressed as nanomoles of 4-methylumbelliferone released per milliliter plasma. Under standard conditions, the activity in plasma activated with chloroform-ellagic acid was 127.2 +/- 23.1 in 7 controls, 70.4 +/- 11.7 in 11 obligate heterozygotes, and 48.7 +/- 16.6 in 12 patients with cystic fibrosis. Identical results were obtained when unactivated plasma was used. These data demonstrate that the judicious use of specific inhibitors such as benzamidine might be useful in assaying low levels of protease activity in crude systems.


A new spectrophotometric assay for enzymes of purine metabolism. I. Determination of xanthine oxidase activity.

A new method for the determination of xanthine oxidase activity with xanthine or hypoxanthine is described. The hydrogen peroxide produced by the oxidation of the substrates is reduced by catalase in the presence of high concentrations of ethanol. The acetaldehyde formed is further oxidized by aldehyde dehydrogenase NAD or NADP-dependent. The reduction rate of the coenzymes were measured at 334 nm and utilized as indicators for the xanthine oxidase. The sensitivity of the method with xanthine as substrate can be doubled by the addition of uricase, which oxidizes uric acid to allantoin.


A new spectrophotometric assay for enzymes of purine metabolism. II. Determination of guanase activity.

A new method for the determination of guanase is described. Xanthine, the product of the guanase reaction, is oxidized by xanthine oxidase, forming uric acid and hydrogen peroxide. Hydrogen peroxide is further reduced to water by catalase in the presence of ethanol. The acetaldehyde formed in this reaction step is dehydrogenated NAD or NADP dependent by aldehyde dehydrogenase. The NADH or NADPH production is measured and utilized for the calculation of the guanase activity. The sensitivity of the method can be doubled by the addition of uricase, which oxidizes uric acid to permit the formation of another mole of hydrogen peroxide.


Cystathionase deficiency: the effect of cofactor on the stability of normal and abnormal enzyme from lymphoid cell lines.

Heating at 70 degrees C with and without added pyridoxal phosphate (PLP) had strikingly different effects on cystathionase protein from normal long-term lymphoid cell lines and on the enzymes from cells derived from patients with vitamin-B6-responsive cystathioninuria. PLP added to extracts of normal cells afforded complete protection against heat inactivation, whereas inactivation of the cystathionase protein in extracts obtained from two cystathioninuric lines was greater in the presence of PLP than in its absence.


Activity of arylamidases in serum during normal pregnancy.

Activity of arylamidases toward 6 beta-naphthylamides of L-amino acids was studied in the blood serum from 110 healthy women between the 6th and 36th weeks of pregnancy. A regular increase in the enzymatic activity was demonstrated toward all substrates under examination, particularly in the presence of alanyl-, leucyl- and lysyl-beta-naphthylamides, most pronounced in the last trimester of gestation. A correlation between oxytocinase and arylamidases activity was demonstrated, suggesting a possibility of using the enzymatic measurement as a diagnostic test in the normal course of pregnancy.


Differential heat sensitivity of human creatine kinase isoenzymes.

Creatine kinase isoenzymes (MM, MB and BB) were isolated from human tissue by ion-exchange chromatography. The B subunit was found to be more heat sensitive than the M subunit. BB and MB isoenzymes respond similarly to heat inactivation. Our results are in contrast with the body temperature inactivation of the brain isoenzyme reported by Lindsey and Diamond.


Endoscopic observations on the deglutition reflex in the horse.

Cine-endoscopy was used to study the deglutition reflex in 2 apparently normal horses. Closure of the nasopharyngeal sphincter was brought about by a lowering of the pharyngeal roof and an elevation of the soft palate caudal to the pharyngeal ostia of the auditory tubes. The medial cartilages of the ostia were not directly involved in bringing about closure of the sphincter. It is postulated that the opening of the ostia of the auditory tubes is brought about during deglutition by the combined action of the palatopharyngeus, pterygopharyngeus and tensor veli palatini muscles.


Observations on the mechanism of functional obstruction of the nasopharyngeal airway in the horse.

Fibreoptic endoscopy was used to study the movements of the larynx and pharynx during nasal occlusion in 10 horses, which showed signs consistent with functional pharyngeal obstruction (FPO) on exercise. Cine-endoscopic films were made on 3 such horses. Consideration of the anatomy of the region indicates that FPO may best be regarded as a subluxation of the nasopharyngeal and larygneal airways and it was found that a constant component of the movements which brought about this subluxation was a marked caudal retraction of the larynx. It is suggested that this caudal retraction of the larynx occurred as a result of the action of th sternothyrohyoid and omohyoid muscles and that removal of a portion of these muscles might prevent subsequent airway subluxation in horses prone to FPO on exercise.


Fluoroscopic investigation of pharyngeal function in the horse.

Videofluoroscopy was used to study the deglutition reflexin 2 horses believed, on the basis of endoscopic and clinical examination, to have normal pharyngeal and laryngeal function. The reflex was found to be the same as that described in man and in the rabbit. A feature of deglutition in the horse was the temporary increase in size of the auditory tube diverticuli (gluttural pouches) as a result of contraction of the pharyngeal muscles.


The effect of laryngoplasty on pharyngeal function in the horse.

Using fluoroscopic techniques and videotape recordings, a study of normal deglutition was made in 2 ponies. Paryngeal function was studied at intervals after laryngoplasty on 1 animal and after a sham technique on the other. Two clinical cases of laryngeal paralysis, which had been treated by laryngoplasty, were also examined. In the 2 experimental ponies, liquid food passed into the lower respiratory tract post-operatively. One of the clinical cases appeared to swallow solid food normally, but some food material entered the larynx of the other horse. It is suggested that pharyngeal dysfunction, as well as over-abduction of the arytenoid cartilage, might be involved in causing the chronic post-operative cough which the experiment was designed to investigate.


Chronic obstructive pulmonary disease (COPD): factors influencing the occurrence.

Breed, age, weight, type of work performed, seasonal onset, poor ventilation and exposure to moulds in the habitat were investigated in relation to the occurrence of chronic obstructive pulmonary disease (COPD). COPD was most commonly detected in showjumping and hacking horses. The older a horse, the more likely it was to become affected although most were 6 to 10 years of age. Of the horses in this sample of the population, which was not a random one, thoroughbred horses were affected least and ponies most often. The high incidence in ponies was related to their more frequent exposure to poor quality fodder and bedding. Most horses are exposed to the hazard of moulds, but more affected horses were so exposed than those not affected with COPD. Poor ventilation of the stable increased the chance of a horse becoming affected. Sex, body weight and season of onset of coughing had no influence on the occurrence of the disease.


Phonocardiography in the horse: 2. The relationship of the external phonocardiogram to intracardiac pressure and sound.

The paper describes observations during individual cardiac cycles on the changing pressures and sounds recorded from the various chambers of the heart and aorta, related to phonocardiograms from an external microphone hand-held over the cardiac area. The majority of the first sound appears to arise from the left side of the heart after the AV valves have closed and the second sound is a consequence of sudden deceleration of blood against already closed semilunar valves. Systolic murmurs at the aortic root may be benign from increased flow and turbulence at the peak of ejection but these may not be recorded externally.


A preliminary report on the use of warfarin in the treatment of navicular disease.

Twenty horses suffering from navicular disease were treated with warfarin given orally. The dosage was to effect, to give a 2 to 4 second prolongation of the one stage prothrombin time (OSPT). Dosage was initially at a rate of 0.018 mg/kg, changing the dose by amounts of 20 per cent until the required dose was achieved. Final dose rates varied from 0.012 mg/kg to 0.75 mg/kg. All the cases treated received warfarin daily throughout the trial. Seventeen of the animals became sound and the remaining 3 showed a marked improvement in their gait. The mean of the ages of the horses was 7.5 years, the mean of the periods of lameness 9 months and the mean of the time taken to recovery one treatment was commenced was 7 weeks.


Indirect measurement of mean blood pressure in the normotensive and hypotensive horse.

Indirect mean arterial pressure was obtained from the tails of 78 anaesthetised and unanaesthetised normotensive and hypotensive horses. Data were obtained to determine the optimum cuff width in relation to tail circumference. A cuff that is too wide under-estimates and a cuff that is too narrow over-estimates blood pressure. With the optimum cuff width, which is slightly in excess of about one-fifth of the tail circumference, indirect mean blood pressure was obtained consistently in normotensive and hypotensive animals with mean blood pressures of 40 mmHg and frequently in animals with pressure as low as 30 mmHg.


Analysis of rapid oscillations of glucose and free fatty acids in plasma.

The authors analyzed rapid oscillations of blood sugar (GL) and free fatty acid levels (FFA) in serum of healthy subjects. They investigated a series of blood samples taken under conditions of absolute rest from the cubital vein at 15-s intervals for a period of 6 min. In addition to common statistical parameters, they calculated the course of autocorrelation and cross-correlation functions and periodograms. The magnitude of oscillations is significantly higher than the error of the biochemical methods. In some sequences periodicities were detected which were statistically significant in 23.8% of GL and in 38.1% of FFA. 24-point series of GL collected in parallel from both arms correlate in 36.3% positively, in 27.3% negatively, and in 36.4% they do not correlate. Series of FFA and GL collected simultaneously from one site correlate mutaually in almost all instances either positively or negatively, frequently with a time shift. The oscillations may be due to (a) feedback regulations of the levels of the two metabolites, (b) permanent mutual interaction between the FFA and glucose level and (c) an uneven concentration of the two metabolites in different parts of the circulation. The above factors may combine, and the list of possible factors may not be complete.


Perception of effort in isometric and dynamic muscular contraction.

The perception of muscular effort was studied using estimation and production methods in the adductor pollicis and quadriceps. A psychometric scale (percentage magnitude) was used. Static contractions were studied in the adductor pollicis, and both dynamic (isokinetic) and static contractions were studied in quadriceps. Linear and logarithmic equations were fitted for the perceived effort as a percentage of the maximum in relation to the produced percentage maximal force or torque. The logarithmic exponent was around or above 1.0. No significant difference was found between mean exponent and intercept values for the adductor pollicis and the quadriceps, or when estimated or produced values for the two muscles were compared. There was no difference in the same subjects between the equations for static and dynamic contractions with low angular velocity of the quadriceps.


Plasma noradrenaline response to sustained handgrip in patients with essential hypertension.

The responses of plasma noradrenaline, arterial blood pressure, and heart rate to sustained handgrip at 30% maximal voluntary contraction were studied in untreated patients with essential hypertension and in healthy subjects of comparable age. There were no significant differences between these two groups in the intensity and duration of handgrip. Increases in heart rate and blood pressure induced by the effort were similar in hypertensive patients and normotensive control subjects, whereas the absolute levels of blood pressure were considerably higher in the patients. In the first 1-2 min of exercise the increases in plasma noradrenaline concentration were similar in both groups. Subsequently, plasma noradrenaline concentration tended to plateau in hypertensive patients while in control subjects it continued to increase. The elevation of plasma noradrenaline in the last minute of effort was, therefore, significantly smaller in hypertensive patients than in the control group.


Cardiovascular response to interval and continuous training in women.

Three groups of five women (age = 18--25 years) participated in a 12-week training program. Cardiovascular responses up to 85% VO2 max to interval (ITG) and continuous (CTG) training were studied in two groups, before training and after 4, 8, and 12 weeks of training four times per week. A control group was assessed before and after 6 and 12 weeks. Both exercise groups demonstrated significant increases in Cao2--Cvo2 after 8 weeks with only slight further increases after 12 weeks (CTG = 8.9%, ITG = 20.0% at 85% VO2 max). No significant changes were noted in either group in SV (+ 5 ml ITG, + 9 ml CTG) or in their Qc. These results indicated that, in response to high intensity training, women may demonstrate similar cardiovascular adaptations to training as have been observed for men.


Physical training under the influence of beta blockade in rats: effect on adrenergic responses.

Rats were trained by daily swimming or running exercises with and without daily propranolol injections. Both training methods resulted in cardiac enlargement, but only swimming exercise caused hypertrophy of the brown adipose tissue. These changes were antagonized by beta blockade. The size of the adrenals reflected the stress of the treatments, but other known stress parameters, such as the size of the thymus or sexual organs dit not. Only swimming training without beta blockade sensitized the rats to the calorigenic action of noradrenaline. The cooling rate of the rats in water, when taking into account the insulative capacity of the body, was decreased in swimming-trained as well as in propranolol-treated rats but increased in running-trained rats. The latter two changes may be due to circulatory alterations, while the delayed body cooling in swimming-trained rats probably results from increased heat production capacity. Training-induced resting bradycardia and enhanced tachycardic response to isoprenaline were observable only in the animal groups trained without beta blockade. The pressor response to noradrenaline tended to be higher in the trained groups and the propranolol-treated group than in the controls and was smaller in the animal groups trained under the influence of beta blockade. On the other hand, the hypotonic response to isoprenaline was smaller in the propranolol-treated and running-trained animals. The results emphasize the importance of the sympathetic nervous system in the adaptation of an organism to physical training.


Effect of training on hormonal responses to exercise in competitive swimmers.

The effects of 9 weeks of training on responses of plasma hormones to swimming were studied in eight competitive swimmers who had not trained for several months. Two types of swimming tests were used: (1) 200 yd, a high intensity, exhausting type of exercise in which maximal effort was required both before and after training, and (2) 1000 yd, a pace type of exercise in which subjects swam as fast as possible prior to training and at the same rate after training. Plasma levels of glucagon increased and of insulin decreased during 1000 yd of swimming, but were not altered by 200 yd of swimming. No training effects were apparent in responses of plasma insulin and glucagon to these shortterm, high intensity exercise tests. During the 1000 yd swim, plasma adrenaline was 0.8 ng/ml before vs. 0.1 ng/ml after training. Plasma noradrenaline response decreased from 3.4 to 1.2 ng/ml as a result of training. In the 200 yd swim, adrenaline, but not noradrenaline, was lower after training.


Transient-phase studies on the creatine kinase reaction. The analysis of a reaction pathway with three intermediates.

1. The initial formation of creatine phosphate by creatine kinase was studied in the millisecond range and the effect of temperature on the transient and steady-state phases exploited. 2. At 25 degrees C and 35 degrees C there was no transient phase. This is in agreement with the results of Gutfreund [Engelborghs, Y., Marsh, A., and Gutfreund, H. (1975) Biochem. J. 151, 47--50]. 3. At 4 degrees C the time course of creatine phosphate formation was complex and consisted of three transient phases: a lag phase, a burst phase and a steady-state phase. Based on this result a reaction scheme for creatine kinase which includes three intermediates was proposed. Despite the completeness of the time course, the extraction of estimates for the rate constants was difficult and computer simulation and iterative methods had to be resorted to. 4. Attempts were made to provide evidence for the complex enzyme.ADP.metaphosphate.creatine on the creatine kinase reaction pathway [cf. Milner-White, E.J. and Watts, D.C. (1971) Biochem. J. 122, 727--740]. Under the conditions used these attempts were unsuccessful at times down to 2.5 ms, at 4 degrees C or 35 degrees C.


[Characterisation of a new endopeptidase from sporulating Bacillus sphaericus which is specific for the gamma-D-glutamyl-L-lysine and gamma-D-glutamyl-(L)meso-diaminopimelate linkages of peptidoglycan substrates (author's transl)].

A new peptidase which splits substrates related to the peptidic chains of peptidoglycans was found in the cell cytoplasm of sporulating Bacillus sphaericus. This is a gamma-D-glutamyl-L-diaminoacid endopeptidase (endopeptidase II). It was shown to have substrate requirements different from those of the previously described gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase (endopeptidase I). The substrates for endopeptidase II are peptides of the general type: formula: (see text). Unsubstituted N-terminal L-alanine was a strict requirement for endopeptidase II activity. Specific activities were variable with the nature and the substitution of the diaminoacid C-terminal groups. The role of endopeptidase II in the biosynthesis of the spore cortex is discussed.


Fructose-1,6-bisphosphate aldolase from rabbit liver. Reaction mechanism and physiological function.

Liver and muscle aldolase display similar reaction mechanisms. Both the enzymes, by reacting with dihydroxyacetone phosphate, form an acid-labile intermediate which is in rapid equilibrium with an eneamine intermediate. Differences are found in the equilibrium concentration of the acid-labile intermediate, which represents approximately 25% of the total intermediates in the liver (this paper) and 60% in the muscle enzyme [E. Grazi and G. Trombetta, Biochem. J. 175, 361 (1978)] and in the rate of formation of the eneamine intermediate which is much slower in the liver enzyme. Furthermore, with liver aldolase, the rate by which the C-3H bond of dihydroxyacetone phosphate is cleaved is increased by 60 times in the presence of glyceraldehyde 3-phosphate. This, mechanistically, indicates that glyceraldehyde 3-phosphate is bound to the enzyme before the formation of the eneamine from dihydroxyacetone phosphate, and, physiologically, that in liever aldolase the gluconeogenetic activity is favoured over the glycolytic activity.


Carbon magnetic resonance studies of the self-aggregation of calf thymus histones.

Self-aggregation of calf thymus histones H2A and H4 was studied by means of 13C magnetic resonance. Analyses of the changes in the intensities of several side-chain resonances, induced by added NaCl, confirm that carbon magnetic resonance can be used to monitor aggregation phenomena more accurately than other physicochemical methods. In particular the validity of an original computer-based method to treat 13C intensities is confirmed. The details of these aggregation phenomena are critically discussed in the light of a simple experiment on a 13C-enriched sample of H2B.


Rearrangement of chromatin structure induced by increasing ionic strength and temperature.

Native rat liver chromatin fragments exposed to 600 mM NaCl at 37 degrees C for 45 min exhibit substantial modification of their original (approximately 200 base pairs) repeating subunit structure: a new repeat of 140 base pairs, superimposed on a high background, is observed after micrococcal nuclease digestion. The same material appears, in the electron microscope, as clusters of tightly packed beads connected by stretches of 'free' DNA. These modifications are not observed when the native chromatin is incubated at 37 degrees C at NaCl concentrations up to 400 mM. When native rat liver chromatin depleted of histone H1 by tRNA extraction is exposed to ionic strengths up to 600 mM NaCl at 4 degrees C, almost no modifications of the original native repeating structure are observed. However, when the incubation is carried out at 37 degrees C in 150, 300 or 400 mM NaCl, rearrangements of the native structure occur as indicated by micrococcal nuclease digestion and electron microscopic studies. Incubation of H1-depleted chromatin at 600 mM NaCl for 45 min at 37 degrees C induces, as for the native chromatin, a complete rearrangement characterized by the appearance of a 140-base-pair repeat superimposed on a high background upon digestion by micrococcal nuclease. It is suggested that these rearrangements are mediated by hydrophobic interactions between the histone cores and are prevented at ionic strengths lower than 500 mM by the presence of histone H1.


A restriction map of cauliflower mosaic virus DNA (strain PV 147). Mapping of the cleavage sites of HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III.

The virion-extracted DNA (Mr5 x 10(6)) of cauliflower mosaic virus (CaMV) has three single-stranded interruptions. The mapping of this DNA using eleven restriction endonucleases (HhaI, SacI, AvaI, PvuII, PstI, XbaI, EcoRI, Bg/II, HincII, HpaII and HindII + III) is reported here. The existence of the three single-stranded breaks complicates the identification and the molecular weight determination of fragments produced by HpaII, HindIII and HindII + III. Indeed the electrophoretic mobility of some fragments in which a single-stranded discontinuity is located is modified, and the fluorescence of ethidium bromide complexed with these fragments is reduced as compared to that observed for the other fragments existing in a molar ratio. These drawbacks were overcome by performing experiments of nick-translation of CaMV DNA with Escherichia coli DNA polymerase I. FRom the data it follows that the CaMV DNA molecule bears bears 1 site for HhaI and SacI, 2 for AvaI and PvuII, 3 for PstI, 4 for XbaI, 5 for EcoRI, 6 for Bg/II and HincII, 11 for HpaII and 15 for HindII + III. The corresponding fragments have all been ordered and precisely located providing a suitable map for further investigations connected with the study of the fine structure and the function of the CaMV genome.


Messenger ribonucleoprotein complexes in human KB cells infected with adenovirus type 5 contain tightly bound viral-coded '100K' proteins.

Late after infection of KB cells with adenovirus 5 an extra protein becomes associated with messenger ribonucleoprotein particles present in the polysomes. This protein has a molecular weight of 100000 and is identical to the virus coded '100K' protein found previously. The extra protein is firmly attached to the messenger ribonucleoprotein complexes. Its binding resists exposure to high salt concentrations as used in puromycin/high-salt dissociation and equilibrium centrifugation in Cs2SO4 gradients. In this respect it resembles the binding of two other proteins of Mr 74000 and 48000 which are commonly found in messenger ribonucleoprotein particles of various eukaryotic cells. The identity between the messenger ribonucleoprotein protein of Mr 100000 and the "100K' protein present in the soluble part of the cytoplasm was established by sodium dodecylsulphate/polyacrylamide gel electrophoresis, isoelectric focusing and peptide mapping after limited proteolysis with Staphylococcus aureus protease.


Purification and properties of alanine dehydrogenase from Bacillus sphaericus.

1. The bacterial distribution of alanine dehydrogenase (L-alanine:NAD+ oxidoreductase, deaminating, EC 1.4.1.1) was investigated, and high activity was found in Bacillus species. The enzyme has been purified to homogeneity and crystallized from B. sphaericus (IFO 3525), in which the highest activity occurs. 2. The enzyme has a molecular weight of about 230 000, and is composed of six identical subunits (Mr 38 000). 3. The enzyme acts almost specifically on L-alanine, but shows low amino-acceptor specificity; pyruvate and 2-oxobutyrate are the most preferable substrates, and 2-oxovalerate is also animated. The enzyme requires NAD+ as a cofactor, which cannot be replaced by NADP+. 4. The enzyme is stable over a wide pH range (pH 6.0--10.0), and shows maximum reactivity at approximately pH 10.5 and 9.0 for the deamination and amination reactions, respectively. 5. Alanine dehydrogenase is inhibited significantly by HgCl2, p-chloromercuribenzoate and other metals, but none of purine and pyrimidine bases, nucleosides, nucleotides, flavine compounds and pyridoxal 5'-phosphate influence the activity. 6. The reductive amination proceeds through a sequential ordered ternary-binary mechanism. NADH binds first to the enzyme followed by ammonia and pyruvate, and the products are released in the order of L-ALANINE AND NAD+. The Michaelis constants are as follows: NADH (10 microM), ammonia (28.2 mM), pyruvate (1.7 mM), L-alanine (18.9 mM) and NAD+ (0.23 mM). 7. The pro-R hydrogen at C-4 of the reduced nicotinamide ring of NADH is exclusively transferred to pyruvate; the enzyme is A-stereospecific.


Kinetic and binding properties of the oxoglutarate translocator of rat-heart mitochondria.

The kinetic study of the oxoglutarateout/malatein exchange through the inner mitochondrial membrane of rat-heart mitochondria has been compelted and extended to higher external-oxoglutarate and to lower internal-malate concentrations. It has been found that the external oxoglutarate inhibits the exchange at high concentration. This excess-substrate inhibition is preceded by four jumps. The kinetic-saturation curve by the internal malate presents an apparent positive cooperativity that may be interpreted in different ways. The independence of the effects of the two substrates on the initial rate has been observed again and supports the conclusions reached in previous work. A method for the determination of oxoglutarate binding to the external face of the inner membrane is described. The binding curve shows four intermediary plateau regions that reflect significant apparent K-effects, alternatively negative and positive. For external-oxoglutarate concentrations below the region of excess-substrate inhibition, the binding-saturation curve and the kinetic-saturation curve are similar, demonstrating that K-effects are predominant. A particularly wide intermediary plateau that seems to correspond to half saturation of the active sites is common to both saturation curves. A clear lack of proportionality between the two curves at low oxoglutarate concentrations seems to indicate that more than one catalytic-rate constant is implied in the exchange kinetics. Two models of the oxoglutarate carrier are presented. Both lead to a minimum degree of 10:10 for the equation of the binding of oxoglutarate to the catalytic sites. In the first model this corresponds to ten subunits associated into a single oligomer while in the second model this results from a mixture of monomeric, dimeric, trimeric and tetrameric associations.


Cysteinyl-tRNA synthetase from Bacillus stearothermophilus. A structural and functional monomer.

A procedure is described for the purification of cysteinyl-tRNA synthetase as a side product of a multi-enzyme isolation from Bacillus stearothermophilus. The native and denatured enzyme are both shown to have a molecular weight of 54000 by gel filtration and sodium dodecyl sulphate/polyacrylamide gel electrophoresis respectively. Fingerprinting and peptide counting indicate that the polypeptide chain has a nonrepeating primary structure. The enzyme has only one binding site for each of its substrates (cysteine, ATP and tRNACys) as judged by equilibrium dialysis, active-site titration and fluorescence quenching. No evidence for the dimerisation of the enzyme in the presence of these substrates could be found. We conclude that cysteinyl-tRNA synthetase, which is the smallest aminoacyl-tRNA synthetase yet described, is both structurally and functionally monomeric.


Turnover rates of the molecular species of alkenyl ether phospholipids of Ehrlich ascites tumor cells.

[1-14C]Glycerol was injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor cells and the lipids were extracted from the cells after selected periods. 1-O-Alkenyl-2-acyl-3-acetylglycerols were prepared from the ethanolamine phosphoglycerides and fractionated by AgNO3-impregnated thin-layer chromatography into five molecular species. Bands 1--5 obtained in this way can be designated as saturated, monoene, diene, tetraene and hexaene species, respectively. Band 5, composed mainly of hexaene species was predominant among those derived from alkenyl ether phospholipids. The specific radioactivities of the fractionated species were determined. Hexaene molecular species turned over most rapidly and disaturated and tetraene species turned over at a high rate, while monoene and diene molecular species turned over very slowly.


Structure of Helix pomatia oxy-beta-hemocyanin and deoxy-beta-hemocyanin tubular polymers.

Mild trypsinolysis of Helix pomatia beta-hemocyanin leads to the formation of tubular polymers after removal of the collar part [van Breemen, J.F.L., Wichertjes, T., Muller, M.F.J., van Driel, R., and van Bruggen, E.F.J. (1975) Eur. J. Biochem. 60, 129--135]. Three-dimensional image reconstruction from electron micrographs of negatively stained tubular polymers showed: (a) alternating deep and shallow grooves in between the 10 helical chains, (b) the presence and position of two domains within each morphological wall-unit of the Mellema and Klug model [Mellema, J. E. and Klug, A. (1972) Nature (Lond.) 239, 146--150]. Optical diffraction of oxy and deoxygenated tubular polymers indicate a significant decrease in diameter with a concomitant increase in length upon deoxygenation.


Diversity and complexity of uterine mRNA from rats of differing hormonal states.

The diversity and complexity of the uterine mRNA population has been compared at two different stages of uterine growth and development in the rat. Analysis by cDNA hybridization to homologous mRNA indicates that there are 8000 different sequences expressed in the immature rat uterus responding to just 4 h of oestradiol-17 beta-induced growth while the fully developed uterus expresses 36 000 sequences. As a check on our results, the complexity is re-analysed by hybridization of mercurated mRNA to total unique DNA. Analysis by this method indicates that the hormone-stimulated immature rat uterus contains 12 000 poly(A)-containing mRNA sequences while the fully differentiated adult tissue contains 53 000 diverse sequences.


Qualitative and quantitative changes in uterine mRNA populations in response to oestradiol treatment of rats.

The mRNA population of immature rat uteri, responding to different lengths of oestradiol-induced differentiation, have been compared by the technique of DNA hybridization with heterogeneous RNA. Over the first 4 h of hormone induction there are continuing qualitative and quantitative changes in the poly(A)-containing mRNA such that, by 4 h after oestradiol administration, the population bears little resemblance to that of the unstimulated animal. Between 2 h and 4 h after oestradiol administration, the most striking changes appear to be in sequences of intermediate abundance. The possible significance of these findings is discussed.


A comparison of the initiation factors of eukaryotic protein synthesis from ribosomes and from the postribosomal supernatant.

A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes: (a) from the ribosomal wash and (b) from the postribosomal supernantant. A comparison is made between these preparations with respect to yield and specific activity. eIF-4A and eIF-4D occur mainly in the postribosomal supernatant; eIF-2, eIF-4C and eIF-5 are more evenly divided over both fractions, whereas eIF-1, eIF-3 and eIF-4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF-4A and eIF-4D.


A centrifugation study of rat-liver mitochondria, lysosomes and peroxisomes during the perinatal period.

We have investigated the intracellular distribution of several enzymes on homogenates of late foetal, early postnatal and adult rat livers. Homogenates were subjected to differential centrifugations in 0.25 M sucrose and four fractions were isolated which corresponded to the N (nuclear) ML (total mitochondrial) P (microsomal) and S (soluble) fractions of de Duve et al. (1955). In general the age of the animal did not significantly affect the distribution pattern. Reference enzymes of mitochondria, lysosomes and peroxisomes were mainly recovered in the total mitochondrial fraction (ML). Glucose-6-phosphatase and esterase, both located in the endoplasmic reticulum, were chiefly associated with the microsomal fraction P together with galactosyltransferase (a reference enzyme of the Golgi apparatus). 5'-Nucleotidase, (a plasma membrane enzyme) exhibits a bimodal distribution and is mainly recovered in the N and the P fractions. Such results indicate that the membrane composition of the fractions isolated by the fractionation scheme was used, does not appreciably differ for the late foetal, early postnatal and adult rat livers. An analytical fractionation of the mitochondrial (ML) fraction of livers at different stages of development was performed by isopycnic centrifugation in sucrose gradients and in glycogen gradients using sucrose solutions of various concentrations as the solvents. The distribution of mitochondria, lysosomes and peroxisomes were assessed by establishing the distribution of their reference enzymes. Some physical characteristics of the particles were deduced from the manner in which the distributions were influenced by the sucrose concentration of the centrifugation medium. The distribution of liver mitochondrial enzymes one day prenatal differs strikingly from that of enzymes one day postnatal; foetal mitochondria seem characterized by a high osmotic space and a high hydrated matrix density; neonatal mitochondria seem devoid of an osmotic space and the density of their hydrated matrix is markedly lower than that of the foetal mitochondria. As ascertained by the distribution of mitochondrial enzymes in a sucrose 2H2O gradient, the high density of a foetal mitochondria matrix does not mainly originate from a lower amount of hydration water. The behavior of lysosomal enzymes in media with increasing concentrations of sucrose suggests that lysosomes originating from late foetal rat liver are endowed with a very small osmotic space. As for the peroxisomes, our results do not display significant behavior differences in centrifugations that would indicate physicochemical changes of these particles during the perinatal period.


Stereochemistry of reactions catalysed by mammalian-brain L-glutamate 1-carboxy-lyase and 4-aminobutyrate: 2-oxoglutarate aminotransferase.

Deamination of 4-aminobutyrate by mammalian or bacterial 4-aminobutyrate aminotransferases involves the abstraction of the pro-S hydrogen on C-4 of 4-aminobutyrate. Decarboxylation of L-glutamate by rat brain glutamate decarboxylase occurs with retention of configuration. Inhibition of this enzyme by (S)-4-aminohex-5-ynoic acid involves the abstraction of the proton at C-4 of the inhibitor. On the basis of this finding, we postulate the existence of an abnormal reaction of glutamate decarboxylase in which the proton at C-4 of (S)-4-aminohex-5-ynoic acid is removed in a manner similar to the one which normally occurs in enzymatic transaminations of L-amino acids. This reaction is presumably facilitated by the acetylenic group adjacent to the eliminated proton.


Deuterium isotope effects on papain acylation. Evidence for lack of general base catalysis and for enzyme--leaving-group interaction.

The experimental data presented in this paper comprise kinetic deuterium isotope effects on acylation of papain with various substrates when conducted in H2O and 2H2O. With alkyl esters of N-acylamino acids there is no or very little isotope effect, whereas with N-acylamino acid amides the ratio kappa H2O/kappa 2H2O is less than 1, i.e. there is an inverse isotope effect. Similarly, alkylation of papain with methyl bromoacetate exhibits no kinetic isotope effect, whereas for the analogous alkylation with bromoacetamide an inverse isotope effect is observed. It is concluded that (a) general base catalysis does not occur in the acylation of papain and (b) kinetic deuterium isotope effects can be affected substantially by interaction between the substrate leaving group and the enzyme, which has not been considered in previous mechanistic investigations.


Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state.

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives. In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier. Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 microM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing role of the catalytic metal ions. The enzyme immobilised on agarose, with unchanged content of active sites [Schneider-Bernlöhr et al. (1978) Eur. J. Biochem. 41, 475--484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogenous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.


Purification and characterization of a protein factor that reverses the inhibition of protein synthesis by the heme-regulated translational inhibitor in rabbit reticulocyte lysates.

We have purified and partially characterized a supernatant factor which reverses the effect of the heme-regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti-inhibitor restores protein synthesis activity in heme deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme. The factor has no effect on the phosphorylation of eIF-2 by the inhibitor nor on any reaction carried out with purified initiation factors. The anti-inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF-2. A possible mechanism of action is discussed.


Sodium and calcium binding to Panulirus interruptus hemocyanin as studied by 23Na nuclear magnetic resonance.

Addition of Panulirus hemocyanin to NaCl solutions produces marked changes in the 23Na relaxation parameters; they show that sodium ions interact with binding sites on the protein and exchange rapidly with the bulk. The observed non-lorentzian lineshapes and the non-exponential decay of the transverse magnetization indicate that non-extreme narrowing conditions apply and give information on the dynamics of the interaction. Panulirus hemocyanin has at least two classes of Na+ binding sites; the binding constant of the more strongly bound sodium ions is in the order of 1 X 10(2) M-1. Competition between Na+ and Ca2+ for protein binding sites is demonstrated by the effect of Ca2+ on the 23Na relaxation parameters. However, only the more strongly bound Na+ are displaced by Ca2+. The number of Ca2+ needed to displace these sodium ions is 3--5 per oxygen binding site. The 23Na relaxation parameters are influenced also by the state of oxygenation of the protein, indicating a linkage between Na+ and oxygen binding. The simplest interpretation of the data is that sodium ions bind more strongly to oxyhemocyanin in agreement with oxygen equilibrium experiments.


Chloroplast phosphoproteins. Phosphorylation of polypeptides of the light-harvesting chlorophyll protein complex.

When isolated, intact chloroplasts of pea (Pisum sativum) are incubated in the light with [32P]-orthophosphate, isotope is incorporated into several polypeptides. Among the most conspicuous phosphoproteins are two which form a very closely spaced doublet on dodecyl sulphate/polyacrylamide gels and co-electrophorese with the major polypeptide component of the light-harvesting chlorophyll a/b binding complex. Like the light-harvesting polypeptide, the phosphoprotein doublet is bound to thylakoids, sediments with the heavy particles released from thylakoids after digitonin treatment, is soluble in chloroform/methanol and has an apparent molecular weight of about 26 000. The doublet also appears in the highly purified light-harvesting chlorophyll a/b binding complex isolated from thylakoids by hydrosylapatite chromatography. I conclude that two polypeptide components of the complex are phosphorylated. One of these components may be the major light-harvesting chlorophyll a/b binding protein.


THE DNA components of the chicken genome.

The organization of the chicken genome was investigated by centrifuging chicken DNA (Mr = 57 X 10(6) in preparative Cs2SO4/Ag+ and Cs2SO4/BAMD density gradients [BAMD = 3.6-bis(acetato-mercurimethyl)dioxane]. An analysis by CsCl density gradient of the DNA fractions obtained from the preparative experiments revealed that 88% of the genome is made up of four DNA components, characterized by buoyant densities of 1.699, 1.702(5), 1.704(5) and 1.708 g/cm3 and representing 39%, 25%, 15%, and 9%, respectively, of the total DNA. The remaining 12% of the genome is formed by seven minor and/or satellite components. The distribution of the ovalbumin gene in a Cs2CO4/BAMD density gradient, as tested with a cloned cDNA probe, coincides with the distribution of the 1.702(5)-g/cm3 component. This shows that the DNA regions flanking the ovalbumin gene are homogeneous in base composition over along distances and that the gene is located on a DNA segment belonging to the 1.702(5)-g/cm3 component.


Isolation and purification of a neurodepressing hormone from the eyestalk of Procambarus bouvieri (Ortmann).

1. A neurodepressing hormone has been isolated and purified to homogeneity from aqueous extracts of 2000 eyestalks of the Mexican crayfish Procambarus bouvieri (Ortmann). 2. Purification was achieved by gel filtration on Sephadex G-25 and G-15, and preparative paper electrophoresis at four pH valueimately 1200 molecular weight and composed of neutral amino acids. 5. No N-terminal group could be found. From its electrophoretic behavior it is concluded that the C-terminal group is also blocked.


Enrichment and characterization of the DNA coding for vitellogenin in Xenopus laevis.

Purified vitellogenin mRNA of Xenopus laevis was incubated with mechanically sheared DNA in high concentrations of formamide and the resulting R-loops (i.e. RNA . DNA hybrid fragments) separated from the bulk DNA by caesium chloride buoyant density centrifugation. Hybridization with 125I-labeled vitellogenin mRNA revealed a 15--30-fold enrichment of the DNA coding for vitellogenin. Restriction analysis of the R-loop-enriched DNA demonstrated that all known endonuclease HindIII fragments coding for vitellogenin of unfractionated Xenopus DNA were also present in the enriched material, including the specific fragments for the oligo(A)-containing segment of the RNA. Comparison of these restriction data with the structure found in cloned vitellogenin cDNA, indicates the presence of at least one intervening sequence in the genomic DNA coding for vitellogenin.


Amino acid sequence of the aminoterminal segment of dermatosparactic calf-skin procollagen type I.

The N-terminal procollagen peptide of the pN alpha 1(I) chain from dermatosparactic calf skin contains 139 amino acid residues. For the determination of the amino acid sequence the procollagen peptide was treated with pyroglutamate aminopeptidase, protease from Staphylococcus aureus V8 and  trypsin. The fragments obtained were separated by molecular sieve and ion-exchange chromatography and submitted to automated Edman degradation. The procollagen peptide consists of three segments, an N-terminal globular domain which contains all the cysteine residues and most of the hydrophobic residues present in the entire peptide, a triple helical part with a relatively high content of proline and hydroxyproline, and a short nonhelical region which forms the connection to the nonhelical region of the alpha 1(I) chain and which contains the proline-glutamine bond specifically split by the N-terminal procollagen peptidase during conversion of procollagen to collagen.


A rare genetically determined variant of psuedocholinesterase in two German families with high plasma enzyme activity.

Activity of pseudocholinesterase (acylcholine-acyl-hydrolase) elevated up to four times has been detected in sera of members of two German families. The catalytic concentrations of the pseudocholinesterase of the afflicted members of both families (male and female) varied between 4800 U/l and 10 200 U/o (acetylthiocholine iodide substrate). The pseudocholinesterase of the propositi exhibits isoenzyme separation patterns in polyacrylamide electrophoresis as well as in electrofocussing which are different from those of pseudocholinesterase from normal persons. No differences could be seen as regards the Km of substrates or the inhibition by dibucaine, fluoride or succinyldiocholine.


Characterization of human platelet proteins solubilized with Triton X-100 and examined by crossed immunoelectrophoresis. Reference patterns of extracts from whole platelets and isolated membranes.

Whole human platelets and platelet membranes have been solubilized in 1% Triton X-100, and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies raised against either whole platelets or isolated membranes. 90% of the platelet proteins were solubilized by this extraction. About twenty immunoprecipitates were observed using the extracts obtained from whole platelets, whereas normally eight immunoprecipitates were seen with extracts from isolated membranes. Albumin, factor VIII and fibrinogen were identified with monospecific antibodies. Correlation of the patterns obtained for platelets or membranes was obtained by addition experiments, by crossed-line immunoelectrophoresis and by crossed immunoelectrophoresis of a mixture of extracts from unlabeled whole platelets and membranes isolated from platelets labeled by lactoperoxidase-catalyzed 125I iodination. Four sialoglycoproteins were identified by their reduced electrophoretic migration after neuraminidase treatment, and six proteins interacted with various lectins, indicating them to be glycosylated. Seven amphiphilic proteins were identified by charge-shift crossed immunoelectrophoresis, and nine by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose. The topographical arrangement of the membrane proteins was examined with lactoperoxidase-catalyzed 125I-labeled platelets as antigens, and by antibodies absorbed with a suspension of whole platelets. Four and six radioactively labeled precipitates could be identified using the platelet and membrane extracts, respectively, indicating them to be exposed at the outer platelet surface. This was confirmed by the use of antibodies absorbed with intact platelets.


Growth patterns of rat hepatocytes during postnatal development.

During postnatal growth in the liver of the rat, a characteristic shift towards binuclear cells and cells of higher ploidy class occurs. When the protein content of individual isolated hepatocytes of different ploidy classes is analysed cytophotometrically using the specific protein stain Naphthol Yellow S, it appears that the growth in mass in the period 30-99 days is due mainly to increase of protein content of binuclear diploid (BD) and mononuclear tetraploid (MT) cells. The mononuclear diploid (MD) cells play a quickly diminishing role in the parenchymal population after the initial growth phase and cells of highest ploidy degree remain unimportant quantitatively. The quickly growing BD and MT cells only reach a Naphthol Yellow S protein value twice that of MD cells after a certain period of growth, whereas changes in protein content are slight or absent from 99 days onwards in all cell types investigated.


[The ultrastructure of epithelial and myoepithelial oncocytes in the human parotid gland (author's transl)].

Oncocytes are well known to occur in the tissue of many organs for over 30 years. Their origin and function are less well known. We observed different types of oncocytes in a human parotid gland with the electron microscope. Besides a hyperplasia of mitochondria they show more or less advanced signs of membrane degeneration of these organelles. On the basis of our findings we discuss oncocyte formation as a special form of cellular aging in an otherwise healthy gland.


Stereo-electron microscopy and energy-dispersive x-ray analysis of avian reticulocytes.

Utilizing the techniques of stereo-electron microscopy (stereo-EM) and energy-dispersive x-ray analysis (EDX), we have studied aspects of the ultrastructure of avian reticulocytes. Stereo-EM of thin sections (0.10 to 0.25 mum thick) stained with uranyl and lead revealed the three-dimensional arrangement of 25 nm chromatin fibers on the tangential surfaces of nuclei. Use of the Bernhard staining procedure in combination with stereo-EM permitted a three-dimensional view of the interchromatin spaces and channels leading to the nuclear pores, and of cytoplasmic polyribosomes. With either staining technique we frequently encountered in the cytoplasm clusters and paracrystalline arrays of electron-dense granules with granule diameters approximately 1/2 that of monomer ribosomes. These granules were highly electron-dense in unstained specimens and have been identified as intracellular ferritin on the basis of the similarity of their ultrastructural morphology to that of horse spleen ferritin and their high content of iron as determined by EDX. The possibility that these granules represent toxic products of phenylhydrazine treatment (Heinz bodies) is considered unlikely, since we have demonstrated in this study that Heinz bodies cannot be visualized in unstained preparations, do not reveal the same granular structure, and do not contain significant amounts of iron above background. The occurrence of intracellular ferritin is discussed in light of current concepts of iron transport and storage during erythropoiesis.


The effects of nicotine on cell division of HeLa cells.

A time lapse cinemicrographic study shows that, at low concentrations, nicotine can speed up cytokinesis and, at high concentrations, prolong the duration of metaphase in HeLa cells. Based upon the known effects of the alkaloid in other systems, it appears that it may stimulate microfilament activity and inhibit microtubule function by increasing the level of cytosolic calcium ions.


Cylindrical laminated bodies in nickel-subsulphide-induced rhabdomyosarcoma in rabbits.

The induction of rabbit rhabdomyosarcoma was obtained after intramuscular implantation of a large quantity of very pure nickel subsulphide, though until the present time the rabbit was considered refractory to Ni3S2 tumorigenesis. These tumors are similar to those induced in rats under the same conditions. Four different cell types were observed: small polygonal cells, small elongated cells, giant cells, and mature myofibers. Electron microscopy reveals a complete disorientation of myofibrils in mature myoblasts. Giant cells appear by pluripolar endomitosis and always contain myofibrillar structures, but M-lines and Z-lines are not present in these cells. Cylindrical laminated bodies were observed very often in all four cell types. They are formed of 4 nm fibrils arranged in crossed position in each lamella. Some of these paracrystalline structures were also observed in nuclei. The laminated bodies are considered to be abnormal formations of contractile proteins produced during tumoral myofibrillar differentiation.


Go and Go,2 cells as identified by their chromatin pattern in dormant and proliferating meristems.

Microdensitometry at different density thresholds of Feulgen-stained nuclei shows that both 2C and 4C dormant cells of the unsprouted root of Allium cepa L. bulbs have a smaller nuclear volume and a larger amount of dense chromatin than their counterpart G1 and G2 cells of the proliferating root. The analysis seems to differentiate between cells in the proliferate compartment from those in the non-proliferative compartment (Go and Go,2 cells).


Effects of oxybarbiturates on the germination behaviour of Impatiens bolstii pollen.

Swelling of and cytoplasmic streaming within the grain, germination pore expansion, and pollen tube emergence, in that order, constitute the normal germination pattern of Impatiens holstii pollen grains. This normal sequence of events becomes manifest when the pollen grains are placed on water-agar, barbituric acid-agar, and on barbital-agar media whereas this sequence of events is interfered with when the pollen grains are incubated on amytal-agar or on seconal-agar media. Evidence is presented which demonstrates that the normal sequence of events which make up the normal germination pattern of Impatiens holstii pollen grains can be separated from each other by varying the concentrations of the oxybarbiturates amytal and seconal used. Evidence is also presented which indicates that the actions of barbituric acid and the oxybarbiturates barbital, amytal, and seconal on Impatiens holstii pollen germination behaviour correlate well with certain aspects of the pharmacological literature concerning the actions of these organic compounds on the central nervous system (CNS).


Helically twisted filaments in giant neurons of a whip spider.

In giant neurons of whip spider legs several filament types are detectable: filaments of 5 to 6 nm thickness as dense masses within the soma of the neuron, an intermediate-sized filament type limited to the dendritic processes forming irregularly wound bundles and finally twisted double filaments in the soma as well as in peripheral regions. The latter are usually aggregated in paracristalloid lattices of different length and diameter.


Effect of other drugs and chemicals on the degradation of aspirin in vitro: possible extrapolation to in vivo metabolism of aspirin.

To assess the possible influence of other drugs and chemicals on the metabolic degradation of aspirin in man, their effect on the serum aspirin esterase activity was determined in vitro. The activation or inhibition of the enzyme as observed with these compounds suggest the possibility that simultaneous ingestion of these drugs with aspirin may influence the pharmacology and toxicity of the analgesic.


The metabolism of saddamine in man: the fate of the benzylamine moiety of saddamine.

The metabolism of 14C-saddamine radiolabelled in the benzylamine moiety has been investigated in man after oral administration. 14C-Hippuric acid was found to be the only radiolabelled urinary metabolite, the overall amount of which was equivalent to over 95% of the dose. The time taken for elimination of 50% of the dose in the urine was approximately five hours and the corresponding value for 14C-benzylamine hydrochloride was less than one hour.


Pharmacokinetic parameters of 14C oxithiopurinol in rat.

The study with labelled compound has shown that in rat, as in man, thiopurinol is primarily and rapidly converted into oxithiopurinol. More than 80% of the radioactivity of plasma was identified as oxithiopurinol less than 10 minutes after intravenous or oral administration. The parameters of oxithiopurinol were estimated from the decline of radioactivity in blood from 5 minutes to 8 hours after intravenous administration of 4 microCi/100 g of thiopurinol (8 animals) or oxithiopurinol (5 animals). Half lives of oxithiopurinol t 1/2 (beta) were 2.52 +/- 0.78 hr and 2.90 +/- 1.00 hr and clearance 2.00 +/- 0.36 ml/min. and 3.38 +/- 0.69 respectively. Estimate of renal clearance for oxithiopurinol in rat was 4,17 ml/min. The rapidity of hydroxylation and of elimination of oxithiopurinol might be assumed to be the cause of the apparent ineffectiveness of thiopurinol in vivo on xanthine oxidase activity.


In vivo studied on the metabolism of dibenzo [c,f]-[1,2] diazepine.

The in vivo metabolism of dibenzo [c,f]-[1,2] diazepine (I) was studied after i.p. administration to rats, in order to observe the influence of the heteroatom on the metabolic pathway. Metabolites were separated by chromatography and their structures elucidated by mass spectrometry, by both gas chromatography coupled with mass spectrometry and the direct inlet system. Six metabolites were identified, some of them by direct comparison with authentic samples.


Prednisolone absorption in coeliac disease.

The pharmacokinetic disposition of prednisolone was studied following oral administration (10 mg) to eight patients with untreated coeliac disease of mild or moderate severity, seven coeliac patients on a strict gluten-free diet and ten normal subjects. No significant differences were shown in the peak prednisolone level, the time of the peak level, the area under the concentration versus time plot, the plasma half-life and the 24 h urinary recovery of prednisolone in the three subject groups. There was however considerable variability within each group. It is concluded that the presence of coeliac disease does not significantly alter the absorption or elimination of prednisolone.


The fate of adenosine monophosphate in the rat after a single oral administration. Influence of a simultaneous treatment by papaverine.

The fate in the organism of Adenosine Monophosphate after a single oral administration of 10 mg/kg, was studied in the Rat, using 14C-labelled AMP. This paper deals more specially with the study of the plasmatic kinetics of AMP, since we proved at first that the AMP molecule goes intact through the intestinal barrier into the blood. The effects on these kinetics of a joint administration of Papaverine are considered.


CT evaluation in patients with transient ischemic attack. Correlation between clinical and angiographic findings.

35 patients clinically diagnosed with transient ischemic attack (TIA) and 15 patients clinically diagnosed with reversible ischemic neurological deficits (RIND) were examined by computerized tomography (CT). 34% of TIA patients showed positive CT scans consisting of small hypodenase areas: 60% of RIND patients had larger lesions. In 20% of the overall group the only pathological finding consisted of unilateral focal atophy or ventricular asymmetry. Correlation between angiographically demonstrated atherosclerotic lesions and positive CT was high. On the contrary, no correlation was found between either the risk factors or the natural history of ischemic attack and positive CT findings.


Does anoxemia play a role in the effects of neonatal seizures on brain growth? An experimental study in the rat.

Groups of 4 Wistar rat littermates of matched sex and weight received the following daily treatment between the ages of 2 and 11 days: handling (untreated controls, UC), succinylcholine paralysis and ventilation with 100% O2 (respirator control, RC), electroconvulsive seizures (ECS) and ECS while paralyzed and ventilated with O2 (respirator seizures, RS). Analysis of heart blood O2 content showed that mechanical ventilation with 100% O2 effectively prevented the anoxemia observed during convulsive seizures. In the ECS and RS groups, several behavioral milestones (e.g. swimming) matured later than they did in either control group (UC, RC). No difference was observed between the two groups of seizure-treated rats. At the age of 30 days, both ECS and RS groups had smaller brains with a reduced DNA, RNA, protein and cholesterol content in both forebrain and hindbrain, suggesting that seizures curtailed the number of brain cells. The lack of any difference between the two seizure groups suggests that at least part of the adverse effects of experimental neonatal seizures on brain and behaviour are independent of anoxemia.


Post-convulsive spinal epidural haematoma in ankylosing spondylitis.

A case of post-convulsive spinal spidural haematoma in a patient with ankylosing spondylitis is presented. As acute tetraplegia developed, surgery was performed with finding of blood clots in the extradural space from C6 to D8. Lethal evolution due to late referral to medical care stresses the need for prompt decompression of the injured spine.


Evoked potentials in neuronal ceroid lipofuscinosis.

Nerve conduction, EEG, visual evoked potentials, electroretinograms and somatosensory evoked potentials were investigated in 3 children with the Bielschowsky-Jansky-type and in 1 child diagnosed as Spielmeyer-Vogt-type of neuronal ceroid lipofuscinosis. Electroretinographic responses were abolished in all of them. Electroencephalograms showed high amplitude, irregular delta-theta activity and spike- or polyspike-wave discharges without localized preponderance. As a characteristic feature for the Bielschowsky-Jansky type grossly enlarged evoked responses to single light flashes were recorded. Somatosensory evoked potentials were increased in amplitude in 2 patients. Myoclonic jerks of the pyramidal type could be elicited by electrical stimuli to the median nerve. The possibility to differentiate certain neurometabolic disorders of childhood by simple electrophysiological parameters is discussed.


Diagnostic validity for different components of pattern shift visual evoked potentials in multiple sclerosis.

Visual evoked potentials, elicited by checkerboard-pattern reversal were recorded from multiple sclerosis patients with different probabilities of diagnosis. Latencies and amplitudes of the different components were analyzed for diagnostic validity. It was demonstrated that the latency of the main positive peak (P2) was the most sensitive parameter. But in a few cases determination of P2 latency and interocular difference of this peak alone failed to detect involvement of the visual system. Additional evaluation of absolute latencies, asymmetries in latency and amplitudes of other components would give evidence in predicting abnormalities of the visual pathways.


Capillary circulation and morphology in Duchenne muscular dystrophy.

Muscle blood flow (MBF) and capillary diffusion capacity (CDC) were determined in 8 patients with Duchenne muscular dystrophy and 5 age-matched control males by measuring the simultaneous clearance of 133Xe and 131I-. MBF was significantly decreased in older patients with severe dystrophy: 35.1 +/- 5.8 ml/100 g/min in 4 advanced Duchenne patients and 45.8 +/- 6.5 ml/100 g/min in controls (p less than 0.005). MBF in 4 early cases did not differ from controls (44.0 +/- 7.9 ml/100 g/min). There was no significant difference in CDC in Duchenne patients and controls. No structural abnormalities in muscle microvasculature were found by light microscopy. In the electron microscope, the Duchenne basement membranes had duplicated or multiple layers of electron-dense material in 63.9% of muscle capillaries while only 6.6% of the control capillaries showed this. These results do not support an ischemic etiology of Duchenne muscular dystrophy.


Ephaptic recurrent response in man.

The antidromic impulse associated to the ephaptic response can produce a recurrent wave (Fe). Its latency, which is longer than that of the F wave, will augment if the site of stimulation is remote from the muscle. It is therefore a direct-indirect response. The 'Fe' wave is similar to the 'S' response described by Thorne, but the gamma-loop is not implicated. It can be misinterpreted as a desynchronized F wave.


Ephaptic response in man.

Following the direct M response, peripheral nerve stimulation frequently evokes a second wave which shows all characteristics of an ephaptic response. The antidromic impulse or back response, which is always associated, is detected by a double stimulation technique. The ephaptic response seems not to be related to any specific nerve or muscle pathology, as it is also observed in normal subjects. It is therefore important to differentiate the ephaptic response from a late potential or a double discharge, the latter two having pathological significance.


Lithium therapy in cluster headache.

The authors used lithium carbonate in the treatment of 15 patients with cluster headache (chronic and episodice form). Disappearance of attacks was achieved in 5 patients (1 chronic and 4 episodic), significant improvement in 5 (4 chronic and 1 episodic) and in 4 cases (2 chronic and 2 episodic) treatment was ineffective.


Free fatty acid and glucose metabolism during hypothermic perfusion of canine kidneys.

Canine kidneys were subjected to continuous nonpulsatile perfusion using 200 ml of a perfusate containing 50 g/l albumin. When optimal oxygenation was achieved, perfusate K+ contents were unchanged for 24 h, indicating adequate membrane function but tended to increase thereafter. Lowered oxygen pressures resulted in significant cellular K+ loss during the first hours of perfusion. During oxygenated perfusion, glucose and free fatty acids (FFA) were oxidized in considerable amounts with a preferential consumption of octanoate. A capacity for long-chain FFA oxidation became obvious when the octanoate had been used up, but the amount of these FFA in the perfusate depended preferentially on FFA being liberated from tissue lipids during the 1st day of perfusion. Glucose consumption rates were highest during the first 2 days of perfusion but the subsequent reduction of the metabolic rate was not accompanied by an accumulation of lactate. Thus medium-chain FFA and glucose should be supplied to the continuously perfused kidney in hypothermia and optimal oxygenation of the perfusate should be guaranteed. However, it seems to be unnecessary to supply exogenous long-chain FFA.


Healing of enterotomies with preexisting peritonitis. An experimental study in guinea pigs.

The question of whether the presence of peritonitis has any influence upon the process of healing and the load capacity of a standard enterotomy was studied in guinea pigs. Laparotomy was performed 4 h after induction of peritonitis by a standardized method. Fecal peritonitis could be confirmed clinically, histologically and bacteriogically in all cases. A standardized enterotomy was carried out. The animals were killed 3 or 5 days later, anastomoses inspected, and the intraluminal pressures for anastomotic rupture measured. Comparison with a control group showed: (1) there can be normal healing of an enterotomy even with coexisting peritonitis; (2) anastomotic breakdown takes place at the same pressure as in the control group (p less than 0.01), and (3) primary lethality is significantly higher in cases operated in the presence of peritonitis (33 vs. 20%).


Homologous blood syndrome in dogs. Effect of erythrocyte and platelet antibodies.

Isovolemic perfusion of 50% of the recipient circulating blood volume was carried out in intact mongrel dogs using whole donor blood as well as plasma or platelet suspensions in saline. Perfusion against the background of circulating red blood cells (RBC) or platelets antibodies in the donor or in the recipient was followed by severe and sometimes fatal homologous blood syndrome in a majority of the dogs. Weak and short-term reactions were seen in a few dogs if red blood cells and platelet antibodies were absent.


Repreoducibility of a method for a graded stenosis in tubes and vessels of small calibres. An in vitro and in vivo experiment.

The reproducibility of a previously described method for graded stenosis has been tested in a model experiment and by repeated stenosis of the portal vein in rats. In the model experiment, the method was tested in terms of flow variations; in the animal experiment, in terms of prestenotic pressure variations. In the model experiment, the coefficient of variation in repeated stenosis was less than 3%. In the animal experiment, repeated stenosis constantly produced the same high level of pressure. The present method is discussed in relation to a theoretical optimal standard and found to be satisfactory.


Prehepatic portal hypertension in the rat. Immediate and long-term effects on portal vein and aortic pressure of a graded portal vein stenosis, followed by occlusion of the portal vein and spleno-renal collaterals.

The creation of a portal vein stenosis with a diameter of 1.2 mm in Wistar rats resulted in a sustained elevation of the portal vein pressure to approximately twice the control value lasting for at least 8 weeks. The alterations in portal vein pressure following occlusion of the stenosed portal vein and splenorenal collaterals, either separately or in combination, indicate that a considerable quantity of portal blood is diverted into the inferior caval vein through the splenorenal collaterals, and that the development of these collaterals occurs during the first 3-4 weeks after the establishment of the stenosis. The concomitant reduction in aortic pressure and increase in heart rate following total occlusion of the portal vein were most pronounced during the first weeks after stenosis, and were probably due to diminished venous return to the heart.


Lysolecithin analogs as adjuvants in delayed-type hypersensitivity in mice. II. Studies on the mode of action.

Lysolecithin analogs (LLA) possess adjuvant activity in delayed-type hypersensitivity (DTH). To elucidate their mode of action, we studied the influence of LLA on the recruitment of hemopoietic progenitor cells and on the induction and transfer of DTH. Whereas the number and composition of colony-forming cells (CFC) in the bone marrow remained unaltered in LLA-treated mice, the number of CFC in the spleen was augmented severalfold, and the content of macrophage progenitors was remarkably increased. DTH was induced with macrophages from peritoneal cells (PC) pulsed with keyhole limpet hemocyanin (KLH). A more marked DTH response was obtained with KLH-pulsed PC from LLA-treated mice than from mice given phosphate-buffered saline (PBS). On the other hand, there was no difference in the DTH response following adoptive transfer of sensitized cells to naive mice pretreated either with PBS or LLA. It is concluded that the adjuvant effect of LLA can be accounted for partly in terms of their effects on macrophage progenitors and partly in terms of a more effective T cell activation by antigens associated with LLA-derived macrophages.


Ischemic-induced alterations in cardiac sensitivity to digitalis.

Intravenous infusion of acetylstrophanthidin to 6 dogs, after a 60 min left anterior descending coronary artery occlusion, was associated with a 43.0 +/- 10.5% decrease in the dose of digitalis needed to produce ventricular arrhythmias as compared to the pre-ischemic dose (97.5 +/- 8.0 microgram/kg). Reperfusion of the ischemic region for 2 h after a 90 min occlusion resulted in a 54.4 +/- 6.7% decrease in the arrhythmogenic dose. Direct intracoronary infusions of digitalis into the ischemic region, after a 90 min coronary occlusion followed by 2 h of reperfusion, was associated with a 47.7 +/- 6.4% decrease in the dose of digitalis needed to produce arrhythmias. The pre-ischemic (control) arrhythmogenic dose of digitalis via the intracoronary infusion method was 1.5 +/- 0.3 microgram/kg (mean +/- S.E.M. of 7 dogs). Sodium pump activity, estimated from the ouabain-sensitive 86Rb uptake in sodium-loaded ventricular slices, was significantly higher in slices obtained from the ischemic regions (6.84 +/- 0.30 nmoles 86Rb/mg dry wt. (mean +/- S.E.M.), than from the non-ischemic regions (3.43 +/- 0.64 nmoles 86Rb/mg dry wt.). Sensitivity of the sodium pump activity to the inhibitory effect of ouabain also was increased in the ischemic regions as indicated by a shift in the log dose--response curve to the left. Thus, it appears that there is an increase in myocardial sensitivity to the toxic effect of digitalis after temporary ischemia and it appears to be related to an increase in the sensitivity of the Na+,K+-ATPase or sodium pump to the inhibitory effect of digitalis.


Immobility induced by forced swimming in rats: effects of agents which modify central catecholamine and serotonin activity.

Rats were forced to swim in a restricted space will rapidly cease apparent attempts to escape and adopt a characteristic posture which we have termed "immobility". We show in previous experiments that immobility was reduced by a variety of antidepressant agents and thus suggested that the method could serve as a screening model for antidepressants. The present experiments showed that immobility was reduced by drugs which increase central dopaminergic and alpha-adrenergic activity but was less affected by drugs which act mainly on central serotonin. Conversely, immobility could be increased by drugs which diminish central catecholamine activity but not by drugs which inhibit central serotonin. It was concluded that immobility depended primarily on the activity of central catecholamines but that caution was required before ascribing immobility exclusively to activity within a single system.


Effect of various antidepressant drugs on the spontaneous firing rate of locus coeruleus and dorsal raphe neurons of the rat.

The spontaneous firing rate of the noradrenergic neurons of the locus coeruleus and of the serotonergic neurons of the dorsal raphe was recorded with extracellular microelectrodes in chloral hydrate-anesthetized rats. A quantitative comparison of the effect of five tricyclic antidepressants, of tranylcypromine and of mianserin on the spontaneous activity of these two types of cells was performed. All drugs tested, except mianserin reduced the frequency of discharge of the noradrenergic neurons. Intravenous perfusion of the drugs allowed the doses required for inhibition of firing to 50% of the baseline rate (ID50) to be determined. Secondary aminated antidepressants (desipramine and nortriptyline) were more potent inhibitors than their tertiary aminated analogues (imipramine, chlorimipramine and amitriptyline). All drugs tested, except desipramine decreased the rate of firing of the serotonergic cells. In this case, the tertiary aminated antidepressants were much more potent than their secondary analogues. Mianserin was only active at very high doses. These results are in good agreement with the relative potencies of the tricyclic antidepressants for blocking the uptake of noradrenaline and serotonin into central and peripheral neurons.


Pre- and postsynaptic localization of central muscarinic receptors.

3H-Quinuclidinyl benzylate and dimethyl 14C-d-tubocurarine were used to localize muscarinic and nicotinic receptors in synaptosomal membranes of the rat cerebral cortex. The results obtained, after the action of different concentrations of Triton X-100, suggest that, while nicotinic receptors are postsynaptic, the muscarinic ones are both pre- and postsynaptic.


Antagonism by droperidol of dopamine-induced relaxation in isolated dog arteries.

Dopamine caused a dose-related relaxation in helically cut strips of dog coronary and renal arteries treated with phenoxybenzamine andcontracted with prostaglandin F2 alpha. The dose-response curve to dopamine was shifted to the right by droperidol in concentrations above 3 X 10(-5) M. Adenosine-induced relaxations were not attenuated by droperidol. The dose--relaxation curve to isoproterenol was also shifted to the right by droperidol. Propranolol (10(-6) M) failed to significantly alter the dose response curve to dopamine, and in propranolol-treated preparations the antagonism by droperidol of dopamine actions was practically identical with that in control preparations. Droperidol appears to act as a reversible, surmountable antagonist to dopamine actions on dog arterial smooth muscles, and such evidence supports the hypothesis of specific dopamine receptors in dog arteries.


gamma-Glutamyl DOPA and gamma-glutamyl dopamine: effect on plasma glucose levels.

gamma-Glutamyl-L-3,4-dihydroxyphenylalanine (gamma-glutamyl DOPA) and gamma-glutamyldopamine (gamma-glutamyl DA) are kidney specific prodrugs. Their effect on plasma glucose levels in the rat was compared to that of L-DOPA and dopamine (DA) after a 30 min intravenous infusion. L-DOPA and DA induced hyperglycemia after 15 min of druginfusion. A more marked and protracted elevation of plasma glucose was observed after infusion of gamma-glutamyl DA. By gamma-glutamyl DOPA had no effect on plasma glucose levels in spite of the high accumulation of DA in the pancreas after this prodrug. Of the various dopamine produrgs studied only gamma-glutamyl DOPA was not hyperglycemic in doses that are known to increase renal plasma flow in the rat. A simplified new procedure for the synthesis of gamma-glutamyl DA is described.


Influence of prazosin on cardiac reflex function.

Prazosin administration significantly decreased arterial blood pressure without affecting heart rate in pentobarbital-anesthetized dogs. Wile pressor and reflex bradycardic responses to angiotensin II were not altered by prazosin, reflex tachycardia produced produced by histamine and acetylcholine were significantly attenuated by prazosin. The failure of prazosin administration to cause an increase in heart rate may be due to the ability of the agent to inhibit reflex tachycardic responses to depressor stimuli, the mechanism of whcih needs to be further investigated.


The actions of 5-hydroxytryptamine on isolated skeletal muscle preparations.

The effects of 5-hydroxytryptamine (5HT) on the mouse phrenic nerve--hemidiaphragm and frog sartorius muscle preparations were studied using twitch tension and intracellular recording methods. Between 1--7.5 mM 5HT transiently augmented the twitch response of the diaphragm to indirect, but not direct, stimulation. Above 5 mM 5HT the augmentation was followed by a concentration-dependent twitch blockade. The blockade was more apparent in anticholinesterase-treated preparations, and was seen to a lesser extent in directly stimulated muscles. Intracellular recording from diaphragm muscles revealed that 5HT depressed the amplitudes of both miniature endplate potentials (m.e.p.p.s.) and endplate potentials (e.p.p.s.), this effect being more pronounced in the presence of neostigmine. 5HT also greatly prolonged the duration of the e.p.p.s. In the sartorius muscles, 1--5 mM 5HT slowed and depressed action potentials. It is suggested that 5HT facilitates transmission by an anticholinesterase action and inhibits by blockade of receptor ion channels.


Trazodone and imipramine: comparative effects on canine cardiac conduction.

Lead II EKG and His bundle electrograms were recorded in closed-chest pentobarbital-anesthetized dogs. Trazodone, a structurally distinct, new antidepressant agent, had no effect on His--Purkinje (HV interval) or intraventricular (HS interval) conduction following intravenous injection at graded doses between 1 and 30 mg/kg. In contrast, the tricyclic psychotropic agent imipramine (0.5--5 mg/kg) significantly slowed impulse conduction as indicated by increases in both the HV and HS intervals. Imipramine (5 mg/kg) also slowed impulse transmission across the atrium (PA interval). These responses to imipramine were associated with a concurrent prolongation of both the PR interval and QRS duration. In the anesthetized dog, trazodone administration promoted no evidence of heart block or sign of rhythm disturbances other than slowing in normal sinus rhythm.


Differential binding of antidepressants to noradrenaline and serotonin transport sites in central nerve endings.

Imipramine and mianserin are equipotent inhibitors of noradrenaline (NA) uptake in synaptosomes. However, after in vivo administration, NA uptake was inhibited only in synaptosomes from imipramine-treated rats, suggesting that imipramine, or its metabolite desipramine, binds to the NA carrier in a manner outlasting the preparation of synaptosomes, whereas mianserin is washed away. To evaluate binding to the NA carrier, synaptosomes prelabeled with 3H-NA were pretreated with an antidepressant and the release of 3H-NA was then stimulated with unlabeled NA. Any reduction of release was taken as an indication of binding. Pretreatment with desipramine, but not with imipramine or mianserin, reduced 3H-NA release suggesting that desipramine is responsible for NA uptake inhibition in synaptosomes from imipramine-treated rats. Transformation of tertiary into secondary amines seems to be crucial for binding to the NA carrier, as confirmed by the stronger binding of nortriptyline and chlordesipramine compared to amitryptiline and chlorimipramine, respectively. In contrast, tertiary amines bound more strongly than secondary amines to the serotonin carrier. Adult and 8-day old synaptosomes showed similar binding properties towards imipramine and desipraine.


Lack of influence of histamine antagonists on digitalis cardiotoxicity.

Experiments were carried out in order to study the interaction between cardioactive glycosides and histamine or histamine H1- AND/OR H2-receptor antagonists in anaesthetized cats or guinea pigs. The toxicity of ouabain or digoxin was tested by infusing the glycosides until idioventricular rhythm or death occurred. Histamine increased the toxicity of ouabain, but the histamine H1- and/or H2-receptor antagonists (metiamide and mepyramine)did not influence the toxicity of digitalis. In cats the increase in pulmonary arterial blood pressure caused by digoxin was not affectedby histamine H1- and/or H2-receptor antagonism in spite of the fact that the increase of pulmonary blood pressure caused by histamine could be abolished by H1-receptor antagonists. Our results indicate that histamine is not involved in the toxic effect of cardiac glycosides.


Serotonin release is modulated by presynaptic autoreceptors.

The existence of presynaptic autoreceptors controlling the release of 5-hydroxytryptamine (5HT) from serotonergic nerve endings was investigated utilizing superfused hypothalamic synaptosomes. Extracellular 5HT reduced the high K+-induced release of previously accumulated 3H-5HT. The central 5HT receptor blocker methiothepin counteracted the inhibitory effect of 5HT. Other 5HT antagonists (cyproheptadine, methysergide and mianserin) were inactive and may therefore act preferentially at the postsynaptic receptors.


Assay for thrombopoietin: a comparison of time of isotope incorporation into platelets and the effects of different strains and sexes of mice.

Several workers have used mice for thrombocytopoiesis-stimulating factor (TSF) assays, but the methods have differed. In quest of the optimum TSF assay conditions, we investigated the effects of interval between isotope injection and measurement of 35S incorporation into platelets, different mouse strains and sexes of mice. The 35S incorporation into platelets of mice increased with increase of the interval after isotope injection. However, the greatest difference in radioactivity between control and TSF-injected mice occurred at 16-40 h for normal mice and 24 h for rebound-thrombocytotic mice. When C3H, BALB/c or B6D2F1 mice were injected with platelet specific antisera, similar degrees of thrombocytopenia and rebound-thrombocytosis occurred. After injection of a standard dose of TSF, C3H and B6D2F1 mice showed greater isotopic incorporation levels, compared to suitable controls, than did BALB/c mice. Female and male mice exhibited essentially the same response to a standard dose of TSF. The data show that the mouse strain and isotopic incorporation time influence the sensitivity of the TSF assay but sex of test animals does not.


Growth patterns, cell composition, and mitotic activity of granulocytic aggregates developing in methyl cellulose from human normal marrow cells.

Marrow cells from five normal healthy subjects were cultured in methyl cellulose with and without leucocyte conditioned medium. Cultures were examined daily from day 3 to day 14 for the number of granulocytic aggregates and for their cell composition and mitotic activity. Aggregates of different size (small and large clusters, small and large colonies) displayed four distinct and characteristic patterns of growth. Unidentifiable blast cells were very rare throughout culture. The average proportion of recognizable granulocyte precursors (promyelocytes and myelocytes) (PMC + MC) declined during culture from 60% to 34%. There was a negative correlation between the size of the aggregates and their PMC + MC content, but on any day of culture some aggregates consisted mainly or uniquely of PMC + MC, while some others consisted mainly or uniquely of non-proliferating granulocytes. The mitotic index of PMC + MC was much higher than the one previously determined in vivo, and was significantly higher in large colonies than in other aggregates. These results are consistent with the recent demonstration of different CFU-C (sub)populations. It is suggested that differentiation of the aggregates according to their size and kinetics, coupled with cytological examination, can provide a more refined assessment of the assay, which could be useful for further comparison of normal, abnormal, and leukaemic granulocytopoiesis.


Classification of animal lymphomas: the implications of applying Rappaport's classification for human lymphomas to experimental tumors.

One hundred and seventy animal lymphomas (species ranging from molluses to monkeys) were reclassified histologically according to the modified Rappaport classification for human lymphomas. The results were correlated with the etiology of the lymphomas, their clinical course, and in selected cases with their immunological type. The study stresses the value of such a procedure for comparative reasons, allowing a more adequate selection of animal models for human lymphomas.


Trochanter bone marrow: a source of normal human colony forming cells.

In vitro studies of human hemopoiesis are often limited by the availability of normal bone marrow. We have overcome this difficulty by taking advantage of the bone marrow fragments removed during total hip replacement. We report here a comparative study of the colony forming capacity of trochanter and sternal or iliac crest marrow from five hematologically normal donors. Our data indicate that trochanter marrow is a reliable source of normal in vitro granulocyte/macrophage colony forming cells.


Hydroxyurea suicide study of the kinetic heterogeneity of colony forming cells in human bone marrow.

The kinetics of granulomonocyte colony forming cells have been studied in unfractionated normal human bone marrow by hydroxyurea suicide and in cells separated by velocity sedimentation. Sequential studies revealed two subpopulations of colony forming cells, having different sizes and different multiplication potentialities. There are large cells with a high suicide rate which develop small granulocyte and monocyte colonies during the first week of culture in semi-solid agar. Smaller cells develop larger colonies of granulocytes, monocytes and eosinophils between 2 and 3 weeks of culture. Only granulocyte progenitors have a substantial suicide rate in this small cell population. This population is also less responsive to stimulation than is the large cell class, which is a more highly differentiated progeny. The role of these different kinetics of colony forming cells is discussed in the context of the heterogeneity of the in vitro differentiation of neutrophil, monocyte and eosinophil lines.


An improved plasma culture system for the production of megakaryocyte colonies in vitro.

A modified culture system has been developed to grow and quantitate megakaryocyte colonies from mouse bone marrow more efficiently than described in other reports. Using this method, it was shown that 30% of CFU-M in normal marrow cells and 70 to 90% of CFU-M in regenerating marrow cells were killed by high specific activity 3H-TdR in vitro. These results indicate that CFU-M are proliferating even in normal adult hemopoietic tissue, but that the proportion of cells that are proliferating is greater in regenerating marrow than in normal intact mice.


Antiserum to mouse hematopoietic pluripotent stem cells (CFU-S): further investigation of its functional properties.

Treatment of mouse hematopoietic cells with heterologous antiserum raised against mouse brain markedly reduces the capacity of pluripotent stem cells (CFU-S) to form surface spleen colonies in lethally irradiated mice. To exclude the possibility that such treatment interferes only with the capacity of CFU-S to form surface spleen colonies, we evaluated the capacity of CFU-S which were treated with rabbit anti-mouse brain serum (RAMBS) to restore hematopoiesis and rescue lethally irradiated mice, and to form microscopic spleen colonies. Marrow cells were treated with RAMBS or control rabbit serum. Fifty thousand treated nucleated cells were injected i.v. into lethally irradiated mice, and hematopoietic reconstitution was studied between days 8 and 15; separate groups of mice were observed for survival. We found that treatment with RAMBS impairs the capacity of marrow cells to repopulate the marrow and spleen with CFU-S, to restore blood RBC, to effect an overshoot in spleen weight, and to prolong survival; in addition it reduces the number of microscopic spleen colonies to the same extent that it reduces the number of macroscopic colonies. Hence, RAMBS appears to effect a general inactivation of CFU-S and should prove to be a useful tool in further investigations of mouse hematopoiesis.