Endoscopic lithotripsy in the common bile duct.

After appropriate preliminary experiments in animals and in corpses, electrohydraulic lithotripsy has been used for the first time in humans in an attempt to destroy stones in the bile duct. For this purpose, a lithotripsy probe combined with a Dormia basket was constructed. To date, this method has been successfully applied to break fragments off large calculi in three patients, without any complications arising. In one case, subsequent extraction of the remaining part of the stone was possible. The probe must, however, be improved to ensure that the stone is always held centrally in the basket.


A snare for the endoscopic extraction of coins.

A special snare is described, by which especially coins can be easily removed from the upper GI tract of children and adults without general anesthesia.


The influences of phenytoin on the fundamental electrical properties of simple neural systems.

The effects of phenytoin on some neurophysiological properties of simple neuronal systems are reviewed. From all the available data phenytoin decreases or has no effect on post-tetanic hyperpolarization, which is interpreted as an expression of the electrogenic pump. Although in some neurons the membrane conductance is increased, the resting membrane potential is minimally affected. The effect on the action potential varies with different preparations and with different neurons of the same ganglion. If an effect is present, the overshoot is decreased or the falling phase is prolonged, or both. Post-synaptic potentials are also affected by phenytoin. EPSPs are decreased in size, while the chloride-dependent, GABA-mediated IPSPs of the crayfish stretch receptor are prolonged. No effect was seen on chloride-dependent, ACh-mediated IPSPs in the abdominal ganglion of the Aplysia. Finally, phenytoin arrests endogenous or pharmacologically induced bursting. Most of the described effects are consistent with the antiarrhythmic and antiepileptic properties of the drug.


Effects of phenytoin on the release of 14C-adenine derivatives.

The release of 14C-containing compounds from rat cortical slices prelabeled with 14C-adenine consisted largely of adenosine (6-7%), inosine (13-18%), and hypoxanthine (70-74%), with small amounts of nucleotides including cyclic AMP and adenine. This efflux was increased by both ouabain (0.1 mM) and veratridine (0.05 mM), the increment in released radioactivity consisting almost entirely of these three compounds. However, relatively more inosine than adenosine output was evoked by ouabain while the reverse was true with veratridine. Phenytoin partially reversed the effect of both depolarizing agents. After prelabeling, the efflux from astrocytoma cell cultures contained predominantly inosine (74%) and hypoxanthine (23%) with little adenosine. Ouabain increased the release of 14C-adenine derivatives, and this increase was diminished by phenytoin. Preliminary studies with neuroblastoma cell cultures have shown considerable variability in the composition of the effluent, with hypoxanthine the prevalent compound and almost no adenosine. Ouabain enhanced the efflux from these cells, and this effect was apparently reversed by phenytoin.


Evaluation of a new immunoassay for determination of phenytoin and phenobarbital: results of a European collaborative control study.

The performance of a new enzyme multiplied immunoassay technique (EMIT) was compared with other current methods, namely gas-liquid chromatography and thin-layer chromatography, for the determination of phenytoin and phenobarbital in serum. Forty-three serum samples were sent as unknowns to the participating laboratories for determination. The precision of repeated determinations was very similar for EMIT and chromatography. There was good agreement among gas chromatography, thin-layer chromatography, and EMIT results. Interlaboratory variability was lower for EMIT determinations of both antiepileptic drugs. The rapid analysis of small samples, made possible by the EMIT system, could have beneficial effects on the treatment of epilepsies.


Cerebellar atrophy in phenytoin-treated mentally retarded epileptics.

The relationship among the serum concentration of phenytoin, pneumoencephalographic measurements describing, in particular, cerebellar atrophy, and various other clinical variables was analyzed statistically in a series of 131 phenytoin-treated mentally retarded epileptics. Phenytoin intoxication was diagnosed retrospectively in 73 patients (56%), of whom 18 had persistent loss of locomotion. The mean duration of phenytoin intoxication until locomotion was lost was 22.8 +/- 23.6 months. There was a temporal relationship between the high serum level of phenytoin and the loss of locomotion. The degree of brain atrophy in the posterior fossa was most severe in these 18 patients with severe phenytoin intoxication. The frequency of cerebellar and/or brain stem atrophy in the present series was 28%, the same as in mentally retarded epileptics without phenytoin treatment from the same institution. That phenytoin levels in serum correlated significantly with the heights of the fourth ventricle suggests that an overdosage of phenytoin or an underlying disease, or both, were the probable causes of cerebellar impairment and atrophy. Thus brain-damaged mentally retarded epileptics appear to be unusually susceptible to the side effects of phenytoin. This antiepiliptic drug is therefore not recommended for patients with no locomotor ability or with marked cerebellar signs and symptoms. To prevent phenytoin intoxication in susceptible patients, careful observation of the patients and routine monitoring of phenytoin levels in blood are stressed.


Phenytoin: an evaluation of several potential teratogenic mechanisms.

The effect of pharmacological doses of phenytoin (DPH) administered for a maximum of 28 days was studied in pregnant and nonpregnant rats as well as in 14- and 21-day fetuses. The following parameters were monitored in the adult rats: maximal electroshock seizures, serum DPH and folate, liver microsomal P-450, and 14C-DPH tissue distribution. 14C-DPH distribution was also evaluated in fetal tissues. Pregnant animals demonstrated an increase in anticonvulsant activity as well as increased serum concentrations of DPH throughout pregnancy and on the 7th post-partum day. Brain concentrations of DPH increased during pregnancy but had returned to the values in the nonpregnant group at the 7th post-partum day. Liver microsomal P-450 was decreased in pregnant animals receiving DPH at days 7 and 14 of pregnancy. Serum folate also decreased at day 14 of pregnancy in animals receiving DPH. Fetal tissue binding of DPH appeared to be related to serum concentrations of the drug at day 14. Teratogenic effects of DPH could be related to the increased serum and tissue concentrations of the drug observed during pregnancy as well as its effect on serum folate at day 14 of gestation.


Phenobarbital and phenytoin effects on somatosensory evoked potentials and spontaneous EEG in normal cat brain.

Chronic oral administration of phenobarbital (PB) and phentyoin was studied in chronically implanted cats. The effects of two dosages (PB, 3 mg/kg and 15 mg/kg; phenytoin, 5 mg/kg and 8 mg/kg) were analyzed with two physiological measures: somatosensory evoked potentials (SEPs) and power spectral analysis of the EEG. The low dosage of PB had no effect on SEPs except those recorded in the mesencephalic reticular formation; high dosage resulted in general SEP attenuation. PB had little effect on the EEG. Phenytoin at both dosages produced three main effects: (1) A negative component in the SEP which appears to be dominant in the dorsal hippocampus was significantly enhanced. (2) EEG changes occurred in the dentatothalamic pathway but not at those electrode locations which recorded fastigiobulbar activity. (3) The EEG changes which did occur were Peak Frequency shifts comparable to those previously reported in humans. The results are interpreted as demonstrating (1) minimal EEG and SEP modifications at the cerebral cortical level due to nontoxic dosages of PB and phenytoin, (2) a selective phenytoin effect on the dentatothalamic outflow of the cerebellum and on the dorsal hippocampus, and (3) a potential usefulness of Peak Frequency analysis as a measure of EEG response to anticonvulsant drugs.


Phenytoin encephalopathy as probable idiosyncratic reaction: case report.

A case of phenytoin (DPH) encephalopathy with increasing seizures and EEG and mental changes is described. Despite adequate oral dosage of DPH (5 mg/kg/daily) the plasma level was very low (2.8 microgramg/ml). The encephalopathy was probably an idiosyncratic and not toxic or allergic reaction. In fact the concentration of free DPH was normal, the patient presented a retarded morbilliform rash during DPH treatment, the protidogram was normal, and an intradermic DPH injection had no local effect. The authors conclude that in a patient starting DPH treatment an unexpected increase in seizures, with EEG and mental changes occurring simultaneously, should alert the physician to the possible need for eliminating DPH from the therapeutic regimen, even if plasma concentrations are low.


Phenytoin-induced hyperkinesia.

Previously described cases of phenytoin-induced hyperkinesia are reviewed. Three new cases are reported, in one of which hyperkinesia persisted for 5 years, although serum phenytoin on several occasions was in the therapeutic range. Similarities between phenytoin-induced hyperkinesia and hyperkinesia in Huntington's chorea and in neuroleptic- and L-DOPA-treated patients are discussed. It is pointed out that the frequency of organic cerebral damage seems to be high among patients with phenytoin-induced hyperkinesia. The hypothesis is presented that phenytoin induces hyperkinesia by increasing dopaminergic and serotonergic activity in the basal ganglia, and that patients with preexisting basal ganglia damage are the most susceptible.


Activity changes of chicken enzymes involved in glutamate and 2-oxoglutarate metabolism at the time of hatching.

At the time of hatching, activities of both KDH and succinate thiokinase increased in heart, while in liver only an enhanced activity of KDH was observed. Glutamate injected into egg exerted a stimulatory effect on KDH activity in chicken embryos but not in newborn chickens. There seems to be an age-dependent response of ASAT and GDH to stimulating effectors. In chicken embryos, 2-oxoglutarate induced GDH activity and in newly hatched chickens, ASAT activity. Concomitantly, liver protein content increased also. Administration of glutamate, however, was not followed by a significant activity increase of ASAT or GDH. Thus, the stimulation of ASAT and GDH activities seems to be favoured when they support glutamate formation out of 2-oxoglutarate.


Citrate synthase activity in human skeletal muscle.

Citrate synthase activity in soluble human muscle extracts (KCl-containing triethanolamine buffer) amounts to 17.5+/-6.97 U/g wet weight at 37 degrees C (n=36 healthy male subjects). Double determinations, both using two procedures and with muscle samples divided into two pieces and analyzed separately, gave very good reproducibilities. The possible causes of the comparatively high values are discussed. Significant correlations of citrate synthase activity with NADP-linked isocitrate dehydrogenase (r=+0.826) and hexose phosphate isomerase (r=-0.582) were found. The distribution of citrate synthase activity in the samples studied is not of the normal type but appears to be binomial.


Some problems associated with intestinal surgery in the horse.

The paper considers the general principles of intestinal surgery in the horse and illustrates a theatre layout suitable for this purpose. The problems of locating and reducing obstructions of the small and large intestine are discussed, with particular emphasis on incarcerations of the small bowel and the techniques of jejunal and ileal re-implantation after bowel resection.


Bone growth in foals and epiphyseal compression.

Limb angulation in foals may be due to defects in epiphyseal growth plates. The present state of knowledge concerning rate of growth in foals and differences in growth of different epiphyseal plates is reviewed and the importance of accurate knowledge of these parameters in treatment of angulation by unilateral retardation of an epiphyseal growth plate is stressed. Retardation of epiphyseal plate growth by compression wiring is described and its advantages in comparison with staplings are suggested. Compression wiring was used in 3 cases in which age, bone width and the degree of angulation were known so that the probable rate of bone growth could be assessed and the necessary unilateral bone increment could be calculated. It is suggested that further measurements of these parameters prior to growth plate retardation would give a more accurate comparison of the benefits of stapling and compression wiring.


Plasma biochemistry alterations in horses during an endurance ride.

The effects of prolonged strenous exercise on the plasma concentrations of sodium, potassium, calcium, chloride, bicarbonate, phosphate, albumin, cholesterol, glucose, creatinine, bilirubin, alkaline phosphatase, creatine phosphokinase, lactic dehydrogenase and asparate amino transferase were studied in a group of 26 horses competing in an endurance ride. There were significant changes in most parameters, when control values were compared with those taken immediately after the ride. There was also a significant correlation between several biochemical parameters and heart rate taken 30 minutes after the ride. When faster and slower horses were compared, significant differences were found only in phosphate and glucose values.


Some metabolic effects of maximal exercise in the horse and adaptations with training.

The effects of intermittent maximal exercise (galloping) before and after a 10 week training programme were studied in 6 horses. Determinations were carried out on venous blood for packed cell volume, total plasma protein, glucose, glycerol, free fatty acids, lactate, 11-hydroxycorticosteroids, blood gases and pH. There were marked changes associated with galloping and some of these could be modified with training. The major findings included (i) an elevated blood glucose, (ii) a large increase in glycerol, which was greatest at 30 min post-exercise and was higher following training, (iii) smaller increases in free fatty acids following training, (iv) higher levels of lactate after training, (v) a marked fall in pH which was less after training, (vi) an increase in 11-hydroxycorticosteroids with possibly a more rapid return to resting levels following training. It was concluded that with maximal exercise in the horse both glycogen and free fatty acids served as important substrates for working muscle, and following training greater utilisation of both these substrates occurred.


The arterial supply of the navicular bone and its variations in navicular disease.

Macroscopic, microscopic and radiological examinations of the navicular bones of 83 horses and ponies showed that the navicular bone in the adult horse has 2 principal routes of blood supply. One supply is present at birth, the other develops within the first 2 to 3 years of life, in response to increasing activity. The size of the nutrient foramina, as seen on radiographs, is related to the type, frequency and regularity of work done by the horse. These foramina are normally conical in shape, alteration to a circular, or mushroom-shape being evidence of occlusive vascular disease in the navicular bone. These changes are visible on a standard antero-posterior radiograph of the navicular bone. A common cause of lameness in the horse is ischaemia of the navicular bone, due to progressive arterial occlusion. Occlusion of at least 2 primary arteries, and some involvement of the compensating collateral blood supply must be present before lameness results.


Binding of polylysine to chromatin subunits and cleavage by micrococcal nuclease. A comparison of accessible sites.

Native chromatin and chromatin subunits (nucleosomes) were titrated with polylysine and digested with micrococcal nuclease and deoxyribonuclease I at individual lysine/nucleotide ratios. In contrast to earlier reports, which had been obtained using mechanically sheared chromatin, a comparison of the sites accessible for micrococcal nuclease and polylysine reveals that polylysine does not preferentially protect the micrococcal-nuclease-susceptible sites in chromatin. Similar results were obtained in digestion experiments with DNase I. From the experimental data presented we conclude that polylysine does not preferentially bind to the internucleosomal DNA, which is the prime target site for micrococcal nuclease, but rather to the total nucleosomal DNA moiety.


Comparative study of the effect of 5' AMP and its analogs on rabbit glycogen phosphorylase b isoenzymes.

A comparative study of the effect of AMP and other nucleotides on the different rabbit glycogen phosphorylase b isoenzymes has been made: muscle, brain, liver isoenzymes, and the hybrid species between muscle and brain isoenzyme. We have studied either the direct kinetic effect of the different nucleotides or the action of the nucleotides on the b to a conversion rate. Muscle, brain, and muscle--brain isoenzymes are very strongly and very specifically activated by AMP and its analogs, whereas liver isoenzyme is not very sensitive to the nucleotides. However, muscle, brain and muscle--brain isoenzymes show slight but very interesting differences in their kinetic properties. These differences allow some speculations about the conformational state of these different isoenzymes.


Water-soluble glycosphingolipids of dog gastric mucosa. Characterization of a branched ceramide heptasaccharide.

Two glycosphingolipids, the carbohydrate portion of which consisted of galactose, glucose and N-acetylgalactosamine, have been isolated from the aqueous phase of buffered tetrahydrofuran extract of dog gastric mucosa. The structures of these glycolipids were identified by partial acid hydrolysis, sequential degradation with specific glycosidases and methylation analysis. The structure of glycolipid I (GalNAcalpha1 leads to 3GalNAcbeta1 leads to 3Galalpha1 leads to 4Galbeta1 leads to 4Glc leads to ceramide) was found to be identical to that of Forssman hapten. The branched structure of glycolipid II, as determined by the combination of enzymatic degradation and comparison of the permethylation products of the enzyme-degraded compounds, is proposed to be: See Source.


The allosteric inhibition by calcium of soluble and partially purified adenylate cyclase from turkey erythrocytes.

Adenylate cyclase from turkey erythrocyte membranes was solubilized in Lubrol-PX and partially purified (22-fold) by molecular sieve chromatography on Biogel A5M. The molecular weight of the enzyme was found to be 316000. The partially purified solubilized enzyme was found to retain all the kinetic and regulatory properties of the native membrane-bound enzyme except its sensitivity to beta-agonists. The enzyme responds to Mg2+ in a positively cooperative fashion, with a Hill coefficient of nH = 2.0. The enzyme is inhibited by Ca2+ in a positively cooperative fashion with a Hill coefficient of nH = 2.0. The calcium effect is only on the kcat of the reaction and not on the binding and kinetic parameters of the enzyme towards the other ligands such as MgATP and Mg2+. The Mn2+-supported adenylate cyclase is not inhibited by Ca2+ as was found for the native membrane-bound enzyme.


Alterations of activities of ribonucleases and polyadenylate polymerase in synchronized mouse L cells.

The activities of the three known catabolic and the one anabolic polyadenylate enzymes have been determined in synchronized L5178y cells: endoribonuclease, exoribonuclease, 5'-nucleotidase and poly(A) polymerase (Mg2+-dependent). These four enzymes were found primarily in the nuclear fraction. The activity of poly(A) polymerase remains essentially constant during the transition from G1 to S phase. However, the poly(A) catabolic enzyme activities increase parallel with DNA synthesis; the endoribonuclease activity increases 4-fold during G1 to S phase, the exoribonuclease and the nucleotidase activities increasing 30-fold and 16-fold. During the S phase the poly(A)-degrading enzymes are far more active than the poly(A)-synthesizing activity of poly(A) polymerase. We conclude that in L5178y cells the poly(A)-degrading enzymes probably function in regulation of the post-transcriptional net-polyadenylation of heterogeneous nuclear RNA during the phase of DNA synthesis.


Gluconeogenesis in the guinea pig. Effect of glucagon on gluconeogenesis from lactate by isolated perfused guinea-pig liver.

Gluconeogenesis was stimulated by glucagon in fed but not fasted isolated perfused guinea pig livers. Both the amount and the rate of incorporation of radioactivity into glucose from L-[U-14C]lactate were increased in fed livers by the addition of glucagon to the perfusate. The glucagon-stimulated increase in gluconeogenesis was accompanied by an increase in oxygen consumption, an increase in the amount of lactate carbon converted to glucose and a decrease in the amount of lactate carbon converted to CO2. The results are interpreted to indicate that glucagon affects gluconeogenesis from lactate in fed livers by redirecting the fate of substrate from other products toward glucose.


Nitrosoalkanes as Fe(II) ligands in the 455-nm-absorbing cytochrome P-450 complexes formed from nitroalkanes in reducing conditions.

Primary and secondary aliphatic nitroalkanes RR'CHNO2, react with microsomal cytochrome p-450 in the presence of dithionite leading to new complexes with a Soret peak at 455 nm. The formation of these complexes is inhibited completely by CO and partially by metyrapone. However, once formed, their exogenous ligand is not displaced by excess CO. By deoxycholate treatment they are transformed into 423-nm-absorbing cytochrome P-420 complexes, which are spectrally similar to the corresponding RR'CHNO2-derived myoglobin complexes. The 455-nm-absorbing complexes are equally produced from RR'CHNO2 reduction, microsomal NADPH-dependent oxidation of the corresponding hydroxylamine RR'CHNHOH, or interaction of the nitrosodimer (RR'CHNO)2 with reduced cytochrome P-450. All the reported results are consistent with the involvement of a new class of ligands of cytochrome P-450-Fe(II), which are the unstable aliphatic nitrosomonomers RR'CHNO, whose nitroso group is isoelectronic with dioxygen and whose stabilisation results from their strong binding to heme-Fe(II), thus explaining the observed inhibition of the hydroxylating function of cytochrome P-450.


Nitrosoalkanes as Fe(II) ligands in the hemoglobin and myoglobin complexes formed from nitroalkanes in reducing conditions.

Primary and secondary aliphatic nitro compounds, R2CHNO2, react with myoglobin and hemoglobin, in the presence of sodium dithionite, leading to new complexes with Soret peaks respectively at 425 and 421 nm. These complexes are very stable even after disappearance of the starting nitro compounds and do not exchange their exogenous ligand after 10 h under 1 atm (101 325 Pa) CO. They are low-spin hexacoordinated myoglobin or hemoglobin complexes, as shown by the resonance Raman spectrum of the nitromethane-derived human hemoglobin complex which is similar to those of the known hemoglobin complexes with O2, CO, NO and nitrosobenzene. Evidence has been produced to show that the nitro compounds themselves do not bind to the hemoproteins; we propose that among the reduction derivatives produced in situ by dithionite, the corresponding unstable nitroso monomers, whose nitroso group is isoelectronic with dioxygen, are the actual ligands of the 425-nm or 421-nm-absorbing complexes.


Transcription fidelity and structural integrity of isolated nucleoli.

1. RNA was transcribed in vitro using isolated nucleoli, the endogenous form A RNA polymerase and mercurated UTP as one of the nucleoside triphosphate substrates. The products were isolated from endogenous nucleolar RNA sequences by chromatography on sulphydryl-Sepharose and analysed by hybridisation in vast DNA excess. The results of competition-hybridisation experiments suggested that a large proportion of the transcript was ribosomal RNA although some sequences had been transcribed from DNA of lower reiteration frequency. 2. Analyses of the constituents of nucleoli following isolation by the sonication procedure suggested that the nucleolar DNA, particularly the ribosomal cistrons, are severely degraded. Furthermore, indications were obtained that the transcription complexes were damaged and many of the nascent RNA chains appeared to have been sheared from near the growing points.


Studies on hemoglobin tryptophanyl contact residues in the haptoglobin-hemoglobin complex.

Hemoglobin and apohemoglobin bind heptoglobin in the same molar ratio. Structural studies on haptoglobin-hemoglobin complex do not suggest any important structural changes in either protein upon binding. However, when apohemoglobin is bound to haptoglobin, a marked reduction in secondary structure, attributed to unfolding of globin chains, has been observed. Here we describe some properties of the haptoglobin-apohemoglobin (Hp-apoHb) complex, prepared by isoelectric focusing in the presence of an excess of haptoglobin. This complex does not exhibit the irreversibility of complexes obtained with hemoglobin in identical experimental conditions. The 'freezing' of the conformation of apohemoglobin upon binding to haptoglobin has been studied by fluorescence quenching experiments carried out in the presence of 8 M acrylamide. Changes in conformation of haptoglobin upon binding to apohemoglobin have been detected by titration of the exposed tryptophans using N-bromosuccinimide. Comparison of the additivity of exposed tryptophans in the complexes reveal that two tryptophans become inaccessible in the complex formation of haptoglobin with hemoglobin but not with apohemoglobin. These tryptophans, probably located on the alpha1beta2 contact interface of hemoglobin, have been tentatively identified as Trp-C3(37)beta.


alpha-Galactosidase from Saccharomyces carlsbergensis. Cellular localization, and purification of the external enzyme.

1. The alpha-galactosidase of Saccharomyces carlsbergensis in an inducible enzyme which is localized mainly outside the cell membrane and which is secreted into the culture medium in increasing amounts during the growth cycle. 2. The soluble form of alpha-galactosidase localized inside the cell appears to have the same characteristics as the external one, contrasting with the different forms found in the case of invertase. Although some activity is membrane-bound, this activity, when solubilized with detergent, has the same characteristics as the external form of the enzyme. 3. A procedure has been developed by which the enzyme has been purified using batch adsorption with DEAE-Sephadex and column chromatography in DEAE-Sephadex, DEAE-cellulose and Sephadex G-200, using the supernatant of a culture of Saccharomyces carlsbergensis grown in yeast/nitrogen base complemented with galactose. 4. The purified enzyme, which is homogeneous by chromatographic criteria and polyacrylamide gel electrophoresis, appears to be glycoprotein. 5. Invertase copurifies with the alpha-galactosidase but because of its lower stability, together with the fact that the synthesis of both enzymes can be controlled separately, it was possible to obtain preparations in which the contaminant activity was approximately 1%.


Interaction of the phosphatidylcholine exchange protein with phospholipids. A fluorescence and circular dichroism study.

The phosphatidylcholine exchange protein from bovine liver has a fluorescence emission maximum at 327 nm. Fluorescence was enhanced in the presence of vesicles containing phosphatidylcholine and various amounts of either phosphatidic acid or phosphatidylglycerol. From the increase in fluorescence it was derived that the apparent dissociation constant of the exchange protein-vesicle complex decreased with an increased vesicle content of acidic phospholipids. Fluorescence indicated that the exchange protein interacted with lysophosphatidylcholine micelles at pH 3.5, 5.9 and 8.5. The increase in fluorescence was most prominent at the acidic pH. Circular dichroism indicated that the alpha-helix content of the native protein was low between pH 3.6 and 8.0. Interaction with lysophosphatidylcholine micelles had a negligible effect on the secondary structure of the protein, except at pH 3.6 where distinct minima at 208 nm and 220 nm in the circular dichroic spectrum became apparent.


Conformational studies on histone H3 and its CNBr peptides.

Histone H3 and H3 peptides 1--120, 1-90, 91-135, 91-120 and 121-135 have been prepared and examined for salt-induced conformational changes by circular dichroism measurements. It was found that reduced histone H3 and the reduced peptides 1-120, 91-135 and 91-120 exhibit biphasic changes with the formation of alpha-helix and beta structures. H3 peptide 1-90, on increasing the ionic strength to moderately high levels, monophasically formed appreciable quantities of alpha-helix and beta structures, while peptide 121-135 remained unfolded under all ionic strengths examined. All the above peptides except 121-135 also aggregate when the ionic strength is raised. The salt-induced near-ultraviolet circular dichroic spectra of histone H3 and peptide 1-90 were found to be very similar, suggesting that the conformational changes induced in the peptide 1-90 are essentially the same as those observed for the intact histone. These results support the contention that the polypeptide segments of this histone interact initially by parallel self-association followed by the formation of even larger aggregates on a longer scale.


Sites of histone/histone interaction in the H3 - H4 complex.

Sites of interaction between histones H3 and h4 have been probed by investigating complex formation, firstly between histone H4 and three peptides cleaved by chemical means from histone H3 (residues 1-90 and 1-120 using cyanogen bromide and residues 42-135 using N-bromosuccinimide), secondly between histone H3 and two peptides cleaved from histone H4 (residues 1 - 84 using cyanogen bromide and residues 38-102 using chymotrypsin) and thirdly between the H4 peptide (residues 38-102) and the three H3 peptides (residues 1-90, 1-120 and 42-135). The criterion for complex formation is the appearance of characteristic perturbed resonances in the aromatic region of the 270 - MHZ proton resonance spectrum of the peptide mixture. It is concluded that loss of 37 N-terminal residues from histone H4 and 41 N-terminal residues from histone H3 does not prevent complex formation, whilst the loss of 18 C-terminal residues from H4 and 45 C-terminal residues from H3 does prevent it; that last 15 C-terminal residues of H3 are, however, not required for forming a complex. The regions important for complex formation are therefore defined as residues 42-120 in histone H3 and residues 38-102 in histone H4.


A DNA polymerase from Ustilago maydis. Evidence of proof-reading by the associated 3' leads to 5' deoxyribonuclease activity.

The 3' leads to 5' deoxyribonuclease activity associated with an Ustilago maydis DNA polymerase hydrolysed non-complementary 3'-primer termini about 12 times more rapidly than complementary termini. An analysis of its substrate specificity suggested that, although it was unable to hydrolyse fully single-stranded polynucleotides, it could hydrolyse such regions less than about four nucleotides in length covalently bound to a primer molecule which was base-paired to a complementary template strand. Template-primer combinations containing complementary or non-complementary primer termini both supported polynucleotide synthesis, but whereas the former were conserved, the latter were hydrolysed during the reaction thus allowing synthesis to occur. No addition of nucleotides onto a conserved non-complementary 3'-primer terminus was detected. The deoxyribonuclease activity therefore fulfilled a proof-reading function during DNA synthesis in vitro.


Sensitivity of hemoglobin thiol groups within red blood cells of rat during oxidation of glutathione.

The intracellular thiol-oxidising diazenes, diazenedicarboxylic acid bis-N,N-dimethylamide and diazenedicarboxylic acid bis-N'-ethylpiperazinide, have been used in the study of red cells. A difference in the consequences of diazene oxidant treatment between the human red cell and rat red cells has been found in respect to the quantity of oxidant needed for glutathione (GSH) oxidation, to the fate of GSH, and to the reactivity of hemoglobin. In the first place, significantly more oxidant is needed for GSH oxidation in the rat red cell than in the human cell. Secondly, in the human cell, all of the GSH is converted to glutathione disulfide (GSSG), from which GSH is regenerated. In the rat cell, GSH disappears without being converted to GSSG, and GSH is not regenerated. Thirdly, a decrease in rat hemoglobin thiol groups, but no change in human hemoglobin, is found. Sterically unhindered thiol groups in the rat hemoglobin are thought to react with the usual adduct intermediate in GSH oxidation by diazene (formed from RCON = NCOR + GSH leads to RCON(SG)NHCOR) to produce mixed disulfides, from which GSH is not easily regenerated. The results support the idea that reduction of mixed disulfides of GSH and protein is not carried out directly by GSSG reductase but necessitates thiol transferase and GSH. The thiol-oxidising diazenes may be of use in mapping of exposed, reactive thiol groups in proteins.


Metabolic behaviour of nonhistone chromosomal proteins in proliferating and in resting fibroblasts.

The metabolism of nonhistone chromosomal proteins was studied in two lines of cells showing a different degree of contact inhibition: human diploid fibroblasts, which are easily contact-inhibited, and Chinese hamster fibroblasts, which had been made to stop proliferating by fasting. By following the 3H414C ratio of [3H]tryptophan-labelled nonhistone chromosomal proteins and [14C]thymidine-labelled DNA in chase experiments three main groups of these proteins could be detected with respect to their metabolic behaviour: (a) a metabolically stable group which is acid-insoluble and represents the bulk of nonhistone chromosomal proteins in proliferating cells; this group is conserved when the cells enter a resting phase; (b) a metabolically labile group which is acid-soluble and is observed as a minor fraction in proliferating cells; (c) a metabolically labile group which is acid-insoluble and accumulates in resting cells; this fraction is much larger in contact-inhibited cells. Stimulation of cell proliferation by trypsinization decreases the amount of nonhistone chromosomal proteins in resting cells to the basic level observed in proliferating cells.


Thermophilic polynucleotide phosphorylase from Thermus thermophilus. Purification and properties of an altered form of enzyme which lacks phosphorolytic activity to polynycleotide.

A thermophilic polynucleotide phosphorylase lacking polynucleotide phosphoryltic activity was purified from Thermus thermophilus HB-8 strain. The enzyme is an altered form of the native polynucleotide phosphorylase, probably attacked by the proteinase(s) of this extreme thermophile during the purification process. This modified enzyme lacks phosphorolytic activity to poly(A) while retaining weak activity to phosphorolyse tetranucleotides or hexanucleotides. The purified enzyme was shown to be homogenous by electrophoretic analysis in polyacrylamide gel. This enzyme had a molecular weight of 190 000 as calculated both from electrophoresis on polyacrylamide gel and from the Stoke's radius derived from the gel filtration pattern and the sedimentation coefficient. The enzyme was separated into three polypeptide chains by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate; their molecular weights were calculated to be 92000, 73000 and 35000. The enzyme was thermophilic and thermotolerant, exhibiting its maximal activity at 70 degrees C. The four ribonucleoside diphosphates (ADP, GDP, UDP and CDP) were polymerized to the extent of 7-S size.


Structure and synthesis of a lipid-containing bacteriophage. An endolysin activity associated with bacteriophage PM2.

Endolysin was induced in Pseudomonas BAL-31 infected with bacteriophage PM2 and was also associated with the purified virion. This enzyme required divalent cations for its activity, Ca2+ being the most effective cation. Endolysin activity in the virion increased up to three-fold upon disruption and the activity could be localized in the viral nucleocapsid. Thus the enzyme is localized within the virion. After purification of the structural proteins of bacteriophage PM2, only the nucleocapsid protein (III) had endolysin activity.


Microbial contamination of radionuclide generators.

Technetium-99m generators were grossly contaminted using 5 different strains of microorganisms. Elution of the generators showed that the number of microorganisms was reduced by a factor of 10(4)-10(6). There were no indications that the generators would support bacterial growth. It is concluded that it would be acceptable from a microbiologic point of view to omit autoclaving and membrane filtration of the final product provided that proper aseptic techniques are applied.


Quantitative analysis of the distribution of 99mTc-labelled ethylene hydroxy diphosphonate in young rats.

The distribution of 99mTc-labelled ethylene 1-hydroxy-1,1-diphosphonate (EHDP) in young rats was studied 1, 3, 6 and 9 h after intraperitoneal injection. The concentration of 99mTc in bone was 8-10 times higher than that in the kidneys and 20-1000 times higher than those in other soft tissues. The concentration in bone remained constant throughout the observation time. In some of the animals sacrificed after 3, 6, or 9 h the uptake in the liver and spleen was higher than that found in the majority of the animals.


Enhancement of incorporation of 131iododeoxyuridine into tumors after application of Clostridium oncolyticum s. butyricum (M 55). Animal experiments on positive scintigraphic localization of tumors.

The application of spores of the bacterium Clostridium oncolyticum s. butyricum (M 55) results in an increased incorporation of 131Iododeoxyuridine in tumors (amelanotic melanoma Fortner III of the golden hamster and Brown-Pearce carcinoma of the rabbit). This leads to an improved scintigraphic demonstration of the tumors.


The dual photopeak-area method applied to scintillation camera measurements of effective depth and activity of in vivo 123I-distributions.

Attenuation curves and photopeak ratios of 159 keV and 28 keV photons emitted in the decay of 123I have been studied using a scintillation camera equipped with an extra ADC for recording the pulse height distribution of the energy signal. Cavities of various sizes containing 123I-solution were placed at different depths in a water phantom in order to vary the effective depth i.e., the thickness of attenuating material above the cavity plus the distance to the centre of activity in the cavity. The centre of activity varies with the distribution of activity in the cavity, size of cavity, and the effective attenuation coefficient derived from the attenuation curves. From the photopeak ratio an average effective depth is determined which can be used for calculating the attenuation correction factor. The dual photopeak ratio has been used in clinical evaluation of thyoid 123I uptake measurements where effective depths ranging from 21 to 43 mm were obtained for the activity in the thyroid. Applications of the photopeak ratio method for renography with 123I-hippuran and blood flow studies using 133Xe or 127Xe and studies with many other radionuclides are also discussed.


A new display technique for computer-processed digital scans.

This article presents a new approach for presenting scintigraphic images. The technique combines the conventional plotting the contour lines and the highlighting, by means of hatching, of the concavities (or convexities) of the 'surface' representative of radioactive distribution. The search for the surface features is achieved generally by the method of normal curvatures. An example with a phantom demonstrates the utility of this representation method.


An analysis of the diagnostic contribution of double-radiocompound renography.

The diagnostic contribution of double-radiocompound renography was tested by applying discriminant analysis to the data obtained in one group of normal subjects and in some groups of selected pathological cases which included glomerulonephritis, pyelonephritis, and hypertension. The results obtained confirm the reliability of the test and give evidence of its usefulness in diagnostic clinical work.


Position dependent renogram changes of the mobile kidney.

The present study evaluates the isotope renograms, obtained in prone and upright position of 87 patients with 93 mobile kidneys. Scintiphotos were made to determine the extent of renal movement. The transient position abnormality of the kidney was compared to the position dependent isotope renogram changes. It was possible to describe three types of floating kidney each with a characteristic motion and position dependent function disturbance. One form of renal ptosis, ptosis with ventral rotation, was commonly associated with a 2nd phase renogram abnormality and with hypertension.


Simplified method of determining left ventricular ejection fraction from a radionuclide bolus.

A method which does not require an online computer has been developed to measure left ventircular ejection fraction from a bolus of 99mTc-pertechnetate. The passage of the bolus through the cardiac chambers was imaged using a gamma camera and recorded on video tape. On replay a region of interest corresponding to the left ventricle was selected directly at the gamma camera console. Counts detected in this region were analysed in 30ms intervals. The data were corrected for the contribution from extra-cardiac activity and were then digitally filtered to remove statistical noise. Ejection fractions measured in 13 patients by this technique agreed well with those determined by contrast ventriculography (r=0.97, P less than 0.001).


Intravenous myocardial imaging performed serially early and late after acute myocardial infarction.

The accuracy and sensitivity of myocardial imaging using intravenous Rb-81 in delineating the extent and subsequent changes in regional myocardial perfusion abnormalities were examined serially in 12 patients one to 510 days after an acute myocardial infaraction. Definite regions of decreased Rb-81 uptake were noted in 10 patients with transmural infarction. There was excellent correlation between the site of perfusion abnormalities, the electrocardiographic infarct location and the region of segmental wall motion disorders. Follow-up studies revealed in 8 of 11 patients a decrease in the extent of the perfusion defect, which was associated with an improvement in the extent and/or severity of regional wall motion disorders. Conversly, wall motion abnormalities did not change in the remaining three patients in whom perfusion abnormalities persisted unchanged. We conclude that intravenous myocardial imaging permits visualization of myocardial perfusion abnormalities early after acute myocardial infaraction. Serial imaging allows detection of changes in the extent of perfusion abnormalities, and, thus, might provide useful information with respect to the patient's prognosis and the effects of therapeutic interventions.


Distribution of intravenously injected 201thallium in the legs duging walking. A new test for assessing arterial insufficiency in the legs.

There is a multitude of method for assessing the efficiency of the arterial circulation in the legs. Most of these methods can give conclusive information about the patency of the main arteries in the leg; the level of an arterial obstruction can also be estimated. However, none of these methods provides a direct estimate of the functional importance of arterial lesions of light to moderate severity. The present report describes preliminary findings of the distribution of intravenously injected 201Thallium, a potassium analogue, in the legs during exercise provoking symptoms of ischemia. From this pilot study the general impression is that the thallium uptake pattern corresponds well with the clinical, physiologic, and angiographic picture.


Comparative study of 111In and 59Fe bone marrow scanning.

The aim of this work is to examine the relative merits of 59Fe and 111In scanning in estimating the functional value of the various erythropoietic areas and in appraising bone marrow distribution and extension. At first we studied a group of patients with heterogeneous distribution of bone marrow secondary to irradiation, ferrokinetics serving a reference for the comparison of 111In and 59Fe scanning. The functional value fo 59Fe scanning was thus proved, in contrast to 111In scanning which was erroneous for 13 of the 29 bone marrow areas studied. A further study of patients presenting polycythemia vera or anemia with 111In demonstrated in some cases false distribution and bone marrow extension. Furthermore, 59Fe scanning permitted evaluation of the efficiency of erythropoiesis and the existence of extramedullary erythropoiesis. Review of the literature revealed that with the activity used there was no dosimetric advantage of 111In over 59Fe.


Isotope scanning of brain tumors in children.

A total of 31 brain tumors (21 verified histologically, 10 by clinical and neuroradiologic methods) in children aged from 7 months to 13 years were investigated by scintigraphy with 99mTc as a radioactive isotope tracer. There were 16 supratentorial and 15 infratentorial tumors. The supratentorial tumors were detected in 88%, and the infratentorial in 13%, of case by brain isotope scanning. The advantages and disadvantages of the method are discussed.


Radiochemical quality control of 99mTc-labeled radiopharmaceuticals. Some daily practice guideliens.

Recognizing the fact that each nuclear medicine facility should be able to perform simple radiochemical quality tests on currently used radiopharmaceuticals, this study was undertaken to evaluate a number of radiochromatographic methods. No single ideal method exists to assess the radiochemical composition of 99mTc-labeled antimony sulphur colloid, sulphur colloid, iron ascorbate, citrate, human serum albumin, EHDP, macroaggregates, and microspheres. It is advisable to include thin layer chromatography with both NaCl and methylethylethylketone for the determination of free pertechnetate in each day's program. More detailed radiochemical analysis can be performed by combining these methods with paper chromatography, paper electrophoresis, and gel filtration. It seems reasonable to regard a constant radiochromatographic pattern as a measure for constant radiochemical quality. The four chromatographic tests lead to consistent results regarding the percentage of free pertechnetate in the radiopharmaceutical preparations. Quantitative analysis shows that the radiochemical purity for each radiopharmaceutical is unique for the chromatographic method used and needs to be defined when stated.


Estimation and prediction of blood volume in infants and children.

Blood volume was studied in 160 infants and children aged from one hour to 14 years. From these data linear and logarithmic regression equations relating blood volume to weight, height and surface area were calculated. Equations utilizing combined weight and logarithmic weight values were found to be the most accurate for predicting blood volume. Therefore, nomograms were constructed for reading blood volume directly from height and weight.


Antibody neutralizing material of factor VII during the first weeks of life.

In a study on 66 newborns and infants, factor VII activity and factor VII related antigen were investigated on 110 occasions. A normal range was calculated by testing plasma from 12 healthy male volunteers at various dilutions. Most of the values in the newborns and infants fell within this range but 18.2% of the factor VII proteins showed a significantly different specific activity.


The influence of perinatal risk factors on the incidence of atypical coagulation factor VII during the first days of life.

The correlation between the appearance of functionally-atypical factor VII and perinatal complications was investigated in 66 newborn infants. The presence of an abnormal clotting factor was assumed if the ratio between clotting activity and antigen-related factor VII material exceeded the normal range for adult plasma. The newborns were divided into a risk group of infants threatened by adverse conditions of labour or post-natal adaptation, and a control group of newborns without perinatal complications. The findings were as follows: The incidence of atypical factor VII was significantly higher in the risk group. There was no difference between prenatal and postnatal complications in this respect. Infants born by caesarian section or with cord complications, as well as those with delayed respiratory adaptation, showed a higher incidence of atypical factor VII than the risk group as a whole. Atypical factor VII was not detected until the third day of life, irrespective of the prenatal or postnatal complications. These findings suggest that perinatal risk factors are associated with an alteration of factor VII synthesis.


A case-report of idiopathic juvenile osteoporosis with particular reference to 47-calcium absorption.

Calcium metabolism was studied in a 12-year-old girl preseting with idiopathic juvenile osteoporosis. Absorption of orally administered 47-Ca was high. Serum calcium and phosphorus, serum immunoreactive PTH and CT and tubular phosphate reabsorption were found to be within normal limits. The data suggest that calcium malabsorption, nutritional calcium deficiency, hyperparathyroidism, a dysfunction related to sex hormones, and Cushing's syndrome cannot be implicated in the aetiology of the osteoporosis in this case who recovered spontaneously with sexual maturation.


Tubular cytoplasmic inclusions in a case of childhood dermatomyositis with migratory subcutaneous nodules.

A boy with dermatomyositis which began at the age of one year and five months showed multiple migratory subcutaneous nodules, which have seldom been described. Histological and electron microscopic studies of muscles and subcutaneous nodules demonstrated the following interesting findings. 1. Light microscopy. The migratory subcutaneous nodules consisted of non-suppurative panniculitis and ischaemic adipo-necrosis as a sequel to vascular lesions. This finding suggests that the nodules may have arisen from the subcutaneous adipose tissue which had been severely damaged by systemic angiopathy. 2. Electron microscopy. Examination of the vessels in muscle and subcutaneous nodules showed tubular cytoplasmic inclusions with a diameter of approximately 250 A in the endoplasmic reticulum of vascular endothelial cells. These observations provide strong support for the concept that the fundamental pathologic process in childhood dermatomyositis is of a vascular nature, and the primary lesion is in the walls of the intramuscular blood vessels.


Sclerosis of the intestinal tract with severe malabsorption.

The clinical findings and pathological changes in a 14 year old boy who died from severe malabsorption are presented. Early signs of the illness were attacks of abdominal pain, diarrhoea, and intermittent periods of constipation. Later, severe malabsorption together with chronic ileus were apparent but no organic obstruction was found at several laparotomies. X-ray examination revealed extremly distended intestinal loops and a very slow transit time of barium. Malnutrition could not be improved by dietary, medical, or surgical therapy. Microscopic examination of the tissues obtained at autopsy showed changes in the small vessels of the lungs, heart, kidneys, and intestine. Increased fibrous tissue combined with atrophy of the muscular coat was observed in the wall of the entire intestine. Although cutaneous changes of scleroderma were not evident the deposition of collagenous material and the vascular changes seem typical of systemic sclerosis.


Effect of E1 type prostaglandin on hypoxemia in a cyanotic congenital cardiac malformation.

Prostaglandin type E1 has been administered on 4 different occasions in a newborn with a ductus-dependent complex congenital cyanotic heart disease. Dramatic improvement of the arterial oxygen concentration followed each prostaglandin infusion. Increased pulmonary circulation and widening of the ductus arteriosus were seen on angiographic examination. Transient flushing of the skin and mild pyrexia were the only complications noted. It is advised that prostaglandin type E1 be used in ductus-dependent heart diseases as an emergency therapy when indicated.


Safe amounts of contrast medium for angiocardiography in neonates and infants.

The results were analysed of 485 (biplane) cineangiocardiograms performed in 200 consecutive cardiac investigations on neonates (under 4 wk) and infants (4 wk to 1 yr). 10 deaths occurred during or within 24 h of cardiac catheterization, all in patients who were profoundly acidemic because of their hemodynamic disturbance. A total dose of Cardio-Conray at any one investigation of 4-5 ml/kg body weight was satisfactory and without undue risk. Single injections of 1.5-2.0 ml/kg body weight were safe whilst providing optimal diagnostic information. The interval between multiple injections did not appear to affect the risk to the patients.


Persistent repetitive supraventricular tachycardia.

A case of persistent repetitive supraventricular tachycardia known to be present for at least 9 yr is reported. Although the patient appeared to tolerate the arrhythmia without any ill effects he was found to have moderate left ventricular dysfunction due to alcoholic abuse and/or significant coronary artery disease. The electrophysiological findings appear to be most consistent with an ectopic focus arising in the low right atrium as the mechanism for this arrhythmia. Although resistant to standard drug therapy, this arhythmia did respond to overdrive suppression.


Development of a highly sensitive radioimmunoassay for digoxin and its application in pediatric practice.

The sensitivity of two established routine digoxin radioimmunoassay methods has been increased to enable the provision of a rapid and relatively atraumatic inpatient and outpatient service for neonates and small children, using capillary blood samples obtained by heel-prick. The methods employ 125I- or 3H-labelled digoxin, a rabbit antiserum raised against a digoxin: bovine serum albumin conjugate and only 10 or 25 microliter of plasma as the sample. The results obtained using these highly sensitive assays correlate closely with those found using conventional assays, requiring larger sample volumes. An apparent difference in sensitivity to digoxin has been demonstrated between infants and children more than 1 yr old. Thus infants appear to tolerate plateau phase plasma levels (mean value for non toxic infants 2.6 +/- 1.8 ng/ml) that in older children or adults would be associated with digoxin toxicity.


Ventricular arrhythmias prior to discharge and one year after acute myocardial infarction.

Ventricular ectopic beats (VEB) were studied in 100 consecutive patients prior to discharge after an acute myocardial infarction and again after 1 yr, on 6-h recordings. VEB were found in 71 patients prior to discharge. Reinfarction and sudden death taken together were significantly more common in the 35 patients who had severe VEB, i.e. multiform, paired, R-on-T or ventricular tachycardia (P less than 0.05). Reinvestigation after 1 yr of 73 survivors who had not reinfarcted revealed a nonsignificant overall increase in patients with VEB from 67 to 78% together with an increase in degree of severity. The intraindividual pattern, however, differed considerably. Several clinical findings including angina pectoris, heart fialure, hypertension, diabetes mellitus, hyperlipidemia, antiarrhythmic therapy, and smoking, failed to differentiate patients with increasing VEB severity from the remainder.


Effect of certain antiadrenergic agents on systolic time intervals in essential hypertension.

Systolic time intervals, including preejection period (PEP) and left ventricular ejection time (LVET), were studied in patients with permanent essential hypertension before and after intra-venous administration of propranolol 0.2 mg/kg (11 patients), clonidine 0.002 mg/kg (10 patients) and methyldopa 2 mg/kg (12 patients). With propranolol, diastolic blood pressure was unchanged and the heart rate decreased, whilst PEP and LVET were significantly prolonged (P less than 0.001). Clonidine caused a fall in blood pressure (P less than 0.001), heart rate was slightly reduced, PEP was prolonged (P less than 0.001) and there was a significant decrease in LVET at 5 min. With methyldopa, no significant effect was observed after intravenous injection, but 7 days oral administration produced the identical effect as clonidine. These observations suggest that certain antihypertensive drugs may impair left ventricular performance and depress myocardial contractility.


Comparison of intravenous ibuterol (KWD 2058), a new bronchodilator, and terbutaline.

The selectivity of the recently developed beta-stimulating drug ibuterol, a terbutaline ester which is rapidly hydrolyzed to terbutaline in vivo, was tested by comparing it with terbutaline after intravenous infusion. Dose-response curves for both drugs for FEV, heart rate, blood pressure, and tremor were recorded. The two drugs had equal bronchodilator effect. Ibuterol caused significantly less increase in heart rate, tremor ratio, and pulse amplitude when doses causing equal bronchodilation were infused. This is probably due to inequality of distribution. The lower tremorogenic activity of ibuterol can also be caused by differences in the concentration of tissue esterases in lung and skeletal muscle. It is concluded that ibuterol is more selective for the bronchi than terbutaline.


The effect of hydroxyethylrutosides on capillary filtration rate in the lower limb of man.

The rate of capillary filtration in the lower limbs after hydroxyethylrutosides administered in various doses and by different routes was studied in patients with chronic venous insufficiency. Capillary filtration rate decreased immediately after intravenous and from 55 to 85 minutes after oral administration, depending on the dose. The slow-release tablets produced a definite falt in capillary filtration 240 minutes after administration.


Adverse drug reactions during treatment of urinary tract infections.

Adverse reactions reported to the Swedish Adverse Drug Reaction Committée concerning Sulfapral (a sulphonamide combination for urinary tract infections) and nitrofurantoin were remarkably similar. The commonest hypersensitivity reactions were in skin and lung. During the 10-year period 1966-75 reactions to the sulphonamide were fewer (n=327) than those reported for nitrofurantoin (n=781), but the former appeared to carry a higher mortality rate - 4.6% of reported sulphonamide reactions were fatal as compared to only 1.0% for nitrofurantoin (P less than 0.001). The number of reactions to the sulphonamide tended to remain constant, whereas those to nitrofurantoin increased rapidly, both absolutely as well as relative to sales of the drug.


Caffeine, tobacco, alcohol and drug consumption among medical students in Barcelona.

A survey of medical students was conducted at the Universitat Autònoma de Barcelona in 1974. Out of 1029 students, 808 present at lectures (78.5%) returned properly completed questionnaires. These showed that mean caffeine consumption was 8.3 g per month and increased with the length of stay at the university. Tobacco consumption (general mean, 190 cigarettes per month, 216 for males and 150 for females) and alcohol consumption (8.8 litres/year for males and 4.1 litres/year for females) also increased with time spent at university. Alcohol consumption was not as high as in the general population. Amphetamine consumption was very high (22% of students had taken amphetamines on more than one occasion in the six months prior to the survey). Marihuana and hashish were by far the most commonly used drugs (9.6%), the use of these drugs being much less common than at other European universities. The use of "harder" drugs was very limited. Appraisal of alcohol, tobacco and amphetamine abuse is necessary, since the authorities have not employed adequate measures to stop or limit them.


Absorption and metabolism in man of 16-epi-gitoxin, a new semi-synthetic cardioactive glycoside.

The pharmacokinetics of 3H-16-epi-gitoxin have been investigated after oral administration: The peak serum level occurred after 30-60 min, and its subsequent decline was in two phases with half-lives of 3.5 and 19.5 h, respectively. Within 3 days 66-70% of the administered radioactivity were eliminated by renal excretion. In the first 16 h 15% of excreted activity was in metabolites, but thereafter the glycoside was excreted in unchanged form. The metabolites were characterized by thin-layer chromatography and radio-scanning; it is assumed that they correspond to the bis- and monodigitoxosides as well as to the genin of 16-epi-gitoxin. The unusual pharmacokinetics of the glycoside are discussed in relation to its special kinetics at the molecular level, i.e. the rapid dissociation rate of the glycoside-(Na+ + K+)-ATPase-complex.


Binding of 16alpha-gitoxin and its 16-acetate to human serum albumin.

The binding of semi-synthetic (16alpha-gitoxin and its 16-acetate) and naturally occurring cardioactive glycosides (digitoxin, ouabain) to human serum albumin was studied using equilibrium dialysis, ultracentrifugation and gel filtration. 16alpha-gitoxin was 75% bound and its 16-acetate 95%. The percentage binding of digitoxin and ouabain was in good agreement with values reported in literature.


Differences in the binding of drugs to plasma proteins from newborn and adult man. I.

The binding of various drugs to plasma proteins from adult and cord plasma was determined by equilibrium dialysis. For almost all the drugs binding to cord plasma was lower than to plasma from adults. No evidence was found that the difference in binding was due to the different protein concentration in cord and adult plasma or to an influence of substances in ultrafiltrates of the plasmas.


Differences in the binding of drugs to plasma proteins from newborn and adult man. II.

The binding of certain drugs to isolated fractions of plasma proteins obtained from newborn and adult man has been studied by equilibrium dialysis. For thiopental, desipramine, nitrofurantoin, sulfamethoxydiazine, meticillin and salicylic acid no difference was found between binding to the albumin fraction from newborns and adults. However, for thiopental, desipramine and promethazine binding to the globulin fraction was smaller in the newborns than in adults. Addition of bilirubin to the albumin fraction decreased the binding of nitrofurantoin, sulfamethoxydiazine and meticillin. No difference in the binding of meticillin to the albumin or globulin fractions from newborns and adults was found. The binding decreased, however, if both fractions were combined. Four mechanisms to explain the difference in binding between newborns and adults are discussed: (1) Displacement of drugs by bilirubin, (2) different binding properties of cord and adult albumin, (3) different properties of the globulins and (4) interaction of albumin with globulins in the newborn.


Pharmacokinetics of salicylate and indomethacin in coeliac disease.

The plasma concentrations of salicylate and indomethacin were measured after a single oral dose of aspirin (600 mg) and indomethacin (50 mg) in twelve starved normal subjects and twelve adult patients with coeliac disease. The absorption of salicylate in the coeliac patients was faster than in the normal subjects. The plasma concentration/time curve of indomethacin in both groups was similar during the absorption phase, but there were significant differences between the groups in its elimination. The abnormal absorption pattern of salicylate in coeliac disease does not appear to be related to its pKa. Possible causes of the difference in salicylate absorption include changes in gastric emptying or altered small intestinal permeability.


Effect of simultaneous treatment with low doses of perphenazine on plasma and urine concentrations of nortriptyline and 10-hydroxynortriptyline.

Plasma levels of nortriptyline and perphenazine were measured in six patients on continuous nortriptyline treatment before, during and after oral administration of perphenazine 4 mg t.i.d. In four patients the plasma levels of the conjugated and unconjugated principal metabolite 10-hydroxynortriptyline were also measured. Urinary excretion of conjugated and unconjugated 10-hydroxynortriptyline and plasma levels of perphenazine were determined in all six patients. During treatment with perphenazine two patients showed a slight increase in the plasma level of nortriptyline. The changes in metabolite excretion rate were inconclusive. Thus, there did not appear to be any important pharmacokinetic interaction between the two drugs at the doses used, which were normal therapeutic doses. The previously reported inhibitory effect of perphenazine on the metabolism of nortriptyline probably depended therefore, either on administration of a higher dose of perphenazine, or on treatment in the reverse sequence--a single dose of nortriptyline was given to patients already receiving perphenazine.


Lymphocytes which differentiate in an allogeneic thymus. II. Evidence for both central and peripheral mechanisms in tolerance to donor strain tissues.

Nude mice which have been grafted with an allogeneic H-2-incompatible thymus are frequently specifically tolerant to skin grafts from the thymus donor strain. The evidence presented here indicates that both clonal deletion and peripheral suppressor cells play a part in maintaining this tolerance.


Embryonic bursa development in vitro.

An in vitro culture system for embryonic chicken bursa is described, in which development, as measured by follicle formation (site of B lymphocyte development), position of large basophilic cells (stem cells), and presence of lymphocytes and cells containing cytoplasmic immunoglobulin (B cells), was comparable to that of the bursa in vivo. Using this system, preliminary studies on B lymphocyte differentiation were made. These support the view that bursal lymphocytes develop from precursors that migrate through the surrounding mesenchyme and into the follicles, but indicate that at least some of these cells may be "pre-B cells" rather than pluripotent stem cells.


Effects of hemicholinium-3 or physostigmine on rat brain noradrenaline depletion induced by foot shocks.

Foot shocks applied to the rat on a grid floor caused brain noradrenaline depletion. Hemicholinium-3 injected intracerebroventricularly (i.c.v.) blocked the noradrenaline depletion and this blockade was reversed by choline (i.c.v.). Physostigmine (i.c.v.) had different effects according to the age of the rats: "young" and "intermediate" groups were depleted but not "adult" groups. Physostigmine plus shocks caused depletion only in "young" groups; the depletion not shown in "intermediat" groups, reappeared after pretreatment with alpha-methyl-p-tyrosine. It is concluded that there probably is a relationship between cholinergic and adrenergic neural systems when brain noradrenaline id depleted by shocks.


Morphine-induced supersensitivity to acetylcholine in skeletal muscle and its mechanism of action.

The effect of in vitro morphine pretreatment (250 micron) on the sensitivity of skeletal muscle to acetylcholine (ACh) was investigated using the isolated rectus abdominis muscle of frog. Morphine (15 to 120 min) significantly increased the sensitivity of the tissue to ACh. This increase in the sensitivity was found to be greatest after 120 min. Morphine significantly increased the sensitivity of the tissue to carbachol only after 120 min. In the presence of physostigmine, morphine did not cause further augmentation of responses to ACh. Potassium chloride-induced contractile responses were not influenced by morphine. In the presence of morphine, maximum contractile responses to ACh were significantly decreased. Highly calcium (1.62 mM) in the medium decreased the magnitude of the morphine-induced supersentivity to ACh at 120 min and antagonised the decrease in maximum contractile responses to ACh. These results suggest that morphine is causing supersensitivity by both its anticholinesterase activity and its inhibitory action on presynaptic nerve terminals.


Neuronal uptake inhibitors, nisoxetine and fluoxetine on rat vascular contractions.

The importance of neuronal uptake processes in rat arteries and veins was compared using nisoxetine and fluoxetine, selective inhibitors for neuronal uptake of norepinephrine and serotonin, respectively. Nisoxetine (10(-7) - 10(-5) M) increased the sensitivity to exogenous norepinephrine in the portal and mesenteric veins (10-fold) and in the mesenteric artery (2.5-fold). Responses to field stimulation were also increased afternisoxetine in all three vessels. After nisoxetine, aortic responses to norepinephrine were unaltered and in all tissues, serotonin-induced contractions were reduced. Fluoxetine did not potentiate responses to norepinephrine or to field stimulation  except in the mesenteric vein where sensitivity to norepinephrine was increased and the relaxation rate after field stimulation was prolonged. Although serotonin has been detected in blood vessels, fluoxetine did not increase vascular responses to serotonin. These studies suggest that rat mesenteric veins have a highly sensitive neuronal uptake mechanism of norepinephrine. Furthermore, these data provide indirect evidence against a functionally important serotonergic uptake process in rat blood vessels and suggest that neuronal uptake of serotonin does not exert a major role in terminating the vascular action of this biogenic amine.


Stimulation of prostaglandin output from rat stomach by hypertonic solutions.

A whole-cell preparation of rat stomach put out appreciable amounts of immunoassayable PGE. Buffers made hypertonic with either sodium chloride or mannitol effected several-fold increases in PGE output, while a comparable osmotic concentration of urea seemed to produce much less stimulation. Enhancement of PGE output by saline--hypertonic buffers was concentration-dependent. It is suggested that the osmolarity of the gastric contents may play a role in regulating gastric PG biosynthesis.


Effects of tetrodotoxin, elevated calcium and calcium antagonists on electrically induced 3H-noradrenaline release from brain slices.

Electrically induced release of 3H-noradrenaline from superfused rat brain cortex slices was completely inhibited by tetrodotoxin (1 microgram/ml) if 0.5 or 1 V stimuli were used, while inhibition was 97% with 2 V, and nil with 12 V stimuli. 20 mM calcium depressed noradrenaline release at all applied potentials, and shifted the release versus voltage curve to higher potentials. Manganese (5 mM) and D-600 (10-100 microgram/ml) also considerably inhibited release. This suggests that stimuli of up to 2 V induce transmitter release by exciting intracortical noradrenergic axons in their non-terminal regions.


Antagonism of smooth muscle responses to oxytocin and vasopressin by salsolinol.

The responses of isolated smooth muscle tissues to the polypeptides oxytocin, vasopressin and bradykinin were evaluated in the presence of the tetrahydroisoquinoline salsolinol. Significant antagonism occurred to oxytocin and vasopressin while the effects of bradykinin were unaltered. These results suggest that the in vivo formation of salsolinol after ethanol consumption could have significant physiological consequences.


The dynamics of dopamine metabolism in various regions of rat brain.

Dopamine metabolism was studied in various regions of rat brain by following the decline of 3,4-dihydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (HVA) from brain after treatment with pargyline, or from the accumulation of the acids after treatment with probenecid. The decline of DOPAC and HVA after pargyline treatment appeared exponential in all regions of brain studied with half-lives of about 13 min for HVA and 6.5 min for DOPAC. DOPAC was the major metabolite of dopamine, with various brain regions producing between 2-5 times more DOPAC than HVA. HVA accumulated after treatment with probenecid but the accumulation in 1 h did ot account for all of the HVA apparently eliminated from brain. DOPAC accumulated in some regions of brain (medulla, hypothalamus and midbrain) and not in others (cerebellum, cortex, striatum and hippocampus) after probenecid treatment. We conclude that dopamine metabolism is not uniform in brain and that the accumulation of DOPAC and HVA in brain after probenecid treatment only accounts for a minor fraction of the dopamine formed in brain.


Anesthesia-induced alteration of small vessel response to norepinephrine.

Closed-circuit television microscopy was used to quantitate the in vivo response of small arteries (approximately 100 micron) and small veins (approximately 150 micron) to topically applied norepinephrine in the rat cremaster muscle. Rats were anesthetized with pentobarbital (50 mg/kg), or urethane (1200 mg/kg) or a combination of urethane (800 mg/kg) and chloralose (60 mg/kg). Complete concentration-response curves were obtained for an artery and vein pair in each rat and pD2 values (-log ED50) were used to evaluate the vascular sensitivity to norepinephrine. Both the artery and the vein in urethane-anesthetized animals had decreased sensitivity to norepinephrine in comparison to the vessels of animals anesthetized with pentobarbital or urethane-chloralose. Pretreatment with cocaine (10(-5) M) significantly increased the sensitivity of both the artery and vein in pentobarbital-anesthetized animals but did not affect the vessels in urethane-chloralose-anesthetized animals. These results are consistent with two opposing effects of the urethane-chloralose combination. The first is an increased sensitivity to norepinephrine via blockade of neuronal uptake and the second is a decreased sensitivity of norepinephrine via a vascular inhibitory effect of urethane.


Histamine, histamine antagonists and regional blood flow.

The effects of mepyramine, a selective histamine H1-receptor antagonist, and metiamide, a selective histamine H2-receptor antagonist, have been determined on the cardiovascular response to intravenous infusions of a large dose of histamine, 1 X 10(-7) mol/kg/min. Pretreatment with either mepyramine, 2.5 X 10(-6) mol/kg, or metiamide, 2 X 10(-6) mol/kg/min, had no significant effect on the fall in blood pressure or fall in total peripheral resistance during histamine infusion. Pretreatment with mepyramine, 2.5 X 10(-6) mol/kg, and metiamide, 2 X 10(-6) mol/kg/min, abolished the fall in blood pressure and total peripheral resistance during infusion of histamine. Histamine-induced vasodilatation in the stomach was unaltered by pretreatment with mepyramine but abolished by pretreatment with metiamide. Histamine-induced coronary vasodilation was reduced slightly by pretreatment with mepyramine and substantially by pretreatment with metiamide. Coronary vasodilatation was abolished by treatment with mepyramine and metiamide.


Effects of amphetamine on brain biogenic amines in isolated and aggregated rats.

d- and 1-amphetamine were injected (15 mg/kg i.p.) into isolated or aggregated rats. Both d- and 1-forms decreased the noradrenaline levels of brain areas in isolated and in aggregated rats. d-Amphetamine decreased 5-hydroxytryptamine (5-HT) and 5-hydroxyindolacetic acid (5-hiaa) levels in aggregated rats only. 1-Amphetamine induced no change in 5-HT and 5-HIAA levels. The greater sensitivity of aggregated rats to d-amphetamine can be ascribed to a change in 5-HT metabolism rather than to a modification of noradrenaline levels.


Effects of 780 SE on lipid metabolism in rabbit aorta.

Pretreatment of rabbits with Benfouorex (780 SE) 50 mg/kg p.o. for 10 days strongly inhibited the incorporation of a 20 muCi tracer dose of 4-14C-cholesterol (given i.v. 24 h earlier) into various segments of the aorta. Incorporation of specific activity into the liver, intestine, lung or plasma was not affected. 1 h after i.v. injection of a 100 muCi tracer dose of 2-14C-acetate, a significant reduction in the radioactivity of total lipids of the aorta and liver  was seen in rabbits pretreated with 780 SE. The inhibition of acetate incorporation into arterial lipids was observed in all lipid fractions (i.e. free fatty acids, free and esterfied cholesterol, phospholipids and especially triglycerides). Incorporation of acetate into triglyceride fractions of liver and intestine and into hepatic cholesterol was also significantly decreased. The results indicate that 780 SE may have anti-atherogenic properties which could be valuable in the clinical treatment of atherosclerosis.


Changes in rat hind limb vascular resistance following intracerebroventricular drug administration.

The right hind limbs of rats (which had previously implanted intraventricular guide cannulae) were isolated from the systemic circulation, but with the nerves to the limb remaining intact, and perfused using a constant output blood pump. Using this preparation, changes in vascular resistance, blood pressure and heart rate were monitored following injection of noradrenaline, phentolamine and propranolol into the lateral cerebral ventricles (i.c.v.) of rats anaesthetised with alpha-chloralose. All three drugs lowered blood pressure. Noradrenaline administered i.c.v. induced a nervously mediated vasocilatation and an insignificant fall in heart rate whereas i.c.v. phentolamine administration was followed by a nervously mediated vasoconstriction in the isolated hind limb and a gradual rise in heart rate. After i.c.v. administration of propranolol there was no evidence of an immediate nervously mediated vasodilatation but heart rate fell significantly. Following i.c.v. phentolamine or propranolol vasodilatation did not occur in the hind limb until after the time taken for circulating blood ro reach the isolated vascular bed. The vasodilatatory and hypotensive responses to i.c.v. noradrenaline were not evident following prior i.c.v. injection of phentolamine. These results indicate the suitability of this preparation for investigations of central actions of other drugs.


Extracellular recording from the guinea-pig myenteric plexus and the action of morphine.

Extracellular recordings were made with glass suction electrodes from neurons in isolated ganglia of the myenteric plexus of the guinea-pig ileum. The activity recorded was considered to arise in neuronal somata as a consequence of the mechanical contact with the recording electrode. The spike activity was depressed by normorphine (10 nM-1 micrometer) and this action was prevented by low concentrations of naloxone. Morphine remained effective in solutions which contained 1 or 12 mM K+. The findings support other evidence that morphine acts by hyperpolarizing the soma membrane of myenteric neurons, but do not elucidate the ionic mechanism of the effect.


Regional vascular resistance and heart rate responses mediated through H1- and H2-histamine receptors in the unanaesthetised rabbit.

The effects of histamine infusions (10-100 microgram/kg/min) on heart rate and hindlimb, carotid, mesenteric and renal vascular resistance were investigated in unanaesthetised rabbits after "total" autonomic effector block to abolish reflex effects. Histamine caused a rise in heart rate that was predominately due to stimulation of H2-receptors (blocked by metiamide). Hindlimb and carotid vascular resistance did not change significantly during histamine infusion. However, after blocking H2-receptors with metiamide histamine infusions produced dose-related vasoconstriction in these beds while after H1-receptor block with mepyramine histamine caused dose-related vasodilatation indicating that H1- and H2-RECEPTORS MEDIATED OPPOSITE VASCULAR EFFECTS WHICH WERE OF SIMILAR MAGNITUDE. By contrast, histamine infusion caused vasodilatation in both the mesenteric and renal vasculature before giving antagonists. This dilatation was mediated by both H1- and H2-RECEPTORS AS EITHER RECEPTOR ANTAGONIST ATTENUATED THE RESPONSE. These studies suggest that H1-receptors in the same species mediate vasoconstriction in som- beds and vasodilatation in others while H2-receptors mediate vasodilatation in all the beds studied and also account for most of the increase in heart rate.


The influence of adrenergic agonists and their antagonists on isolated salivary glands of ixodid ticks.

Various drugs elicit fluid secretion by isolated salivary glands of two species of ixodid ticks (Dermacentor andersoni Stiles and Amblyomma hebraeum Koch). Among catecholamines, the following order of potency was observed: dopamine, epinine, noradrenaline = adrenaline and isoprenaline. The following drugs, in order of potency, were also agonists on this preparation: ergonovine, ergotamine, 6-hydroxydopamine, apomorphine, phenylephrine, norphenylephrine, beta-phenylethylamine, tyramine, D, L-dopa and octopamine. Nialamide increased the response to near-threshold concentrations of dopamine but had no intrinsic activity. Dopamine-induced secretion was depressed by phenoxybenzamine, alpha-flupenthixol, phentolamine, propranolol and dichloroisoprenaline, but only at conce,trations 10- to 1000-fold that of the agonist. Pimozide and spiperone (10(-6) M) augmented the maximum response of dopamine. The tick salivary gland, thus appears to contain one or several receptors differing pharmacologically from mammalian alpha-adrenergic, beta-adrenergic and dopamine receptors.


Noradrenaline reversal in canine subcutaneous adipose tissue.

Although adrenaline reversal is a well known phenomenon, fewer investigations have demonstrated reversal of the vascular response to noradrenaline (NA). The present studies compared subcutaneous adipose tissue vascular responses to intra-arterial NA with those of sympathetic nerve stimulation. Both NA and electrical stimulation produced vasoconstriction in adipose tissue which was reversed to beta-adrenergically mediated vasodilation following local alpha-adrenergic block.


The effect of sodium nitroprusside on the uptake and efflux of 45Ca from rabbit and rat vessels.

In this study we investigated if sodium nitroprusside (NaNP), a vasodilator with direct action on smooth muscle, would have any effect on calcium kinetics in blood vessels. The effect of NaNP, in 0.1-10 micrometer concentration, was studied in vitro on the uptake of 45Ca with the lanthanum method and on the efflux of 45Ca. The drug failed to alter the uptake of 'La-resistant' 45Ca in rabbit mesenteric artery and vein. This was true even if Ca2+ uptake was increased in the vessels by 60 mM KCl. The efflux of 45Ca on the other hand was enhanced from rabbit mesenteric vein and from rat aorta exposed to NaNP, in a dose-related way. The present study, like our previous one on other vasodilators, indicates that NaNP has en effect on vascular calcium kinetics but this is the first time that a relationship could be established between the dose of the drug and the effect on 45Ca efflux.


Vascular actions of histamine H1- and H2-receptor agonists in dogs and cats.

Systemic and pulmonary vascular responses to infusions of histamine, 2-methylhistamine (2-MeH), and 4-methylhistamine (4-MeH) were determined. In dogs, H1-receptor stimulation with 2-MeH induced pulmonary vasoconstriction, and a decrease in cardiac output. H2-receptor stimulation with 4-MeH induced pulmonary and systemic vasodilation, tachycardia, and an increase in cardiac output. In cats, 2-MeH caused slight pulmonary vasoconstriction and 4-MeH caused vasodilation. Both H1- and H2-receptors mediated the systemic vasodilation, tachycardia, and increase in cardiac output observed in cats.


Potentiation of d-tubocurarine (d-Tc) neuromuscular blockade in cats by lithium chloride.

I.v. infusion of LiCl at 1.0 mEq/0.25 ml/min produced significant decrease in plasma Na+ from 158 +/- 4.5 mEq/l (p less than 0.025) and in Ca2+ from 7.8 +/- 0.34 to 6.8 +/- 0.24 mg% (p less than 0.01) with insignificant increase in K+. No significant changes in plasma electrolytes were observed on infusion of NaCl. The d-Tc induced neuromuscular blockade in Li Cl infused animals was significantly greater than that in Na Cl infused ones.


Temporal effects of p-chloroamphetamine on catecholamine synthesis.

Within 0.25 h after the administration of 5 mg/kg of p-chloroamphetamine (PCA), the conversion index of both norepinephrine (NE) and dopamine (DA) was increased. The effect on DA synthesis was similar, but of less magnitude after 1.25 h, while the effect on NE was reversed, i.e. the conversion index was decreased. This biphasic effect of PCA on NE synthesis is discussed the in the context of two opposing regulatory mechanisms for neuronal catecholamine synthesis.


The role of the submandibular glands in extrarenal erythropoietin production in mice.

The plasma erythropoietic activity of anephric mice and anephric mice without submandibular glands was compared. The erythropoietin production was stimulated by anemia (phenylhydrazine) and hypobaric hypoxia (0.4 atm). The activity was measured by the 59Fe incorporation in total circulating red blood cells in mice rendered polycythemic by hypoxia. The results were also calculated in units (IRP) of erythropoietin. The plasma erythropoietic activity of anephric mice was neutralized by antiserum against normal erythropoietin. Extrarenal erythropoietin production was significantly reduced in mice deprived of submandibular glands. These data indicate that this phenomenon is not restricted to the rat and support the concept that the submandibular glands are important extrarenal sources of erythropoietin.


Growth and differentiation of rat bone marrow stem cells in diffusion chamber culture.

The growth of granulopoietic progenitors (CFU-C) and morphologically identifiable haemopoietic cells from rat bone marrow was studied in intraperitoneal diffusion chambers over a 6-day period. The aim of this study was to investigate whether stem cells preceding the granulopoietic progenitors are able to restore and to maintain depleted compartments of CFU-C and immature granulopoietic cells under the conditions of diffusion chamber culture. After treatment of rats with repeated doses of hydroxyurea (HU) a bone marrow cell suspension could be obtained wherein proliferating haemopoietic cells were reduced to a very low level and CFU-C to undetectable levels. During culture in diffusion chambers, growth of CFU-C, immature granulopoietic cells and macrophages could be observed from this HU-treated bone marrow. It is concluded that in diffusion chamber culture stem cells preceding CFU-C are able to rebuild functioning compartments of granulopoietic progenitors and morphologically identifiable haemopoietic cells.


Factors affecting erythroid colony growth (CFU-E) from human marrow.

Human marrow grown in a plasma clot gives rise to discrete colonies of nucleated red cells in response to erythropoietin (EPO). In 10 studies of human marrow with normal erythropoiesis, human EPO in doses up to 1 unit/ml produced a significant increase in colonies when compared to sheep EPO. To achieve 50% of maximum colony growth, only 0.1 units/ml of human EPO was needed in contrast to 0.5 units/ml of sheep EPO. In seven other experiments, nandrolone decanoate, 10(-7) to 10(-9) M, yielded a mean increase in colonies of 38% when compared to cultures without added androgen. This effect was entirely dependent on the presence of EPO. We conclude that the method is useful for the invitro study of factors affecting early events in erythropoiesis.


The interaction of erythropoietin with fetal liver cells. I. Measurement of proliferation by tritiated thymidine incorporation.

Erythropoietin stimulates the erythropoietin responsive cell to undergo DNA synthesis and subsequent mitosis. To define further the physiology of this effect, a liquid suspension microculture utilizing mouse fetal liver cells was developed. Tritiated thymidine incorporation into erythroid precursors was found to parallel radiolabeled iron incorporation with peak DNA synthesis occurring after 24 hours of culture. Both tritiated thymidine and iron incorporation were dependent on erythropoietin concentration. The responsiveness to erythropoietin decreased when erythropoietin was withheld and this diminishment in reactivity paralleled a morphological differentiation of the cells. This observation, together with the finding that erythropoietin activity could be removed by absorption with large numbers of cells, suggests the proliferation induced by erythropoietin depends on a specific stage in the differentiation of the red blood cell and may be mediated through a specific cellular receptor.


Some aspects of iron metabolism during erythropoietic depression in the mouse.

Spleen and bone marrow patterns of response differ in mice subjected to erythropoietic depressors. Radioiron injected a few hours after a high dose of cyclophosphamide or x-irradiation is retained in the bone marrow. The magnitude of medullary retention is closely related to the number of cells able to synthesize hemoglobin at the moment of iron administration, and to the rate of cell death provoked by the cytotoxic agent. Depressors such as Actinomycin and transfusion, that block the differentiation of stem cells while allowing normal maturation of the erythroid cohort, do not induce marrow entrapment of iron. By contrast, retention is never observed in the spleen, where the 59-Fe turnover is not influenced by the mechanism and magnitude of aplasia. A functional lack of homogeneity of splenic and bone marrow erythropoiesis, hemoglobin metabolism and/or handling of iron stores is proposed. These results would be in agreement with other data from the literature reporting physiological differences among the various sectors of the reticuloendothelial system.


The effect of peripheral blood contamination on colony yield from human bone marrow aspirates.

Serial bone marrow aspirates were taken from a single site in each of nine individuals. The colony forming capacity of these samples, measured by culturing the cells in semi-solid agar in diffusion chambers, fell as the aspirate volume increased. Comparison of the results from different individuals showed that this effect was consistent within the group and may be useful as a simple method for standardizing results from human bone marrow cultures.


Hematologic changes and thrombopoietin production in mice after X-irradiation and platelet-specific antisera.

Sera or plasma thrombopoietin (TSF) levels of mice were determined after: (a) no treatment; (b) induction of thrombocytopenia by injection of rabbit anti-mouse platelet serum (RAMPS); (c) exposure to 750 R or 900 R whole-body x-irradiation; or (d) irradiation and injection with RAMPS. Levels of TSF were assayed in thrombocythemic mice, using Na235SO4 uptake. RAMPS produced an immediate, severe thrombocytopenia without altering RBC or WBC counts of mice. Plasma collected from mice 4 hours after RAMPS injection increased both 35S incorporation into platelets (170% of control, P less than 0.005) and platelet counts (P less than 0.025) of TSF-assay mice. Although severe thrombocytopenia persisted, plasma TSF levels decreased thereafter, i.e., 111% of control after 8 hours and 99% of control after 16 hours. Platelet counts in mice exposed to 750 R and 900 R x-rays progressively decreased to severe thrombocytopenia by day 7, but almost normal RBC counts were maintained. Sera or plasma from animals with x-ray-induced thrombocytopenia caused significant increases in 35S incorporation into platelets of TSF-assay mice (196% of control, P less than 0.005 after 750 R and 141% of control, P less than 0.025 after 900 R). A combination of x-irradiation and RAMPS-injection did not produce greater TSF levels in mice than did x-ray or RAMPS treatment alone.


Radiation-induced enlargement of granulocytic and macrophage progenitor cells in mouse bone marrow.

The peak sedimentation velocity of C57BL mouse bone marrow progenitors of granulocytes and macrophages (GM-colony-forming cells, GM-CFC's) increased from 4.3 mm/h to 7-8 mm/h by 2 days after 250 rad whole body irradiation and slowly returned to normal over the next 3 weeks. Ppreliminary irradiation and/or endotoxin injection did not prevent this radiation-induced change. Some change in sedimentation velocity was seen with as little as 100 rad irradiation. Neither buoyant density nor cell cycle changes could account for the sedimentation velocity data which therefore indicate a major volume increase in the GM-CFC's. This size enlargement affected all subpopulations of GM-CFC's which consequently maintained their size relationship with one another.


Attempts to purify hemopoietic stem cell enrichment in bone marrow by use of glass wool filtration.

This study is about enrichment of hemopoietic stem cell concentration in mouse bone marrow by use of a glass wool filtration technique. Maximum stem cell concentration was accomplished when filtration conditions were as follows: (1) support medium supplemented to 15% with fresh serum from syngeneic donors; (2) no incubation time allowed for cells on the columns; and (3) an operational temperature of 24 degrees C. With these conditions, stem cell concentrations were increased 5,4-fold relative to the concentration in untreated marrow.


Induction of proliferation of haemopoietic stem cells in culture.

The behavior of the pluripotent haemopoietic stem cells (HSC) in liquid culture was investigated by determining CFU-S number and cell cyclinc status (by in vitro thymidine suicide) at regular intervals. The influence of fibroblast feeder cells and fibroblast conditioned medium on these parameters was investigated. Evidence was obtained for self replication of HSC in these cultures. Proliferation of HSC was found to be dependent on a humoral factor, released into the medium by fibroblasts. Separation experiments show that this stem cell activating factor (SAF) is distinct from the colony stimulating factor (CSF). Comparison of SAF with a lysate preparation of red blood cells, a product able to sustain an elevated recovery of stem cells in culture, showed a different mode of action on the persistence of HSC. The system offers an approach to the in vitro study of regulation at the level of HSC self generation.


Cerebral evoked responses to skin warming recorded from human scalp.

Reponses evoked by warming the glabrous palmar skin were recorded maximally from a contralateral parietal scalp site that approximated the hand projection area of sensorimotor cortex. A smaller and later occurring response was also seen at the corresponding ipsilateral site. The temperature to which the skin was adapted was critical and was maintained at 35 degrees C rather at 30 degrees C as it was in an earlier study where no responses were seen. Peak latencies ranged from 280 msec to 356 msec for stimulus intensities of 8 degree C presented at a rate of 19 degrees C/sec. This warm evoked response appeared to have its origin in the specifically sensitive primary warm afferents. The presence of an evoked response when warming occurred from the 35 degrees C adapting temperature (AT) and its absence at the 30 degrees C AT coincide with the greater sensitivity of warm receptors at the higher AT. Comparison of these results with those for evoked responses to skin cooling and tactile tap suggest that the cortical organization of temperature (both warm and cool stimuli) in human is similar to that of touch.


Neurones in the spinal trigeminal nucleus of the cat responding to movement of the vibrissae.

1. In chloralose anaesthetised cats the receptive field organisation and response properties of neurones of the spinal trigeminal nucleus were exmained and compared with the discharge characteristics of afferent units from vibrissae. 2. The response properties of the primary afferent discharges were preserved in at least a proportion of the second order cells, 82% of those which responded to movements of the maxillary vibrissae had phasic discharges and 18% had tonic discharges. 3. The discharge characteristics of the two main types of primary afferent slowly-adapting units (St I and St II) were distinguishable in the tonically active cells of the nucleus. These two groups showed (i) substantially different interspike interval distributions, (ii) different adaptive properties and (iii) different directional sesitivity. The two categories were designated nuclear type I and type II in accordance with the classification of primary afferent slowly-adapting units. 4. A loss of stimulus information was deduced from a consistent increase in the variability of the second order discharges compared with their presumed afferent input, but this information loss may be important in allowing the linear summation of discharges at the level of the thalamus.