Report and characterization of a new variant, EB, of human red cell acid phosphatase.

A new variant phenotype of human red cell acid phosphatase, designated EB, was discovered in a male during a survey of blood donors from Copenhagen, Denmark. Electrophoretically, the variant revealed the two isozymes corresponding to the B type as well as two fast moving anodic isozymes. The enzyme activity and thermostability were found to be higher than in any earlier reported type. Isoelectric focusing of the variant type indicated that the isoelectric point of the variant enzyme is lower than in the common types.


Intropin (dopamine hydrochloride) intravenous admixture compatibility. Part 1: stability with common intravenous fluids.

The stability of dopamine hydrochloride (Intropin) in several large-volume parenteral solutions was studied. Admixtures of dopamine were assayed by colorimetric and chromatographic procedures. Admixtures (800 mug dopamine per ml) in the following intravenous fluids in glass bottles at pH 6.85 or below were found to be chemically and physically stable for at least 48 hours at room temperature: dextrose 5%, dextrose 5% and sodium chloride 0.9%, 5% dextrose in 0.45% sodium chloride, dextrose 5% in lactated Ringer's solution, lactated Ringer's injection, 0.9% sodium chloride, 1/6 molar sodium lactate, and 20% mannitol. The admixture of dopamine in 5% dextrose was stable for a minimum of seven days at 5 C. A 5% dextrose-dopamine admixture in a polyvinylchloride bag was stable for at least 24 hours at room temperature. The admixture of dopamine in 5% sodium bicarbonate solution produced an unstable solution of pH 8.20. A chemical and physical change (development of a pink color) was observed in this admixture. It is recommended that dopamine not be added to 5% sodium bicarbonate solution or any alkaline intravenous solution.


Evaluation of a hospital controlled substances distribution system.

The distribution and control system for controlled drugs at a 500-bed hospital was evaluated. The time requirements of the system for pharmacy and nursing, the drug control procedures used, and the income and expenses of the system were evaluated. It was concluded that the system appears to offer full accountability, but it makes a heavy time demand on pharmacy and nursing. Time requirements could be reduced through a computerized system or by use of a certificate of disposition form that incorporates a charge voucher.


Drug diffusion and bioavailability: tetracycline metallic chelation.

The effects of chelation on the in vitro diffusion rate of tetracycline at variable pH values and concentrations was studied. A two-compartment diffusion cell using a semipermeable cellophane membrane was used to determine diffusion rates. Tetracycline in solution was mixed with aluminum, magnesium, copper, calcium and cobalt ions and analyzed by ultraviolet spectrophotometry. Diffusion rates were determined at pH 1, 5.5 and 8. Tetracycline chelate formation is pH-dependent. The diffusion rate of tetracycline chelates is concentration-dependent and is reduced even if the chelate is water soluble.


Evaluation of bacteriological transport systems.

Seven commercially manufactured bacteriological transport systems, including Culturette, Trans-Cul (with Stuart and Amies), Handiswab, Securline (with Amies and Amies without charcoal) and Culture Caddy, were evaluated to determine whether these systems were capable of maintaining the viability and constant numbers of mixtures of hardy, fastidious and anaerobic organisms over 72 hours at 25C and 4C. The results of the study indicated that if specimens are maintained in transport systems at 25C they should be cultured at four hours to minimize loss of viability and to assure accurate quantitation. No one transport system excelled over the other for maintenance of cultures at room temperature. Most organisms survived well in all of the transport systems for 72 hours when refrigerated.


Prolonged fetal bradycardia with recovery--its significance and outcome.

bradycardia lasting more than 21/2 minutes with a decrease in FHR of more than 30 b.p.m. were observed in the course of 15 monitored labors. The mean fetal pH during the 20 minutes following the bradycardia (early recovery) was significantly less than the mean pH obtained 30 minutes or more after the bradycardia (late recovery). Various types of periodic heart rate changes were observed during the early recovery period but few persisted into late recovery. Vaginal delivery occurred in 13 of 15 patients. Two cesarean sections were performed for indications unrelated to the prolonged bradycardia. The 5 minute Apgar score of all 15 neonates was 7 or greater.


Renal excretion of N'1-methylnicotinamide in the rat.

The renal excretion of N'1-methylnicotinamide (NMN) was studied in the rat.  Renal clearance experiments clearly demonstrated that: 1) NMN is secreted; 2)a tubularmaximum (Tm), 7 mumol/min per kg, could be reached; and 3)NMN secretion is inhibitedby a competitive inhibitor, mepiperphenidol.  In free-flow micropuncture experiments, animals were infused with plasma concentrations of NMN ABOVE Tm; the TF/P NMNto TF/P inblin ratio for proximal and distal samples was 2.34 and 2.28, respectively, indicating that NMN is secreted in the proximal tubules and is not secreted orreabsorbed in the distal tubules.  This finding was further confirmed by intratubularmicroinjections of ['14C]NMN into rats.  In diuretic animals approxiamately 10%of the NMN injected into early proximal tubules was reabsorbed, but no reabsorption could be detected after distal injections.  The nondiuretic animals showed no significant reabsorption of NMN.  It was concluded that NMN transport is a carrier-mediated process and that reabsorption, if it occurs, plays only a minor role.


Peritubular pH and PCO'2 in renal tubular acidification.

The influence of peritubular capillary pH and PCO'2 on renal tubular acidification was studied in rats by luminal and peritubular perfusion techniques.  Luminal stopped-flow microperfusions were carried out with bicarbonate or alkaline phosphate solutions and luminal pH continuously measured by antimony micorelectrodes.  Peritubular calpillary microperfusions were carried out with mammalian Ringer solution kept at different pH and PCO'2.  The acidification process was assessed in terms of 1)maximal pH differences, 2)rates of pH change, and 3)rates of bicarbinate reabsorption or H'+ ion secretion.  During peritubular perfusions at physiological pH and PCO'2 tubular  acidifying capacity was maintained at near-normal levels.  Perfusingcapillaries at high pH and low PCO'2, especially with bicarbonate Ringer, acidification was markedly depressed; it was moderately reduced at a peritubular pH of 5.6.  At a capillary pH of 7.4, acidification was similiar at low and physiological PCO'2and enhanced at elevated PCO'2.  Systemic respiratory acidosis enhanced acidification in the proximal tubule, but reduced it in distal segments.


Phospholipase A2 in experimental hypertension.

An assay method has been developed to measure phospholipase A2 (PLA2) in ratserum and to study the possible role of this enzyme in experimental hypertension.  Experiments with rat serum following 48 h of bilateral nephrectomy indicated a decrease inPLA2 activity, suggesting that kidneys might be playing an important role in regulating serum PLA2 activity and that kidneys might be a source of this enzyme.  Experiments with renal hypertensive rats, spontaneously hypertensive rats, and rats receiving a low-salt diet demonstrated that a decrease in PLA2 activity was found only in those conditions in which elevated plasma renin activity was accompanied by elevated blood pressure.  When elevated plasma renin activity was not accompanied by elevated blood pressure, serum PLA2 activity was unchanged.  These observations represent the first biochemical separation between conditions of elevated plasma renin activity without an increase in blood pressure and conditions of elevated plasma renin activity with an increasein blood pressure.


Effect of hyperventilation on dynamics of cerebral energy metabolism.

Hypocapnia of moderate and extreme degree (Paco2 21.1 and 13.5 torr, respectively)was induced by hyperventilation in rats subjected to the closed system of Lowry inorder to evaluate the effects on utilization rate of cerebral energy metabolites.  The tissue levels of high-energy phosphates and calculated intracellular pH did not change, whereas glucose, pyruvate, and lactate increased significantly.  The La/Pyratio and NADH/NAD-+ RATIO BOTH INCREASED IN PROPORTION TO THE DEGREE OF HYPOCAPNIA. Utilization rates of glucose, glycogen, and ATP were all significantly reduced by hypocapnia, whereas the utilization rate of phosphocreatine was increased.  The rate oftotal high-energy phosphate use was also diminished in proportion to the degree of hypocapnia.  The constant value of the energy charge (0.94 plus or minus 0.01) indicates that the energy production rate might also be reduced by hyperventilation; thus the intermediate metabolics and substrates increased.  It is concluded that extreme hypocapnia reduces the rate of cerebral energy metabolism significantly.


Patterns of Plasmodium vivax recurrence in a high-incidence coastal area of El Salvador, C. A.

During field studies carried out in 1971, 1972 and 1973 in a highly malarious coastal area of El Salvador, it was possible to collect information on the patterns of Plasmodium vivax parasite occurrence in a large number of infected individuals. In most of the persons who had experienced a malaria attack during the high transmission period in June, July, August and September, renewed activity occurred 5 to 8 months later, during the low transmission season the next year. Subsequent activity in these same cases occurred after intervals of about 4 to 9 weeks in duration. On epidemiologic grounds, and on the basis of the life patterns of the El Salvador P. vivax strains demonstrated in previous studies in volunteers, this renewal of activity probably represents the occurrence of relapses rather than new infections. This pattern is similar to the "temperature zone" strains of P. vivax, where initial relapses occur after prolonged periods, followed by subsequent relapses after much shorter intervals of inactivity. This type of relapse pattern in a tropical area may enhance the survival of the parasite through a prolonged period of vector inactivity, such as the long dry season experienced in El Salvador.


California encephalitis virus prevalence throughout the Yukon Territory, 1971-1974.

California encephalitis (CE) virus (snowshoe hare subtype) was isolated from 1 of 38 pools comprising 970 unengorged female Aedes canadensis mosquitoes and from 3 of 152 pools containing 5,676 A. communis mosquitoes which were collected in the Yukon Territory, Canada between latitudes 61 and 66 degrees N during June and July 1974. During four summers 1971 through 1974, this virus was recovered from 26 of 648 pools derived from 30,686 mosquitoes of 4 species. Isolation of CE virus from 1 of 109 pools of Aedes sp. larvae collected during May 1974 suggests maintenance of this virus over winter by transovarial transfer. Infectivity has been maintained in Culiseta inornata mosquitoes which were held continuously at 32 degrees F for 138 days. Neutralizing antibodies to CE virus were detected in 705 of 4,913 (14%) mammals collected during summers 1971 through 1974, including 430 of 1,076 (40%) snowshoe hares (Lepus americanus), 266 of 3,610 (7%) ground squirrels (Citellus undulatus) and 9 of 227 (4%) red squirrels (Tamiasciurus hudsonicus).


Nephrotic syndrome in Schistosomiasis mansoni complicated by chronic salmonellosis.

Thirteen patients with concomitant chronic salmonellosis and schistosomiasis and with the nephrotic syndrome were studied. Of the seven patients' sera examined, all had markedly low complement C3 and near normal C4 levels. This indirectly suggests an active role of Salmonella endotoxin in the etiology of the nephrosis.


[Delayed healings of the epithelium of the cornea due to soft lenssolutions].

Six different soft lens solution have been tested to investigate their effect on the healing process of a corneal erosion, experimentally scratched in rabbit-cornea. Healing delays have been observed in every cases; also by three medicaments which are used for "dry" eyes. The latter ones should never have  a conjunctival application. Theexplanation of those  healing delays is given by the concentration properties of HEMA-lenses for disinfectants. The admixtures of soft lens solutions like buffers and liquid polymerscause and additive effect on retardment of coeneal regeneration.


A paradoxical action of minute doses of nondepolarizing muscle relaxants in anaesthetized man.

The administration of minute doses of all currently available nondepolarizing muscle relaxants result in an increase in contractile force of the skeletal muscles. This surprising finding has been obtained by means of mechanograms of the hand muscles after electrical stimulation of the ulnar nerve (100-200 volts, 0.3 msec, 0.2 Hz). Higher doses produce a decrease in contractile force and a paralysis of the skiltalmuscles. The paradoxical action of muscle relaxants can be explained by repetitive firing of the muscle action potential as has been demonstrated in the phrenic nerve-diaphragm preparation with tubocurarine and gallamine.


The effect of artifical ventilation and dehydrobenzperidol on the development of early changes in the respiratory system in dogs with Mendelson's syndrome.

The experiments were carried out to observe certain functional changes in the respiratory system which develop in the early stage of aspiration pneumonia and to study the possibility of influencing these changes by application of artificial ventilation (IPPB) and/or administration of dehydrobenzperidol. The experiments were carried out on 32 mongrel dogs divided into 4 groups. Experimental Mendelson's syndrome was produced by instilling, during anaesthesia, hydrochloric acid solution of pH 1.5 in a dose of 4 ml/kg into the tracheobronchial tree. Immediately after anaesthesia and then 10 times at intervals of one hour after HCl instillation the following determinations were done: mean blood pressure, CVP, haematocrit, PO2 and PCO2 in arterial and mixed venous blood, and minute artifical ventilation. The veno-arterial blood shunt in the lungs, alveolo-arterial difference of oxygen partial pressure and the effective compliance were also determined. The chemical damage to the lungs by acid caused condensation of blood and a fall in CVP. These changes were due to fluid escape from the vessels especially in the pulmonary vascular tree. Disturbances in pulmonary gas exchange were a result of deficient ventilation of lung areas damaged primarily by acid and suffering secondarily from developing disturbances in pulmonary blood flow. Dehydrobenzperidol in a dose of 1 mg/kg applied as the only treatment in Mendelson's syndrome had the same favourable effect on pulmonary changes as controlled IPPB. The combination of controlled artificial ventilation and intravenous dehydrobenzperidol decreased the disturbances in pulmonary gas exchange in Mendelson's syndrome. This was possible because both factors exert a benficial effect on ventilation and perfusion preventing the development of oedema and atelectasis.


Comparison of controlled respiration by the manual technique and with a respirator during general anaesthesia for abdominal operations.

These studies investigating the difference between controlled respiration performed by manual technique and controlled respiration applied by means of a respirator during general anaesthesia, and assessing the influence of these methods on the immediate post-operative condition of the patient were carried out on 75 patients during abdominal operations. Gasometric tests in arterial blood were performed before premedication, at intervals of 30 min from the beginning of operation, and 10 min. after the removal of the endotracheal tube. Controlled respiration with a respirator is a more favourable method of pulmonary ventilation during general anaesthesia given for a long-lasting surgical operation. The manual technique of controlled respiration leads to respiratory alkalosis. The best results were obtained using a respirator with inspiratory volume of 10 ml/kg and respiratory frequency 10/min., which ensured normocapnia. It was observed that oxygenation of arterial blood was reduced postoperatively in all patients but the value of PaO2 was the lowest in patients on manual controlled respiration. Disturbances in acid-base balance and oxygenation had an unfavourable effect and delayed the regaining of consciousness.


Influence of mare uterine tubal fluids on the metabolism of stallion sperm.

Three experiments were conducted on the metabolism of stallion sperm. In experiment 1, whole and washed sperm were incubated under aerobic and anaerobic enviroments and analyzed before and after controlled incubation for motility, pH, lactic acid, glucose, fructose, and O2 comsumption. In experiment 2, whole and washed sperm were incubated aerobically and anaerobically with and without uterine tubal fluids. Experiment 3 was the same as experiment 2, except added substrates of glucose and lactic acid were studied. The same examinations were made in experiments 2 and 3 as for experiment 1. Motility decreased significantly during incubation for all treatments, with the greatest decrease occurring for whole semen where only trace amounts of substrate (fructose) were present. Exogenous glucose plus uterine tubal fluid maintained sperm motility better than did added lactate. However, sperm respiration rates were highest when exogenous lactate was the only substrate in the incubation medium. The mean pH values for gel-free stallion semen at the start of controlled aerobic and anaerobic incubation were 7.08 and 7.34. Lactic acid accummulation for 1 hour increased from 0.05 mg to 0.09 mg/10(9) sperm when uterine tubal fluid was added to the incubation medium. Washed spermatozoa incubated in 0.03 M glucose plus uterine tubal fluid utilized less glucose than did sperm incubated in the glucose medium. These results, along with the increased oxygen utilization (ZO2) values produced by adding uterine tubal fluid to the incubation mediums, might indicate utilization of a uterine tubal substrate. Added uterine tubal fluid resulted in increased ZO2 values (expressed in mul of O2 utilized by 10(8) sperm in 1 hour at 37 C) for whole semen from 10.45 to 12.63. Washed spermatozoa also respired at a significantly greater rate than whole sperm. Respiration rates were greater for sperm incubated with 0.01 M lactic acid than for any other substrate or experiment.


[Alterations of renal function in pyelonephritis (author's transl)].

Renal function was investigated in a group of ten patients aged two months to eleven years, with chronic or recurrent urinary tract infection caused by different malformations of the urinary tract. The following tests were performed: endogenous creatinine clearance, maximal urinary concentrating ability, urinary acidification, maximal urinary diluting ability, free water clearance, index of fractional distal sodium delivery and index of distal tubular reabsorption of sodium. It is concluded that the follow-up of glomerular function by means of creatinine clearance and of tubular function by means of maximal concentrating ability consitute the most sensible way to detect renal functional impairment in children with chronic or recurrent pyelonephritis.


Recurrent wound sinuses after cholecystectomy for salmonella paratyphi.

Several cases in which Salmonella, spread by the haematogenous route, produced localised inflammation have previously been described in the literature. The infecting of an operation wound through direct contamination by Salmonella has earlier been reported only once, in connection with appendectomy. The present case report describes a case in which recurrent sinuses infected by Salmonella were formed in the operative wound.


The effect of drug therapy without volume replacement in experimental traumatic shock. Part I. Phenoxybenzamine and dopamine.

The effect of phenoxybenzamine and dopamine on the respiratory and metabolic changes in experimental traumatic shock was examined in 45 rabbits. Phenoxybenzamine caused a more effective release of the peripheral vasoconstriction than dopamine. The effect of these drugs without similar restoration of blood volume was unfavourable. This should be borne in mind when the use of these drugs is meditated in clinical work.


The effect of drug therapy without volume restoration in experimental traumatic shock. Part II. Pethidine.

Different opinions seem to exist as to the use of analgesies in the treatment of trauma patients. For this reason we investigated experimentally the effect of pethidine in traumatic shock. Pethidine was administrated to eight of sixteen anaesthetized rabbits with lower limb injuries. The effect of pethidine was unfavourable. This was due to the effect of the drug on circulation and not on respiration.


[Asparagine metabolism in mycobacteria. II. -- Asparagine hydrolysis and aspartohydroxamic acid formation and hydrolysis catalysed by M. fortuitum, M. phlei and BCG asparaginases (author's transl)].

Crude extracts of BCG, M. fortuitum and M. phlei, hydrolyse asparagine (I) and L-beta-asparthohydroxamic acid (III), and catalyse the synthesis of aspartohydroxamic acid from asparagine and hydroxylamine (II). The ratio between these enzymatic activities (I:II and I:III) presents a certain stability during the different steps of purification of these mycobacteria asparaginases. In particular, M. fortuitum asparaginase has been purified 90 to 130-fold, with recovery of approximately 10%. Only the fractions of supernatants which have an asparaginase activity catalyse the formation of aspartohydroxamate from asparagine and hydroxylamine. Some differences between the asparaginases of these strains are described. Particularaly, in comparison to reaction I, their abilities to catalyse reactions II and III vary noticeably from one asparaginase to an other. The asparaginase of BCG catalyses very slightly in the reactions II and III and is more specific of L-asparagine hydrolysis than are the asparaginases of M. fortuitum and of M. phlei. Furthermore, in the case of M. phlei, p-chloromercuribenzoate (pCMB) inhibits very stronly the reactions I and III and slightly reaction II, whereas conversely, for M. fortuitum, pCMB does not inhibit reactions I and III but strongly inhibits reaction II. In the case of BCG, these three reactions are not inhibited by pCMB. Moreover, the asparaginases from these strains are more or less sensitive to the ionic strength of the buffer used.


The effect of drug therapy without volume restoration in experimental traumatic shock. Part III. Glucocorticoid.

The effects of methylprednisolone in connexion with a standardized trauma was studied on 30 rabbits. The treatment with glucocorticoid accentuated the hemodilution due to the trauma. Intensified lactacidosis was also demonstrated.


[Remarks on ethanol oxidation by an "Acetobacter xylinum" microbial electrode (author's transl)].

A "microbial electrode" for ethanol assay has been designed using combination of an oxygen probe and cellulosic pellicle of Acetobacter xylinum. Assay is feasible with an ethanol concentration below 0.4 mM on a pH range of 2,5-7. The formation of acetic acid leds to a competitive inhibition of ethanol oxidation as observed with free cells. Pellicle stability at room temperature is good over a ten hours period. At 4 degrees C, film preservation is quite satisfactory over a ten days storage period. The author compares the ethanol oxidation kinetics observed, using both cellulosic pellicles and free cells of A. xylinum.


An inducible beta-lactamase in a strain of Escherichia coli.

The production of beta-lactamase (penicillin/cephalosporin beta-lactam amidohydrolase, E.C.3.5.2.6) was found to be inducible in a clinically isolated strain of Escherichia coli. This is the first report of an inducible beta-lactamase in E. coli. The optimal concentration of inducer was 400 mug/ml of ml of benzylpenicillin, or 800 mug/ml of 6-aminopenicillanic acid. About fiftyfold induction was achieved. Maximum induction took ninety minutes from the time of adding the inducer. Induction was abolished by the presence of chloramphenicol(10 mug/ml). The enzyme has a molecular wieght of 23,000, and is inhibited by rho-chloromercuribenzoate and by iodine. It is active against a wide range of substrates, including cephaloridine and cloxacillin.


A novel inducible formaldehyde dehyrogenase of Pseudomonas sp. (RJ1).

A novel, pyridine-nucleotide-independent, inducible formaldehyde dehydrogenase acitivity was detected in cells of Pseudomonas sp. (RJ1) propagated on methylamine and oxalated. The pH optimum of the dehydrogenase was 7.0. Dichlorophenol-indophenol or potassium ferricyanide served as an electron acceptor. The rate of reduction of these electron acceptors was shown to be stimulated by phenazine methosulfate. The dehydrogenase was inhibited by parahydroxymercuric benzoate and iodoacetamide. This inhibition suggests that the enzyme contains sulfhydryl groups. The stoichiometry of the reaction in terms of oxygen uptake to formate formation was 0.5, which agrees with the theoretical value.


The occurrence of killer character in yeasts of various genera.

Species of 7 of the 28 yeast genera in the National Collection of Yeast Cultures exhibited killing activity against Saccharomyces cerevisiae. The highest incidence of killer yeasts was found in the genus Hansenula (12 of the 29 strains examined). Saccharomyces, the best represented genus in the Collection, showed a low incidence of killer activity and many of the killer strains are hybrids with a common S. cerevisiae parent. The activities of culture filtrates of the 59 killer yeast isolated responded differently to pH and four types of response were recognised.


Properties of D-xylose isomerase from Streptomyces albus.

A partially purified D-xylose isomerase has been isolated from cells of Streptomyces albus NRRL 5778 and some of its properties have been determined. D-Glucose, D-xylose, D-ribose, L-arabinose, and L-rhamnose served as substrates for the enzyme with respective Km values of 86, 93, 350, 153, and 312 mM and Vmax values measuring 1.23, 2.9, 2.63, 0.153, and 0.048 mumol min per mg of protein. The hexose D-allose was also isomerized. The enzyme was strongly activated by 1.0 mM Mg2+ but only partially activated by 1.0 mM Co2+. The respective Km values for Mg2+ and Co2+ were 0.3 and 0.003 mM. Mg2+ and Co2+ appear to have separate binding sites on the isomerase. These cations also protect the enzyme from thermal denaturation and from D-sorbitol inhibition. The optimum temperature for ketose formation was 70 to 80 C at pH values ranging from 7 to 9. D-Sorbitol acts as a competitive inhibitor with a Ki of 5.5 mM against D-glucose, D-xylose, and D-ribose. Induction experiments, Mg2+ activation, and D-sorbitol inhibition indicated that a single enzyme (D-xylose isomerase) was responsible for the isomerization of the pentoses, methyl pentose, and glucose.


Death of Staphylococcus aureus in liquid whole egg near pH 8.

Incubating and shaking Staphylococcus aureus in liquid whole egg causes a decline in viability. During the period of agitation, the natural pH of the egg rises from about 7.2 to between 8.0 and 8.2 as a result of a loss of carbon dioxide. However, if the pH of the egg is prevented from rising, either by not shaking or by addition of a buffer, S. aureus will grow. The cause of death is traced to the presence of lysozyme of egg white. Interestingly, the action of lysozyme is not attributable to its bacterial lytic property but, instead, to the basicity of the lysozyme molecule. This conclusion is supported by the fact that the lytic property of lysozyme is known to have its optimal activity near neutrality and by the finding that protamine sulfate, a nonenzymatic basic polypeptide, also caused death of S. aureus at pH 8.0 but not at 7.0. It was postulated that the rise in pH renders the bacterial cells more negatively charged, so that in the presence of positively charged molecules like lysozyme or protamine sulfate a complex is formed, agglutinating the cells.


Degradation of ethylenediaminetetraacetic acid by mictobial populations from an aerated lagoon.

The ferric chelate of ethylenediaminetetraacetic acid (EDTA) was biologically degraded by a mixed population of microorganisms present in an aerated lagoon receiving this chemical in its feed. As determined radiorespirometrically, 28% of the acetate-2-C and 30% of the ethylene position of the ammonium ferric chelate of [14C]EDTA was recovered as 14CO2 after 5 days. In a separate experiment using gas liquid chromatography and the sodium ferric chelate, as much as 89% disappearance of EDTA (0.1% wt/vol) was observed during a similar time period. Optimum 14CO2 evolution was observed at a pH value between 7 and 8 and at room temperature. Degradation of NH4Fe-[2-14C]EDTA was stimulated by the addition of either unlabeled NaFe-EDTA, nitrilotriacetic acid or ethylenediamine, and inhibited by the addition of a variety of different sugars and amino acids. Consistent with the biological nature of this degradation, little or no 14CO2 evolution was observed after heat treatment of the microorganisms at 100 C for 10 min, or after the addition of antibiotics to the incubation mixtures. Gas-liquid chromatography and mass spectral analyses were performed to demonstrate EDTA disappearance and to identify possible intermediates of EDTA degradation.


Method for detecting small numbers of Vibrio cholerae in very polluted substrates.

A method is presented for the indirect detection of Vibrio cholerae by the multiplication of two specific bacteriophages: phiH74/64 for El-Tor vibrios, and phage group IV (Mukerjee) for classical vibrios. The product to be examined is seeded in alkaline tryptone water for enrichment, as in the classical method, and is then incubated for 6 h at 37 C. Thereafter, a loopful is transferred to each of two nutrient broth (pH 9) tubes. One of these receives a drop of phage phiH74/64; the other receives a drop of phage group IV. The stock phages are diluted so as to contain about 3,800 plaque-forming units in one drop; this is the maximum amount which, when added to 10 ml of broth, will not be detected in a loopful of 1 mm diameter. The tubes containing phage phiH74/64 are incubated at 42 C; those with phage group IV are incubated at 37 C. After 18 h the cultures are killed by agitation with chloroform, and a 1-mm loopful is deposited on a layer seeded with the detector strains: Makassar 757 for El-Tor phage and V. cholerae 154 for classical cholera phage. After 4 to 5 h at 37 C, lysis appears on the spot areas if there has been phage multiplication in the respective broth tubes. With experimentally contaminated sewage water, vegetables, or stools, 1 to 10 cholera vibrios were detected in every sample. In rare cases, false-positive results were obtained by multiplication of the phage on non-cholera vibrios.


Influence of colloidal iron on the respiration of a species of the genus Acinetobacter.

Washed suspensions of Acinetobacter sp. isolated from water caused the precipitation of iron from a suspension of colloidal ferric iron at pH 6.0 and 7.6. Iron-encrusted cells of the bacterium formed large aggregates. The amount of iron removed from the colloidal preparation in the form of aggregates was from 21 to 52% at pH 7.6 and 49% an pH 6.0 by the bacterial cells. Endogenous respiration rates of the iron-encrusted cells were from 32 to 72% lower than the rates for unencrusted cells. Respiration rates, measured polarographically in the presence of glucose, were also greatly reduced by the coating of iron on the cells.


Epidermal nicotinamide adenine dinucleotides in psoriasis and neurodermatitis (lichen simplex hypertrophicus).

Epidermal nicotinamide adenine dinucleotides were measured in subcorneal and basal epidermal layers in patients with psoriasis and neurodermatitis and in healthy controls. Lowry's microtechniques utilizing enzymatic cycling were used. The total NAD content in these groups was except for the involved psoriatic skin about 1.1 mmoles/kg dry weight. Between 40 and 50% of this content consisted of the reduced form. In the involved psoriatic skin the total NAD content was increased to 1.5 mmoles/kg, this increase being mainly due to a rise in NAD+. There was no difference in NAD content between epidermal layers in the various groups studied. The total NADP content was near 0.15 mmoles/kg in healthy controls and in patients with neurodermatitis. The subcorneal layers contained 10% more of the dinucleotide than the basal layers, but in the two layers the reduced form amounted to about 80% of the total. In both non-involved and involved skin of the psoriatic patients the total NADP content was significantly increased above the control level, by up to 20% in the former and up to 65% in the latter. In the two layers studied the NADP+ content was increased by about 75% in both non-involved and involved areas. In contrast, the NADPH content rose only in the basal layers of the lesion, by 55%. The increased levels of NADP+ and NADPH found in psoriasis might suggest an accelerated or differently conducted NADPH dependent biosynthesis in this disease.


Epidermal nicotinamide adenine dinucleotides in psoriasis during treatment with dithranol.

Epidermal nicotinamide adenine dinucleotides were measured in subcorneal and basal epidermal layers in patients with psoriasis and in healthy controls during a 2-week period in which they were treated once a day with 0.15% dithranol in white petrolatum. Lowry's microtechniques utilizing enzymatic cycling were used. In the controls the total NAD contents decreased 25% during the treatment period. The total NADP content did not change, nor did the proportions of NADH and NADPH. In the psoriatic patients effects of the treatment were seen only in the epidermis of the lesions. Both the total NAD and NADP displayed an initial increase on the second day, followed by a steady decrease to the levels found in the non-involved skin. During the first week of treatment parakeratosis was still evident in the lesions. In spite of variation in the total NAD contents during this period, a significant reduction of the percentage of NADH was found. Concomitant with the appearance of an orthokeratotic horny layer, both NAD+ and NADH returned to normal. The percentage contribution of NADPH did not change during these events.


Malaria studies in vitro. IV: Chloroquine resistance and the intracellular pH of erythrocytes parasitised with Plasmodium berghei.

The erythrocytic stages of strains of plasmodia sensitive to chloroquine (CQ) concentrate some three to six times more drug than CQ-resistant strains. A simple, but so far untested hypothesis is that the intracellular pH of drug resistant strains is higher than that for sensitive strains. This would result in a reduced accumulation of any weakly basic drug molecule (e.g. CQ) which is capable of passive diffusion through the cellular membran. The mean intracellular pH of erythrocytes parasitised with either CQ-sensitive or CQ-resistant strains of P. berghei was determined, therefore, from the distribution of 14C-5, 5-dimethyloxazolidine-2,4-dione between intra- and extracellular aqueous phases. The results obtained showed that over the range 6-8--7-2 the intracellular pH is governed directly by the extracellular pH and that the intracellular pH of CQ-sensitive parasites is slightly above that for resistant ones, neither being greatly different from that for uninfected erythrocytes. There was no evidence, therefore, to support, the suggested hypothesis. Elimination of this possibility adds weight to the opinion that changes in drug sensitivity of these plasmodia are the result of changes in the properties of drug-binding sites.


[Concomitant variations in the weight of adrenals and uremia during kidney provocation by high doses of glafenine].

High doses of glafenine given orally to rats induced renal tubular changes and simultaneously raised not only blood urea concentrations but also adrenal volume and weight. These modifications were transient and dose-dependent. Blood urea and adrenal weight returned to normal at the same time. Treated rats showed no alteration in serum K+, Na+, Cl-, uric acid, glucose and cholesterol and only a slight decrease in serum transaminase levels. An indirect relationship between renal lesions and adrenals hypertrophy induced by flafenine may exist.


Sympathetic component of the pressor response to angiotensin II in the pithed rat after pretreatment with disulfiram.

Pressor responses to angiotensin II in the pithed rat were unaffected or slightly increased by procedures such as reserpine pretreatment, alpha and beta adrenoceptor blocking agents or bethanidine which inhibit the sympathetic system. Disulfiram-induced inhibition of angiotensin II pressor responses was unaffected by pretreatment with either alpha-adrenoceptor blockers or bethanidine. Pretreatment with disulfiram prevented infusions of angiotensin II from increasing the responses to spinal sympathetic outflow stimulation. After inhibition of pressor responses to angiotensin II by disulfiram the responses were restored by infusions of noradrenaline, or alpha-methyldopa. The restored responses, unlike control responses, were inhibited by phentolamine. The results suggest that angiotensin II facilitates neuronal release of noradrenaline and that this effect is revealed most clearly when post-synaptic angiotensin II receptors are blocked.


Biologically active substances in oak gall extracts. Part 1: Isolation and chemical identification of a substance exerting antihistamine-like activity (KC-18).

The previously reported antihistamine-like activity of partially purified oak gall extracts has been confirmed. Isolation of the active principle was achieved through the use of organic solvent extractions and column chromatographic (Sephadex LH-20 and silica gel) procedures. Preliminary investigation on the structure of this chemically pure substance using mass spectrometry, thin layer chromatography, base hydrolysis and electrophoresis indicated that it is most probably an ester of piperonylic acid. KC-18, given intraperitoneally in doses of 4 mg/kg to guinea pigs 5 hours prior to an exposure to a 0.15 per cent histamine aerosol, significantly reduced the bronchoconstrictor effect of histamine.


Biologically active substances in oak gall extracts. Part 11. The antihistamine-like activity of a substance (KC-18) isolated from oak gall extracts.

The effects of the newly isolated antihistamine (KC-18) from the Hungarian oak gall extracts on some actions of histamine have been investigated. Intraperitoneally administered KC-18 into guinea pigs and cats in doses of 2.5-16 mg/kg exerted a significant dose-related inhibition of histamine-induced bronchoconstriction, increase in capillary permeability and hypotension. In guinea pigs, actively sensitized with ovalbumin, 4 mg/kg KC-18 administered intraperitoneally prevented the development of anaphylactic shock following antigenic challenge. Histamine-induced gastric acid secretion in the rat was also dose-relatedly reduced by the intravenous administraton of 72 and 120 mg/kg KC-18. However, KC-18 in doses up to 100 mug/ml did not modify the histamine-induced contraction of the isolated guinea pig ileum.


Effects of drugs and sympathetic nerve stimuation  on retrograde coronary blood flow in dogs with experimental coronary occlusion.

Effects of nitroglycerin, dipyridamole, adenosine, reactive hyperemia, propranolol, norepinephrine and sympathetic nerve stimulation on retrograde coronary blood flow were investigated in dogs with experimental coronary occlusion. The retrograde blood flow (RF), pressure and conductance of the occluded left anterior descending coronary artery were measured and compared with the flow (CF), pressure and conductance of the non-occluded circumflex artery. The RF increased during sympathetic nerve stimulation and after the administration of nitroglycerin and norepinephrine, while it decreased during reactive hyperemia and following injection of dipyridamole, adenosine and propranolol. The RF/CF ratio was increased by nitroglycerin and propranolol, whereas it was lowered by dipyridamole and adenosine. The results suggest that nitroglycerin and propranolol may produce a redistribution of myocardial blood flow and that the collateral vessels are little influenced by sympathetic nerve stimulation.


Antihistaminic action of (--)-trans-delta 9-tetrahydrocannabinol.

THC has an antagonistic effect against histamine on the isolated perfused guinea-pig lung and rabbit kidney. This antagonism seems to be a competitive one at the concentrations used and interacts with histamine at H1-receptors. THC also antagonizes the effect of acetylcholine, PGE2, angiotensis II and histamine in the isolated continuously superfused guinea-pig ileum by a non-competitive manner. The antagonism between THC and histamine on the isolated superfused rabbit aortic strips was found to be highly specific, since 100% relaxation was obtained when the muscle contracted by histamine but not by the equipotent doses of angiotensin II and noradrenaline. THC also causes a significant increase in survival time of guinea-pigs when the animals were exposed to histamine aerosol. These results indicate a specific antagonism of THC against histamine in the preparations used in this investigation.


[Peptic ulcer and insular carcinoma of the pancreas with hepatic metastasis (Zollinger-Ellison syndrome)].

A Zollinger-Ellison syndrome case is reported. During arteriography and surgical treatment islet cell carcinoma of the pancreas with metastasis to the liver was found. After surgery, patient has presented several periods of hypoglicemia. Hence it was speculated that this type of neoplasm might have double endocrine function.


Utilization of methanol by rhodospirillaceae.

Enrichment culture of organisms growing anaerobically in the light in methanol-bicarbonate medium resulted in isolation of strains of Rhodopseudomonas gelatinosa and Rhodopseudomonas acidophila. The pH optimum for growth on methanol for all strains tested was approximately one unit higher than for growth on carbon sources containing more than one carbon atom. At the appropriate pH, 17 strains of Rhodospirillaceae out of 39 in a culture collection grew anaerobically in the light on methanol-bicarbonate. Rhodopseudomonas acidophia strain 10050 showed the most abundant growth and was studied in more detail. Its growth on methanol was stimulated by yeast extract or vitamin-free casamino acids. The organism grew on methanol-bicarbonate, methanol-formate or formate alone as the sole carbon sources. No growth was observed on methylamine or fomaldehyde. In the presence of excess bicarbonate a maximum yield of 98 g cell material from 100 g methanol was obtained. Ribulose diphosphate carboxylase was present in the methanol-bicarbonate-grown organism at six times the specific activity of that in the succinate-grown organism.


Study of the regulation of oxidation and CO2 assimilation in intact Nitrobacter winogradskyi cells.

1. Changes of the adenine nucleotides in resting and growing Nitrobacter winogradskyi cells were measured in connection with regulating processes during nitrite oxidation and endogenous respiration. 2. After the addition of nitrite to endogenously respiring cells the ATP pool increased strongly during the first 60 sec at the expense of the ADP pool. At this point the energy charge was approx. 0.55. After the first 90 sec the ATP pool dropped, oscillating, to a lower level. The CO2 assimilation began at this point. 3. Under a nitrogen atmosphere the AMP pool increased and the ATP pool decreased. With a value of approx. 0.17 the energy charge was extremely low. When oxygen was added the Nitrobacter cells began to oxidize stored NADH. The ATP pool increased in a few seconds whereas the AMP pool decreased. The P/O ratio of endogenously respiring cells equaled 0.6 under these conditions. 4. During the changeover from anaerobic to aerobic conditions and in the presence of nitrite the nitrite oxidation and CO2 assimilation, opposed to aerobic conditions, were inhibited at first after the nitrite addition. The changeover of the respiratory chain enzymes from a reduced to an oxidized charge and the ATP increase were delayed in comparison with experiments without nitrite. According to these findings the endogenous respiration must be almost nil while nitrite oxidizing cells are growing.


Comparative biochemistry of alpha-glucan-utilization in Pseudomonas amyloderamosa and Pseudomonas saccharophila: physiological significance of variations in the pathway.

Growth patterns on and utilization of various alpha-glucans were investigated in Pseudomonas amyloderamosa and P. saccharophila. Maltose, maltodextrins (average chain length 7 glycosyl units) and glycogen supported excellent growth of both organisms and were extensively metabolized, although glycogen utilization in P. saccharophila was preceded by a prolonged lag phase. P. amyloderamosa produced limited growth on amylopectin and the carbohydrate was only partly degraded. It seemed likely that many of the unit chains liberated from amylopectin had a length exceeding the substrate range accepted by the maltodextrin permease (transport) system. A correlation was established between the pH of the medium and the utilization of glycogen and amylopectin for growth in P. amyloderamosa. The carbohydrates were at least partly utilizable at pH 6.0, whereas they could not support any growth at pH 6.5. Most likely, the lack of growth at the higher pH reflected the low activity of isolamylase at this pH. The enzyme patterns of maltodextrin catabolism in the two bacteria were established. Intracellularly, maltodextrin phosphorylase and 4-alpha-glucanotransferase occurred in both. Degradation of extracellular alpha-glucans was mediated by a mainly intracellular isoamylase in P. amyloderamosa, whereas P. saccharophila possessed an extracellular alpha-amylase and a firmly cell-bound pullulanase.


Enzymatic analysis of the pathways of glucose catabolism and gluconeogenesis in Pseudomonas citronellolis.

Extracts of Pseudomonas citronellolis cells grown on glucose or gluconate possessed all the enzymes of the Entner-Doudoroff pathway. Gluconokinase and either or both 6-phosphogluconate dehydratase and KDPG aldolase were induced by growth on these substrates. Glucose and gluconate dehydrogenases and 6-phosphofructokinase were not detected. Thus catabolism of glucose proceeds via an inducible Entner-Doudoroff pathway. Metabolism of glyceraldehyde 3-phosphate apparently proceeded via glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase. These same enzymes plus triose phosphate isomerase were present in lactate-grown cells indicating that synthesis of triose phosphates from gluconeogenic substrates also occurs via this pathway. Extracts of lactate grown-cells possessed fructose diphosphatase and phosphohexoisomerase but apparently lacked fructose diphosphate aldolase thus indicating either the presence of an aldolase with unusual properties or requirements or an alternative pathway for the conversion of triose phosphate to fructose disphosphate. Cells contained two species of glyceraldehyde 3-phosphate dehydrogenase, one an NAD-dependent enzyme which predominated when the organism was grown on glycolytic substrates and the other, an NADP-dependent enzyme which predominated when the organism was grown on gluconeogenic substrates.


Aromatic amino acid biosynthesis in Alcaligenes eutrophus H16. I. Properties and regulation of 3-deoxy-d-arabino heptulosonate 7-phosphate synthase.

Properties and regulation of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHP-synthase), EC4.1.2.15, from Alcaligenes eutrophus H16 were investigated. DAHP synthase was unstable during manipulations such as dialysis, dilution, ammonium sulfate fractionation, chromatography on DEAE-cellulose or Sephadex G-200. For kinetic measurements Sephadex G-25 treated crude extracts were used. The enzyme was not affected by thiol reagents, EDTA or divalent metal ions. The activation energy, deltaH, amounted to 16100 cal/mole. Between pH 7.2 and pH 8.2 there was little change of enzyme activity. The Km-values for the two substrates were found to be 0.043 mM phosphoenolpyruvate and 0.055 mM erythrose-4-phosphate. DAHP-synthase was inhibited by 0.5 mM phenylalanine for 60% and by 0.5 mM tyrosine for 20%. In the presence of both amino acids cumulative inhibition occurred amounting to about 70%. No other amino acid exerted inhibitory effects. A repression of DAHP-synthase by the aromatic amino acids was not observed. Some other strains of hydrogen bacteria were included in this study. The DAHP synthase from strain 12/60/X and Corynebacterium autotrophicum 7C was unregulated. The enzyme from strain 33/X was subject to retro-tyrosine inhibition and from strain 3/2, H1 and H20 were subject to cumulative inhibition.


Nitrate reductase of Dunaliella parva: electron donor specificity and heat activation.

Nitrate reductase of the salt tolerant alga Dunaliella parva, in contrast to that of most green algae, can use NADPH as well as NADH as electron donor. Extracts of cells contained various amounts of latent nitrate reductase. The latent enzyme could be activated at 45 degrees C but only in the presence of flavine adenine dinucleotide. The heat activated enzyme did not require flavine adenine dinucleotide for activity and was fully active with NADH, NADPH or reduced flavine mononucleotide as electron donors.


The mechanism of swarming of Vibrio alginolyticus.

Factors leading to swarming of Vibrio alginolyticus cells on solid media were studied.  Polar flagellated rods from liquid medium develop into small colonies on solid medium.  Byproducts, accumulating in the colony area, induce at certain critical concentrations, the formation of peritrichous flagella and development of long heavily flagellated filaments which swarm away form the high by-product concentrations.  Several types of nonswarming mutants were isolated, among them, mutants which lack the capacity to form swarming-inducing pyproducts, but can be induced to swarm by byproducts of other mutants incapable of swarming.  Different compounds could replace the natural metabolic byproducts; at very low concentration these compounds induce peritrichous flagella and swarming in some of the nonswarming mutants mentioned above.  The natural metabolic byproducts accumulating in yeast-extract-peptone medium are suggested to be volatile acids belonging to the valine and isoleucine pathway.  Wild-type V. alginolyticus cells cannot swarm on certain substrates but its mutants, able to swarm on many substrates in minimal media, are easily selected.


Ammonium uptake by nitrogen fixing bacteria I. Azotobacter vinelandii.

Both the changes in the activities of nitrogenase, glutamine synthetase and glutamate dehydrogenase and in the extracellular and intracellular NH4+ concentrations were investigated during the transition from an NH4+ free medium to one containing NH4+ ions for a continuous culture of Azotobacter vinelandii. If added in amounts causing 80-100% repression of nitrogenase, ammonium acetate, lactate and phosphate are absorbed completely, whereas chloride, sulfate and citrate are only taken up to about 80%. After about 1-2 hrs the NH4+ remaining in the medium is absorbed too, indicating the induction or activation of a new NH4+ transport system. One of the new permeases allows the uptake of citrate in the presence of sucrose. Addition of inorganic NH4+ level leads to a reversible rise in the glutamine synthetase activity which is not prevented by chloramphenicol, and to a reversible decrease in nitrogenase activity. During these measurements glutamate dehydrogenase activity remains close to zero. The intracellular NH4+ level of about 0.6 mM does not change when extracellular NH4+ is taken up and repression of nitrogenase starts.


Comparative study on hydrolases in five species of Ochromonas (chrysomonadina).

Acid phosphatase and beta-glucosidase were shown to be present in five species of Ochromonas grown in organic media (O. danica, O. malhanesis, O. munuta, O. sociabilis and Ochromonas sp. 933/4). Acid phosphatase was found to have a pH optimum at 4.0 in O. danica, and at 5.1 in the four other species. No alkaline phosphatase was found in any of the above mentioned species. Beta-glucosidase in the species studied has a pH optimum at 4.6. Low alpha-glucosidase activity was found only in O. danica. Acid phosphatase in all the five species shows an increase in activity during the logarithmic phase of growth and a decrease during the early stationary phase. Beta-glucosidase shows a similar behavior only in O. danica.


Thioridazine stimulates prolactin secretion in man.

Thioridazine, unlike most other effective antipsychotic drugs, appears to be only a weak dopamine antagonist in various regions of the brain. We decided to test, indirectly, thioridazine's effects on another brain dopaminergic system, the tuberoinfundibular tract, which regulates prolactin secretion by stimulating hypothalamic secretion of prolactin-inhibiting factor. Chlorpromazine and several other phenothiazines have been shown to stimulate prolactin secretion. Five healthy men ingested 50 mg of chlorpromazine concentrate on one occasion, and 50 mg of thioridazine concentrate on another. Both drugs noticeably stimulated prolactin secretion within two hours. It is concluded that thioridazine is a potent dopamine antagonist in the tuberoinfundibular system, and it is suggested that this system's regulation of prolactin secretion may provide a useful method for studying antipsychotic drug effects in man.


The long-acting phenothiazines.

Injected intramuscularly, the enanthane and decanoate esters of the phenothiazine fluphenazine are an effective treatment of the disordered behavior and thinking of schizophrenia. The decanoate preparation is not only slightly longer-acting but also has a smaller incidence of side-effects that the enanthate. The major adverse effect of these medications is the high frequency of extrapyramidal system disturbance. Since the 50% rate of failure of schizophrenic outpatients to take prescribed oral medications decreases treatment failure to about 20% with the use of long-acting injectable phenothiazines, this route of administration offers an advantage in patient management particularly applicable to community mental health systems. Moreover, parenteral administration of long-acting fluphenazine may be useful for patients who do not attain effective serum levels with medication taken orally because of metabolic or absorption difficulties.


Butaperazine pharmacokinetics. Effect of dosage regimen on steady state blood levels.

Experimental evidence showed that a higher whole blood steady state drug plasma level was achieved when a patient was given medication three times a day compared to the same total daily dose once a day. In addition, evidence indicated possible enzyme induction by butaperazine in one patient. We have demonstrated that the general stochastic theory developed to investigate the pharmacokinetics of lithium carbonate "in vivo" has more general applicability in that it applies also to butaperazine, and facilitates interpretation of findings (without blood level data) in one other drug study.


Feedback of true heart rate during exposure in vivo.

Ten women with specific chronic animal phobias were treated by graded exposure in vivo in two to four two-hour treatment sessions. Sessions were divided into four balanced half-hour epochs, half with visual feedback of true heart rate plus instructions to lower heart rate during approach to phobic stimulus. All patients improved significantly from the start to end of sessions on heart rate and on subjective anxiety. Feedback of heart rate plus instructions to lower it substantially reduced heart rate during epochs of feedback, compared to non-feedback epochs without instructions, but this effect did not generalize to skin conductance or to subjective anxiety in our short-term experiment.


Comparative studies of psychotherapies.  Is it true that "everywon has one and all must have prizes"?

Tallies were made of outcomes of all resonably controlled comparisons of psychotherapies with each other and with other treatments.  For comparisons of psychotherapy with each other, most studies found insignificant differences in proportions of patients who improved (though most patients benefited).  This "tie score effect" did not apply to psychotherapies vs psychopharmacotherapies compared singly-psychotherapies did better.  Combined treatments often did better than single treatments.  Among the comparisons, only two specially beneficial matches between type of patient and type of treatment were found.  Our explanations for the usual tie score effect emphasize the common components among psychotherapies, especially the helping relationship with a therapist.  However, we believe the research does not justify the conclusion that we should randomly assign patients to treatments-research results are usually based on amount of improvement; "amount" may not disclose differences in quality of improvement from each treatment.


[Constraints matrix for the concept of new cancerostatics with activation in optimally superacidulated carcinomatous tissue (author's transl)].

Maintained immunoreaction -- an essential factor for the prospects of healing in any type of cancer therapy -- can be warranted on principle by developing cancerostatics with substantially increased selectivity. One way to approach this goal is the synthesis of functionally masked cancerostatics the active form of which is released only in optimally superacidulated carcinomatous tissue. Used as a key element for such a liberation is a enzyme contained in the cancer cells in its highly concentrated free form. The activity curve of this enzyme must have a pronounced optimum in a span of pH equal 5 to 6. Bonding the active form of, say, an alkylizing molecular group may be glycosidic by nature. Finally, a model is outlined which duly considers the various pharmaco-kinetic aspects.


Hyperkalemic periodic paralysis. Effects of potassium, exercise, glucose, and acetazolamide on blood chemistry.

Effects of strenous exercise, followed by rest, and of potassium administration on blood chemistry values were studied in two patients with hyperkalemic periodic paralysis and in normal volunteers. These procedures produced attacks of flaccid paralysis that occurred concomitantly with rapid rises in serum potassium concentrations and decreases in blood glucose and inorganic phosphate levels. With the exception of the serum potassium level which rose following exercise and potassium administration, there were no changes in the blood chemistry values of the normal volunteers. During the induced attacks of paralysis, the expired breath of the patients had a very strong odor of ketosis. Results of subsequent glucose tolerance tests were abnormal. Following 24 hour administration of acetazolamide, the studies were repeated. The drug appeared to cause lesser effects of stimuli on serum potassium levels and a stabilizing effect on blood glucose levels.


Intraocular irrigating solutions. Their effect on the corneal endothelium.

The effects of several intraocular irrigating solutions on the corneal endothelium of rabbit and monkey corneas were evaluated, utilizing a specular microscope perfusion system with both scanning and transmission electron microscopy. Corneas perfused with 0.9% sterile isotonic physiological saline swell at a rate of 60mum to 90mum/hr; endothelial cells separate from each other and show extensive degenerative changes. Corneas perfused with lactated Ringer solution swell at a rate of 37mum to 40mum/hr, and the endothelial cells show slower, but progressive degeneration. Corneas perfused with balanced salt solution swell at 24mum to 31mum/hr, and degenerative changes become severe only after two hours. Corneas perfused with Ringer solution containing bicarbonate, reduced glutathione, and adenosine do not increase in thickness, and there is minimal deterioration of endothelial ultrastructure for periods of up to six hours.


The applied surgical anatomy of the peritoneal fascia of the groin and the "secondary" internal inguinal ring.

The preperitoneal fascia of the groin is distinct from the transversalis fascia, although often mistaken for it. This distinction, and other special features of this fascia in the inguen, are more readily appreciated in the course of the preperitoneal approach than by the conventional transinguinal approach. Certain features of this fascia which are of practical surgical significance are emphasized, especially those concerning operations for hernias, hydroceles, or undescended testes in infancy and childhood. These special features include the preperitoneal fascial ring or secondary internal ring, which appears to have been confused in the past with the transversalis fascial opening or internal ring proper. Other features concerning the relative merits of the preperitoneal approach to groin hernias as opposed to the conventional transinguinal approach are discussed.


The effects of cosmic particle radiation on pocket mice aboard Apollo XVII: IV. engineering aspects of the experiment and results of animal tests.

A closed passive system independent of support from the spacecraft or its crew was developed to house five pocket mice for their flight on Apollo XVII. The reaction of potassium superoxide with carbon dioxide and water vapor to produce oxygen provided a habitable atmosphere within the experiment package. The performance of the system and the ability of the mice to survive the key preflight tests gave reasonable assurance that to mice would also withstand the Apollo flight.


Interactions of urdine diphosphate glucose dehydrogenase with the inhibitor urdine diphosphate xylose.

1. UDP-xylose and UDP-glucose both bind to UDP-glucose dehydrogenase in the absence of NAD+, causing an enhancement of protein fluorescence. 2. The binding of UDP-xylose is pH-dependent, tighter binding being observed at pH8.2 than at pH8.7. 3. At low protein concentrations sigmiodal profiles of fluorescence enhancement are obtained on titration of the enzyme with UDP-xylose. As the protein concentration is increased the titration profiles become progressively more hypebolic in shape. 4. The markedly different titration profiles obtained on titrating enzyme and the enzyme-NAD+ complex with UDP-xylose suggests a conformational difference between these two species 5. NAD+ lowere the apparent affinity of the enzyme for UDP-xylose. 6. There is no change in the apparent moleculare weight of UDP-glucose dehydrogenase on binging UDP-xylose. 7. Protein modification by either diethyl pyrocarbonate or 5, 5'-dithiobis-(2-nitrobenzoate) does not "desensitize" the enzyme with respect to the inhibition by UDP-xylose. 8. UDP-xylose lowers the affinity of the enzyme for NADG. 9. It is suggested that UDP-xylose is acting as a substrate analogue of UDP-glucose and causes protein-conformational changes on binding to the enzyme.


Properties of catechol O-methyltransferases from brain and liver of rat and human.

Kinetic and electrophoretic properties of catechol O-methyltransferases (EC 2.1.1.6) from brain and liver were studied. The enzyme of either rat or human tissues exhibited a single molecular form when subjected to electrophoresis at pH7.9. At pH9 a second, apparently oxidized, form was detected. Isoelectric-focusing experiments also indicated only one enzyme form, which was identical from extracts of brain and liver of each species (pI = 5.2 for rat, 5.5 for human). Similarities between brain and liver catechol O-methyltransferase of a given species were also demonstrated by kinetic parameters, meta/para ratios of products, and inhibitor potencies. Human catechol O-methyltransferase exhibited lower Km values than did the rat enzyme for S-adenosyl-L-methionine, dopamine and dihydroxybenzoic acid. Adrenochrome inhibited both rat and human enzyme. It was concluded (1) that only a single enzyme form could be demonstrated in the physiological pH region; (2) that catechol O-methyltransferase of brain could not be distinguished from the liver enzyme of the same species; and (3) that species differences exist between the enzymes of rat and human tissues.


Molecular charcteristics of chicken kidney arginase.

Chicken kidney contains two arginases with different sedimentation coefficients and substrate specificity. The ligher of these arginases, which hydrolyses only L-arginine, has been purified about 3000-fold. Like the "ureotelic" arginase, developed in chicken liver after starvation, it displays many of the properties of the arginase of the "ureotelic" species. This seems to exclude the possibility that ureotelism and uricotelism are characterized by a specific type of arginases. Both liver and kidney arginases are located in the mitochondrial matrix. The rate of hydrolysis of arginine thus not only depends on the arginase activity but also on the rate of transport of arginine into the matrix. This last process therefore is of regulatory significance.


Characterization of a plasmin-digest fragment of rabbit immunoglobulin gamma that binds antigen and complement.

Rabbit immunoglobulin gamma (IgG) was digested with plasmin after being left for 15 min at pH2.5, 30 degrees C followed by a rapid increase in the pH to 7. The fragment antigen and complement binding (Facb) was isolated and characterized chemically and biologically. Sequence studies showed that the C-terminal quarter of the heavy chain had been removed, the split occurring at a lysine-alanine bond in the sequence Thr-Ile-Ser-Lys-Ala-Arg. The fragment Facb retained the capacity to precipitate with antigen and the precipitate caused activation of the first component of complement of the same order as that of acid-treated IgG. Both Facb and acid-treated IgG showed a fall in complement fixation relative to the native molecule of 30-40%.


Deoxyribonucleic acid polymerases of BHK-21/C13 cells. Partial purification and characterization of the enzymes.

DNA polymerase from BHK-21/C13 cells were separated into two species, DNA polymerase I corresponding to the heterogeneous enzyme with sedimentation coefficient of 6-8S, and DNA polymerase II, corresponding to the enzyme with sedimentation coefficient of 3.3S. DNA polymerase I was purified 114-fold and DNA polymerase II 154-fold by a simple extraction procedure followed by column chromatography on phosphocellulose and gel filtration through Sephadex G-100. The purified enzymes differed markedly in respect of pH optimum, stimulation and inhibition by K+, Km for the deoxyribonucleoside 5'-triphosphates, stability to heating at 45 degrees C, and inhibition by N-ethylmaleimide. The preferred primer-template for both enzymes was "activated" DNA (DNA submitted to limited degradation by pancreatic deoxyribonuclease); native or thermally denatured DNA templates were relatively very poorly copied. When certain synthetic templates were tested, substantial differences were revealed between the two enzymes. Poly[d(A-T)] was poorly used by polymerase I but was superior to "activated" DNA for polymerase II. Poly[d(A)]-oligo[d(pT)10] was used efficiently by polymerase I but not by polymerase II. Poly(A)-oligo[d(pT)10] was not an effective primer-template although polymerase I could use it to a limited extent when Mn2+ replaced Mg2+ in the polymerase reaction and when the temperature of incubation was lowered from 37 degrees to 30 degrees C. When only one or two or three triphosphates were supplied in the reaction mixture, the activity of polymerase I was more severly diminished than that of polymerase II.


The electrophoretic mobilities of 5-dimethylaminoaphthalene-1-suphonyl-glycopeptides and their relation molecular weight.

The relationship between the electrophoretic mobility at pH2.1 of dansyl-glycopeptides of known composition and their molecular weight is shown to conform with a model equation previously derived for peptides. A dansyl-glycopeptide prepared from hen's-egg ovotransferrin is degraded sequentially with two glycosidases. The molecular weight of each glycopeptide intermediate formed is determined from its electrophoretic mobility. From successive molecular-weight changes, the number and type of sugar residues lost from the parent glycopeptide can be decided and the probable composition of each intermediate determined. The notion that the method has considerable application and would permit analysis of very small quantities of glycopeptides is discussed.


Isolation and characterization of acidic structural glycoproteins in pulmonary tissues.

1. Extraction of the pleural and parenchymal regions of bovine lungs with salt and organic solvents gave powders that contained glycoproteins in addition to collagen and elastin. The contents of these glycoproteins (g/100 of powder) was 4% in pleura and 15.5% in parenchyma. 2. Attempts were made to purify these glycoproteins fromboth tissues by various methods. 3. Both tissues contained heterogeneous mixtures that were similar solubilities, had similar amino acid and carbohydrate compostions. 4. The compostions of the pulmonary glycoproteins resembled those isolated from other connective tissues by other workers.


The purification of fructose 1,6-diphosphatase from ox liver and its activation by ethylenediaminetetra-acetate.

1.A procedure for the purification of ox liver fructose 1,6-kiphosphatase is described. A number of criteria indicate that the enzyme was not subjected to any significant degree of proteolytic attack during the purification. 2. The molecular weight, amino acid composition and subunit molecular weight are reported. 3. The activation by EDTA was shown to be due to the chelation heavy metals rather than by a more complex interaction with the enzyme as had previously been suggested.


Oxidation of polymines by diamine oxidase from human seminal plasma.

1. Diamine oxidase [amine-oxygen oxidoreductase (deaminating)(pyridoxal-containing), EC 1.4.3.6] was purified from human seminal plasma more than 1,700-fold. The enzyme appeared to be homogeneous on polyacrylamide-gel electrophoresis at two different pH values. 2. The general properties of the enzyme were comparable with those described for other diamine oxidases from different mammalian sources. The molecular weight of the enzyme was calculated to be about 182,000. 3. The enzyme had highest affinity for diamines, but polyamines spermidine and spermine were also degraded at concentrations that can be considered physiological in human semen. 3. The possible degradation of spermine by diamine oxidase in human semen in vivo may give rise to the formation of cytotoxic aldehydes that conceivably can influence the motility and survival of the spermatozoa.


The sialic acid content and isoelectric point of human kininogen.

The sialic acid content of highly purified human kininogen was found to be about 8.6 mol/mol(mol.wt. 50,000). The isoelectric point (pH 4.9 +/- 0.2) is much higher than that of bovine low-molecular-weight kininogen, but is close to that expected from the amino acid and sialic acid analyses.


Protein kinase and phosphatases from human polymorphonuclear leucoytes.

Purified preparations of human polymorphonuclear leucocytes contain a protein kinase in the cytosol which is stimulated by cyclic AMP and cyclic IMP but not by other cyclic nucleotides. The holoenzyme had a molecular weight of 66000 estimated by gel filtration; when it was incubated with histone or cyclic AMP, it dissociated into two smaller subunits of molecular weight 45000 and 30000; the former remained cyclic AMP-sensitive, whereas the latter had become independent of added cyclic AMP. By means of substrate-affinity chromatography on histone-Sepharose 4B, cyclic [3H5AMP-binding activity (regulatory or R subunit) could be resolved into two peaks of enzyme activity, one again independent of added cyclic AMP, with a molecular weight of 30000 (catalytic or C subunit). Also by means of substrate-affinity chromatography it was possible to resolve 'specific' polymorphonuclear leukocyte histone phosphatases from 'non-specific' phosphomonesterases capable of dephosphorylating histone previously phosphorylated by the protein kinase. Specific histone phosphatase displayed greatest affinity for histone-Sepharose 4B, followed by acid p-nitrophenyl phosphatase, and the unretained acid beta-glucerophosphatase. Polymorphonuclear leucocyte histone phosphatase, purified approx. 40-fold, was further resolved from the other phosphatases by gel filtration on Sephadex G-150 from which it was eluted with apparent molecular weights of 45000 and 18700. The apparent Km values for dephosphorylation of histone are 4.3 X 10-6M and 3.6 X 10-6M. Most (69%) of cytoplasmic histone phosphatase was found in the cell sap, whereas 20% remained tightly associated with polymorphonuclear leucocyte lysosomes from which it could not be solubilized by treatments (Triton X-100, freeze-thawing) that released approx. 70% of lysosomal beta-glucuronidase or acid phosphatases. Although both soluble and particulate enzymes required 5-10 mM-Mn2 for maximal activation, and showed a pH maximum of 6.5-7.0, only the particulate enzyme was partly inhibited by ammonium molybdate. Polymorphonuclear leucocyte histone phosphatases were neither inhibited nor stimulated by those cyclic nucleotides that greatly stimulate the protein kinase of the same subcellular fraction


Evolutionary variation between a monomer and a dimer arginine kinase. Purification of the enzyme from Holothuria forskali and a comparison of some properties with that from Homarus vulgaris.

1. A purification procedure for the dimeric arginine kinase of the sea cucumber Holothuria forskali is described. 2. The enzyme has a mean molecular weight of 77250 and is composed of two equal, dissociable subunits. 3. It also shows co-operativity between substrate binding at one catalytic site to a much greater extent than the nomomeric lobster arginine kinase for which such co-operativity could not be detected unambiguously. The constants for substrate binding are reported assuming that the enzyme follows rapid-equilibrium random kinetics. From a comparison with other species, the development of co-operativity between the nucleotide- and guanidine-binding sites on one subunit is suggested to have occurred more than once in the evolution of the phosphagen kinases and is not dependent on subunit aggregation. 4. Both enzymes show similar pH profiles for thermal inactivation at 22 degrees C and have very similar stabilities. Above 40 degrees C the dimeric enzyme is much more stable than the monomer. Rate constants for heat inactivation and Arrhenius activation energies are reported. 5. The dimeric enzyme is also more stable to urea inactivation. Substrates and argininic acid all improve the stability of both enzymes. The effects of individual substrates are more distincitive with the dimeric enzymes and increase its stability to an extent that makes it about as stable as dogfish creatine kinase. In the physiological range dimerization does not seem to confer any particular advantage with respect to stability over the monomer form.


Acetyl-Coenzyme A carboxylase. Role of the prosthetic group in enzyme polymerization.

Apo-(acetyl-CoA carboxylase) completely free from the holoenzyme was prepared from biotin-deficient rat adipose tissue by using affinity chromatography. The apoenzyme does not aggregate under conditions favouring the transition of the holoenzyme to the polymeric form. Such transition is possible after the conversion of the apoenzyme into the holoenzyme in vitro, thus demonstrating the requirement of the prosthetic biotinyl group for enzyme activation.


Purification and properties of the pyruvate kinase of sturgeon muscle.

Pyruvate kinase was purified from sturgeon muscle in yeilds comparable with those obtained from the muscles of other species. In contrast with mammalian muscle pyruvate kinase the enzyme from sturgeon muscle gives a sigmoidal velocity curve with respect to phosphoenolpuruvate saturation, is activated by fructose 1.6-diphosphate, and is inhibited by bivalent copper ions. In these respects it is similar to the enzyme isolated from mammalian liver. The degree of interaction between phosphoenolpyruvate-binding sites is dependent on temperature.


Metal-dependent proteinase of the lens. Assay, purification and properties of the bovine enzyme.

1. Two new assay methods were developed for the lens proteinase. In both, the substrate was alpha2-crystallin (a major lens protein); in the first method, the products were detected by reaction with trinitrobenzenesulphonate in the presence of SO32-, whereas in the second method, 3H-labelled substrate was used, and the products were detected as radioactivity soluble in trichloroacetic acid. 2. The neutral proteinase from bovine lens was partially purified by extraction of the lens at pH5.0 and column chromatography on hydroxyapatite and Sepharose 6B gel. 3. The purified enzyme had no detectable activity against haemoglobin, azo-casein or gamma-crystallin under optimum conditions for alpha2-crystallin. 4. The enzyme showed greatest activity and stability at pH7.5. It was reversibly inhibited by EDTA and 1,10-phenanthroline, and activated by Ca2+ and Mg2+. 5. Molecular weights obtained for the enzyme by chromatography on Sepharose 6B were approx. 500,000 in buffer of I = 0.02, and 250,000 at I = 1.02. 6. The properties of the purified lens proteinase are such as to suggest that this enzyme could account for the entire endopeptidase activity of the lens.


The microbial metabolism of C1 compounds. The cytochromes of Pseudomaonas AM1.

Pseudomonas AM1 contains cytochromes a, b and c and more than one CO-binding pigment (cytochrome a3, cytochrome c and possibly a cytochrome o). The soluble cytochrome c has been purified; its isoelectric point is low and its molecular weight is 20000. This cytochrome is reduced in whole bacteria by all oxidizable substrates at rates determined by the primary dehydrogenases. A mutant lacking cytochrome c oxidizes all substrates except methanol, ethanol and methylamine; these no longer support growth. The role of cytochrome c in electron transport in Pseudomonas AM1 is discussed.


The activities and intracellular distribution of nicotinamide-adenine dinucleotide phosphate-malate dehydrogenase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase in rat, guinea-pig and rabbit tissues.

1. Measurements are presented of the activity and intracellular distribution of phosphoenolypruvate carboxykinase, pyruvate carboxylase and NADP-malate dehydrogenase in rat, guinea-pig and rabbit liver and kidney cortex, together with previously obtained measurements of these enzymes in adipose tissue. 2. In all three tissues pyruvate carboxylase activity was greatest in the rat and lowest in the rabbit. 3. Guinea pig and rabbit were very similar to each other with respect to the extramitochondrial-mitochondrial distribution of phosphoenolpyruvate carboxykinase in all three tissues. 4. NADP-malate dehydrogenase was present in all three tissues in the rat, present in kidney cortex and adipose tissue in the guinea pig and absent from all tissues examines in the rabbit.


The dependence on vitamin E and selenium of drug demethylation in rat liver microsomal fractions.

1. The effects of vitamin E deficiency, and of vitamin E and selenium deficiency, on rat liver microsomal aminopyrine demethylase activity were investigated. It was found that, over a wide range of substrate concentrations, the enzyme activity in preparations from deficient animals was significantly lower than that in controls. 2. Addition of antioxidants in vitro, either to the homogenization or to the assay media, was without significant effect on the depressed enzyme activity. Castration and alteration in dietary protein concentration were also without effect. The rate of oxidation of NADPH was however, lower in preparations from deficient animals. 3. Lineweaver-Burk plots of the reciprocal of enzyme activity and substrate concentration showed a higher Km value in preparations from vitamin E-deficient animals, irrespective of whether selenium was present; the Vmax. was unaffected. These parameters were unchanged when antioxidants were added in vitro. Induction with phenobarbitone and 3-methylcholanthrene showed large changes in Km value which, for preparations from vitamin E-deficient animals, was higher than that for corresponding controls. 4. Examination of the synergism between NADH and NADPH as donors of reducing equivalents for aminopyrine demethylation showed that vitamin E and selenium were only minimally involved in the phenomenon. However, both the initial rate and the extent of demethylation were significantly lower in vitamin E- and selenium-deficient preparations and both nutrients were required for the restoration of full activity. 5. The significance of these results is discussed in the light of our working hypothesis.


Incorporation of L-[1-14C]leucine into protein by liver postmitochondrial supernatant: opposing effects of preincubated nicotinamide-adenine dinucleotide phosphate and 4-dimethylamino-3'-methylazobenzene.

Combination of preincubated drug-metabolizing medium containing NADP+ with a cell-free protein-synthesizing system resulted in marked stimulation of incorporation of L-[1-14C]leucine into protein. Addition of 4-dimethylamino-3'-methylazobenzene, present and previously preincubated in the drug-metabolizing medium, decreased this effect.


Purification and heterogeneity of inorganic pyrophosphatase of pig scapula cartilage.

Inorganic pyrophosphatase (pyrophosphate phosphohydrolase; EC 3.6.1.1) was purified from pig scapula cartilage by fractionation with N-cetylpyridinium chloride and (NH4)2SO4, followed by ion-exchange and gel-filtration chromatography. Enzyme preparations of high purity were obtained, with specific activities (100-400 units/mg) higher than those previously described for mammalian pyrophosphatases. The enzyme activity could be separated into several subfractions on ion-exchange columns.


Properties of inorganic pyrophosphatase of pig scapula cartilage.

The properties of a highly purified inorganic pyrophosphatase (pyrophosphate phosphohydrolase; EC 3.6.1.1) from pig scapula cartilage were studied. The enzyme had a molecular weight of 66 000 and a pH optimum of 7-8. It was markedly activated by magnesium, but not, or only to a much smaller degree, by other metal ions. PP1 was the only substrate found and had a Km value of 11 muM. The enzyme was not inhibited by phosphate and other inhibitors of alkaline phosphatase such as CN- minus, amino acids and theophylline; it was slightly inhibited by tartrate, formaldehyde and ammonium molybdate and strongly inhibited by F- minus, Ca2+ and other metal ions. The properties of the enzyme in the presence of concentrations of PP1 present in plasma (3.5 muM) were similar to those found at higher (2 mM) concentrations of PP1. The diphosphonates ethane-1-hydroxy-1,1-diphosphonate and dichloromethylenediphosphonate inhibited the enzyme in the presence of low PP1 concentrations. The characteristics of this enzyme are therefore similar to pyrophosphatases from other sources, such as from yeast and erythrocytes, and do not support a specific role of this enzyme in the calcification process.


Poly(adenosine diphosphate ribose) polymerase in Physarum polycephalum.

1. The isolated nuclei of the slime mould Physarum polycephalum contain an enzyme that will incorporated [adenine-3H] NAD+ into an acid-insoluble product, which is shown to be poly(ADP-ribose). 2. This incorporation has an optimum pH of 8.2 and a temperature optimum below 10degreesC. 3. Optimum stimulation is given by 15 mM-Mg2+. 4. 2-Mercaptoethanol or dithiothreitol also stimulates the incorporation, the latter at an optimum concentration of about 1 mM. 5. Under optimum conditions the Km value for the reaction is 0.28 mM at 15degreesC. Nicotinamide inhibits the incorporation with a Ki of 5.7 muM. 6. Exogenous DNA stimulates the incorporation by about 100%. 7. Preincubation of the nuclei with deoxyribonuclease, but not with ribonuclease, almost completely inactivates the incorporation of NAD+. 8. The enzyme is unstable at both 0degrees and 15degreesC in the absence of dithiothreitol. The presence of dithiothreitol at a concentration of 1 mM stabilizes the enzyme at both these temperatures. 9. The activity of this enzyme per nucleus was shown in three separate experiments to fall by about one-half in early S phase and then to rise to its pre-mitotic value after about 3 h, that is in late S phase. 10. The possible physiological function of this enzyme system is discussed.


Transamidase kinetics. Amide formation in the enzymic reactions of thiol esters with amines.

1. Beta-Phenylpropionylthiocholine and N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylcadaverine) serve as a pair of water-soluble (pH7.5) model substrates for transamidating enzymes. Amide formation could be followed directly through fluorescence measurements by monitoring the continuous extraction of the water-soluble coupling product, N-(beta-phenylpropionyl)dansylcadaverine, into n-heptane. By this procedure, the steady-state kinetics of glutamine-lysine endo-gamma-glutamyltransferase from human plasma (fibrinoligase, thrombin- and Ca2+-activated blood coagulation Factor XII) and from guinea-pig liver (liver transglutaminase) were investigated at 25 degrees C. 2. With beta-phenylpropionylthiocholine as the varied substrate, Lineweaver-Burk plots with various concentrations of dansylcadaverine intercept on the horizontal axis, suggesting that formation of the acyl-enzyme is rate limiting. 3. On the basis of functional normality of active sites, kcat. values of 1.8 s(-1) and 0.9 s(-1) were obtained for the plasma and liver gamma-glutamyltransferase respectively. The two enzymes show identical affinities for the first substrate, beta-phenylpropionylthiocholine, with Ka 4 times 10(-4) M. 4. Utilization of the second substrate, dansylcadaverine, appears to be an order of magnitude more efficient with the liver enzyme. 5. N-(5-Amino-3-thiapentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylthiacadaverine) could be used instead of dansylcadaverine in the fluorescent kinetic system. 6. Competitive inhibition by a non-fluorescent amine substrate histamine was also evaluated.


Subcellular localization and properties of mouse adrenal C19-steroid 5beta-reductase.

The localization and some characteristics of mouse adrenal C19-steroid 5 beta-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 alpha-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3alpha-, 3beta- and a small amount of 17 beta-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 beta-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 beta products first had to be oxidized to 5 beta-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 beta-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 beta-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 beta reduction of [7 alpha-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) "/- 0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration.


A comparative study of some kinetic and spectral properties of guanidinated and native cytochrome c.

An investigation of the spectral and some kinetic properties of a chemically modified cytochrome c is presented. The kinetics of reduction by chromous ion and ascorbate are shown to be unchanged from that of the native molecule, as are the kinetics of NO binding. The effects of pH on the visible spectrum are discussed in terms of a possible change in the pattern of co-ordination of the molecule with changing pH.


A deactivating conformational change induced by reduced nicotinamide-adenine dinucleotide phosphate in a Neurospora glutamate dehydrogenase.

Stopped-flow fluorescence techniques have been used to observe the formation of the binary comples of E-NADPH. At pH 7.5 there is a protein conformational change after the formation of the binary complex. This conformational change can be detected by a decrease in the fluorescence intensity of the complex at 350 nm and by an increase in its fluorescence intensity at 450 nm.


Aspergillus oryzae acid proteinase. Purification and properties, and formation of pi-chymotrypsin.

An acid proteinase from Aspergillus oryzae was isolated from a commercial powder by successive (NH4)2SO4 fractionation, acetone precipitation, and ion-exchange chromatography on phosphate- and DEAE-cellulose columns. The purified enzyme was found to be homogeneous by ultracentrifuge-sedimentation analysis (S20, W equal 3.63S), but electrofocusing in polyacrylamide gels and electrophoresis at pH 3.2 revealed that it consists of two very closely migrating bands. No difference in the amino acid composition and enzymic activities of the two partially separated bands could be detected, and it was concluded that the acid proteinase exists in two molecular forms. The enzyme activates bovine trypsinogen and chymotrypsinogen at pH 3.5 (the kappacat. and Km values at 35degrees C are 11.3S- minus 1, 0.10mM and 1.14S- minus 1, 0.18mM respectively). It hydrolyses the Phe-Phe bond of the synthetic pepsin substrates Z-His-Phe-Phe-OEt (kappacat. equal 1.65S- minus 1, Km equal 0.640mM at pH 3.5, 30degrees C) and Z-Ala-Ala-Phe-Phe-OPy4Pr (kappacat. equal 0.37S- minus 1, Km equal 0.037 mM at pH2.9, 39degrees C), where Z represents benzyloxycarbonyl and OPy4Pr represents 3-(4-pyridyl)-propyl 1-ester. Activation of bovine chymotrypsinogen results from the cleavage of the Arg(15)-Ile(16) bond in the zymogen. No other cleavages were observed. The use of A. oryzae proteinase provides a simple tool for the production of pi-chymotrypsin in good yield and purity.


Physical-chemical properties of C-phycocyanin isolated from an acido-thermophilic eukaryote, Cyanidium caldarium.

C-Phycocyanin from an acido-thermophilic eukaryotic alga, Cyanidium caldarium, was characterized with respect to subunit structure, absorption spectrum and fluorescence properties and was found to be similar to C-phycocyanins from mesophilic sources. The pH-dependence of fluorescence polarization and the changes in sedimentation velocity as a function of pH, concentration and temperature indicate the presence of extremely large amounts of unusually stable 19S aggregates. It was not possible to disaggregate this phycocyanin completely to monomer under normal conditions. The amino acid composition is similar to that of phycocyanins from other thermophilic and halophilic sources. The isoelectric point of this C-phycocyanin was 5.11, an unusually high value. The properties of this C-phycocyanin suggest an increase in protein stability as its mode of adaptation to the environmental stress of high temperature.


Chemical modification of amino groups and guanidino groups of trypsin. Preparation of stable and soluble derivatives.

1. Isoionic chemical modification of amino groups of trypsin (EC 3.4.21.4) was studied for the purpose of obtaining a well-defined modified trypsin with minimum changes in physicochemical properties and with sufficient stability at neutral pH. Acetamidination with methyl acetimidate hydrochloride proceeded very rapidly at pH9.8 and 5degrees C and all 14 epsilon-amino groups were modified in 2h. The reaction was limited to epsilon-amino groups. The alpha-amino group of N-terminal isoleucine was modified only by repeated reactions in the presence of 5.5 M-guanidine or 8 M-urea. 2. The epsilon-acetamidinated derivative of beta-trypsin retained enzymic activity at values comparable with those of native enzyme tested with alpha-N-benzoyl-L-arginine ethyl ester and alpha-N-benzoyl-L-arginine p-nitroanilide as substrates; it also showed substrate activation comparable with that of native enzyme. The acetamidination of alpha-trypsin resulted in approx. 50% decrease in its esterolytic activity. 3. The epsilon-acetamidinated beta-trypsin was very stable at pH8 and 25degrees C in the absence of Ca2+. The activity of 0.04% (W/V) enzyme solution remained practically unchanged for 10h, and after 24h 90% of the activity was still retained. Possible autolytic cleavage of peptide bonds of acetamidinated enzymes was followed by N-terminal analysis by using automated Edman degradation. Only the Arg(105)-Val(106) bond was found to be cleaved to an appreciable extent. Thus beta-trypsin can be stabilized simply by complete acetamidination of epsilon-amino groups without modifying guanidino groups of arginine residues. Acetamidinated alpha-trypsin was unstable, but its inactivation at a neutral pH could not be attributed to the cleavage of a single specific peptide bond. 4. The acetamidination of the alpha-amino group of the N-terminal isoleucine results in the inactivation of esterolytic activity. However, this enzyme retained the ability to react with p-nitrophenyl p'-guanidinobenzoate. 5. It was concluded that acetamidination of beta-trypsin is a convenient method for preparing a well-defined stable and soluble trypsin derivative without appreciable change in its physical properties.


The formation of oligoglucans linked to lipid during synthesis of beta-glucan by characterized membrane fractions isolated from peas.

Membrane fractions were obtained from peas roots by using a method that permitted the isolation of a fraction rich in relatively intact dictyosome stacks. No chemical fixatives were used. The method involved incubation of the roots with cellulase, followed by gentle homogenization and sucrose-density-gradient fractionation of the homogenate. The fractions were characterized by electron microscopy. All fractions were enzymically active in incorporating glucose from UDP-glucose into water-insoluble glycolipids containing both single glucose residues and glucose oligosaccharides. Some or all of the linkages of glucose to lipid were through phosphate esters. A substance containing glucose oligosaccharides attached to or very strongly adsorbed on to protein was also formed. The membrane fractions also incorporated glucose from UDP-glucose into alkali-soluble and alkali-insoluble beta-glucans, which like the oligosaccharides contained beta(1leads to 3) and beta-(1leads to4) linkages. The distribution of the enzymic activities and the chemical properties of the lipid-linked and protein-linked oligosaccharides suggest that they may be intermediates in beta-glucan synthesis. The synthetic activity is associated with smooth-membrane vesicles which may be derived from the plasma membrane.


The effects of halothane on hepatic microsomal electron transfer.

1. The effects of halothane (CF3CHBrCl), a volatile anaesthetic agent, on electron transfer in isolated rat liver microsomal preparations were examined. 2. At halothane concentrations achieved in tissues during clinical anaesthesia (1-2mM), halothane shifts the redox equilibrium of microsomal cytochrome b5 in the presence of NADPH towards the oxidized form. Halothane accelerates stoicheiometric consumption of NADPH and O2, increases the rate of reoxidation of NADH-reduced microsomal ferrocytochrom b5, but does not affect NADPH- or NADH-cytochrome c reductase activity. The enhanced microsomal electron flow seen in the presence of halothane is not diminished by CO nor is it increased by pretreatment of the animals with phenobarbital. 3. The effects of halothane are maximum in microsomal preparations isolated from animals fed on a high-carbohydrate diet to induce stearate desaturase activity. Changes in microsomal electron transfer caused by halothane are in all cases abolished by low concentrations (1-2mM) of cyanide. Microsomal stearate desaturase activity is unaffected by halothane. 4. The first-order rate constant for oxidation of membrane-bound ferrocytochrome b5 in the absence of added substrate (k1 equals 1.5 times 10(-3)A-1) is similar to that for autoxidation of purified ferrocytochrome b5(k1 equals 7 times 10(-3)S-1) the rate of autoxidation of soluble ferrocytochrome b5 is unaffected by halothane. 5. It is concluded that the effects of halothane on microsomal electron transfer are not related to cytochrome P-450 linked metabolism but rather arise from the interaction of halothane with the cyanide-sensitive factor of the stearate desaturase pathway.


The stimulation by transmitter substances and putative transmitter substances of the net activity of phospholipase A2 of synaptic membranes of cortex of guinea-pig brain.

1. The distribution of the hydrolyses of phosphatidylcholine by phospholipase A2 and phospholipase A1, and the hydrolysis of lysophosphatidylcholine by lysophospholipase, in subcellular and subsynaptosomal fractions of cerebral cortices of guinea-pig brain, was determined. 2. Noradrenaline stimulated hydrolysis by phospholipase A2 in whole synaptosomes, synaptic membranes and fractions containing synaptic vesicles. 3. Stimulation of hydrolysis by phospholipase A2 in synaptic membranes by noradrenaline was enhanced by CaCl2, and by a mixture of ATP and MgCl2. The optimum concentration of CaCl2, in the presence of ATP and MgCl2, for stimulation by 10 muM-noradrenaline was in the range 1-10muM. The optimum concentration for ATP-2MgCl2 in the presence of 1 muM-CaCl2 was in the range 0.1-1mM. 4. Hydrolysis by phospholipase A2 of synaptic membranes was also stimulated by acetylcholine, carbamoylcholine, 5-hydroxytryptamine, dopamine (3,4-dihydroxyphenethylamine), histamine, psi-aminobutyric acid, glutamic acid and aspartic acid. With appropriate concentrations of cofactors, sigmoidal dose-response curves were obtained, half-maximum stimulations being obtained with concentrations of stimulant in the range 0.1-1muM. 5. Taurine also stimulated hydrolysis of phosphatidylcholine by phospholipase A2. There were only slight stimulations with methylamine, ethylenediamine or spermidine. No stimulation was obtained with glucagon.