The covalent modification of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate. Reaction at a binding site other than the active site.

A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfide bonds between the 6 thiol group on the purine ring and certain key cysteines on myosin, heavy meromyosin, and subfragment one. The EDTA ATPase activities of myosin and heavy meromyosin were completely inactivated when 4 mol of thiopurine nucleotide was bound. When similarly inactivated, subfragment one, depending on its method of preparation, incorporated either 1 or 2 mol of thiopurine nucleotide. Modification of a single cysteine on subfragment one resulted in an inhibition of both the Ca2+ and the EDTA ATPase activities, but the latter always to a greater extent. Modification of two cysteines per head of heavy meromyosin had the same effect suggesting that the active sites were not blocked by the thiopurine nucleotides. Direct evidence for this suggestion was provided by equilibrium dialysis experiments. Heavy meromyosin and subfragment one bound 1.9 and 0.8 mol of [8-3H]adenylyl imidodiphosphate per mol of enzyme, respectively, with an average dissociation constant of 5 X 10(-7) M. Heavy meromyosin with four thiopurine nucleotides bound or subfragment one with two thiopurine nucleotides bound retained 65-80% of these tight adenylyl imidodiphosphate binding sites confirming the above suggestion. Thus previous work assuming reaction of thiopurine nucleotide analogs at the active site of myosin must be reevaluated. Ultracentrifugation studies showed that heavy meromyosin which had incorporated four thiopurine nucleotides did not bind to F-actin while subfragment one with one thiopurine nucleotide bound interacted only very weakly with F-actin. Thus reaction of 6,6'-dithiobis(inosinyl imidodiphosphate) at nucleotide binding sites other than the active sites reduces the rate of ATP hydrolysis and inhibits actin binding. It is suggested that these second sites may function as regulatory sites on myosin.


Immunological studies of bovine nasal cartilage proteoglycan "link proteins".

Bovine nasal cartilage proteoglycan aggregates are dissociated and separated by density gradient centrifugation in 4 M guanidine into proteoglycan subunit (PGS) and glycoprotein link (GPL) fractions, the latter containing hyaluronic acid and "link proteins" responsible for aggregate formation. It was previously concluded on the basis of immunodiffusion studies that GPL has two antigenic components, one in common with PGS and one specific for the link proteins. However, in the present study it was found that antisera to PGS, which should lack link proteins, reacted with both "subunit" and "link" components of GPL, and antisera to fragments of PGS derived from the hyaluronic acid-binding portion of the molecule reacted preferentially with the link component. Reduction and alkylation of GPL led to modification of the reactions of both anti-GPL and anti-PGS sera with its link component. These immunodiffusion results indicate that the proteoglycan subunit and the link proteins are immunologically related and suggest that the link proteins may be identical with and derived from the hyaluronic acid binding portion of the proteoglycan subunit.


The role of phospholipid acyl chains in the activation of mitochondrial ATPase complex.

1. The role of length and unsaturation of phospholipid acyl chains in the activation of ATPase complex was studied with synthetic phosphatidylcholines and a phospholipid-dependent preparation obtained after cholate-extraction of submitochondrial particles (Kagawa, Y. and Racker, E. (1966) J. Biol. Chem. 241, 2467--2474). 2. Micelle-forming, short-chain phosphatidylcholines produced activation only at critical micellar concentration. The reactivated complex was cold-stable but the oligomycin sensitivity was low. 3. Bilayer-forming saturated phosphatidylcholines produced activation which was maximal at 9 carbon atoms in each chain but decreased sharply as the chain-length was increased and essentially disappeared at 14 carbon atoms. By contrast the oligomycin-sensitivity increased with the increase in chain length. 4. Activation of ATPase complex reappeared when bilayers were formed with long-chain unsaturated phosphatidylcholines. The activity was oligomycin sensitive. Significant inhibition of activity was observed also after incorporation of cholesterol into the bilayers. 5. By contrast the activation induced by negatively charged liposomes of diacylphosphatidylglycerol was independent on acyl-chain composition and occurred at very low amounts of phospholipid. 6. The discontinuity in the Arrhenius plot of activity of the ATPase complex reactivated with saturated phospholipids was found at temperatures close to the gel-to-liquid crystalline transition of the lipid showing that the activity of ATPase complex was sensitive to the physical state of membrane phospholipids. 7. It is concluded that (a) reactivation of ATPase complex by isoelectric phospholipids is an interfacial activation, the minimum requirement for the lipid effect being micelle formation. (b) In order to gain the properties of the native complex a stable lamellar phase is needed. Both activity and oligomycin sensitivity are regulated by the chain length and degree of unsaturation of phospholipid acyl chains.


Comparative structural studies of urinary glycosaminoglycans in the Hurler and Hunter syndromes.

Some hitherto undetected differences in chemical and macromolecular structure between both dermatan sulphates and heparan sulphates excreted in the Hurler and Hunter syndromes are demonstrated. 1. Of Hunter dermatan sulphate, 37-43% is resistant to periodate oxidation, as opposed to 25% of the corresponding Hurler material. It is likely that the resistance is conferred by the presence of sulphate groups on carbon atoms 2 or 3 of the iduronate residues, correlating with the recently established deficiency of a sulphoiduronate sulphatase in Hunter fibroblasts. 2. Two distinct electrophoretic species of dermatan sulphate are found in Hunter urine, but only one in Hurler preparations. 3. Ion-exchange chromatography and gel filtration reveal that Hurler dermatan sulphate is more heterogeneous with respect to molecular weight distribution than the other. The dermatan sulphates were degraded by hyaluronidase to a limited extent. 4. Hurler heparan sulphate contains a higher proportion of sulphoamino-glucose than material from Hunter urine. Similar high levels in Sanfilippo patients, representing 65-78% of the total glucosamine suggest a direct correlation with mental deficiency.


The immunological specificity of myosins from cross-striated muscles as revealed by quantitative microcomplement fixation and enzyme inhibition by antisera.

The immunological properties of myosins, especially their muscle-type, class and species specificity, are still controversial. It is the opinion of the authors that the lack of agreement might at least in part be due to the use of contaminated myosins as immunogens and inappropriate methods. We, therefore, purified myosins to a very high degree (approximately 99%) and induced antisera in guinea-pigs. Studies of quantitative microcomplement fixation and enzyme-inhibition by antisera yielded the following results: myosins of cross-striated muscle have an absolute class, a very pronounced muscle-type and a low species specificity. It can be shown that even a very small contamination of myosins with other proteins could seriously hamper the experiments and that the results obtained depend significantly on the immunological methods employed.


Studies on the subunits of myosin from muscle layer of Ascaris lumbricoides suum.

1. A purified preparation of Ascaris myosin was obtained from the muscle layer of Ascaris lumbricoides suum, using gel filtration and ion-exchange chromatography. 2. Ascaris myosin whether purified or unpurified, had almost the same ability for ATP-splitting and superprecipitation. 3. Ascaris myosin and rabbit skeletal myosin were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A significant difference in the number of light chains between both myosins was found. Ascaris myosin was found to have one heavy chain and two distinct light chain components (LC1-A and LC2-A), having molecular weights of 18000 and 16000, respectively. These light chains correspond in molecular weight to the light chain 2 (LC2-S) and light chain 3 (LC3-S) in rabbit skeletal myosin. 4. LC1-A could be liberated from the Ascaris myosin molecule reacted with 5,5'-dithio-bis(2-nirobenzoic acid( Nbs2) with recovery of ATPase activity by addition of dithiothreitol. These properties are equivalent to those of the LC2-S in rabbit skeletal myosin, although Ascaris myosin when treated with Nbs2-urea lost its ATPase activity.


Isolation and characterization of a mutant of Neurospora crassa deficient in general amino acid permease activity.

A mutant of Neurospora crassa (pm-nbg27) was isolated on the basis of its resistance of p-fluoro-phenylalanine on ammonium-deficient Vogel's medium. This mutant was found to be devoid of both conidial and post-conidial (after 180 min of preincubation) transport activity of all amino acids. Genetic analysis of pm-nbg27 by crossing it to wild-type (74A) resulted in the predicted segregants exhibiting transport characteristics of pm-n, pm-b, pm-g, pm-nb, pm-ng, pm-bg and parental types. The above observations confirm the postulated general amino acid permease system as well as a single genetic locus control of that activity.


Asymmetric membrane expansion and modification of active and passive cation permeability of human red cells by the fluorescent probe 1-anilino-8-napththalene sulfonate.

1. The membrane perturbations induced by the interaction of the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS) with human red blood cells were studied. 2. ANS below 0.5 mM inhibits partially (20% maximum) the ouabain-insensitive Na+ and K+ influx and efflux. Above 0.5 mM ANS increases both Na+ and K+ leak fluxes. The increased cation leaks are larger for Na+ than K+. 3. The (Na+ +K+)-ATPase and ouabain-sensitive Na+ and K+ fluxes are inhibited by ANS. Ouabain-insensitive, Mg2+-dependent ATPase activity of ghosts is stimulated by [ANS] less than 0.3 mM and inhibited by [ANS] greater than 0.3 mM. 4. ANS also inhibits the Na+-dependent, ouabain-insensitive K+ influx that is inhibited by ethacrynic acid and furosemide. 5. Red cells become crenated with [ANS] less than 1 mM and sphere at [ANS]  greater than 1 mM. In the former conditions hypotonic hemolysis is decreased whereas the latter increase osmotic fragility. 6. It is suggested that ANS expands the membrane asymmetrically by binding preferentially to the external membrane surface. 7. It is concluded that ANS is a general inhibitor of ion transport, particularly of those processes thought to involve facilitated-diffusion mechanisms. The increased cation leaks observed at high ANS concentrations may be related to prehemolytic membrane disruption. 8. The membrane perturbations caused by ANS are compared to those caused by other reversible inhibitors of anion exchange in red blood cells. Their possible modes of action are discussed.


Genetic regulation of phosphate transport system II in Neurospora.

Phosphate transport system II, previously shown to be responsible for high-affinity phosphate uptake under conditions of phosphorus starvation, is regulated by at least three genes: pcon-nuc-2, preg, and nuc-1. nuc-1 and nuc-2 mutants cannot be derepressed for phosphate transport system II, while pconc and pregc mutants are partially constitutive.


Hydrolysis of exogenous ATP by isolated frog gastric mucosa.

ATP, added to the solution bathing the nutrient (serosal) surface of isolated frog gastric mucosa, was found to break down rapidly to ADP, inorganic phosphate and other products. This activity is due to an ectoenzyme, i.e. to an enzyme system easily accessible to the bathing solution. This conclusion follows from experiments which showed that penetration of ATP into the mucosal cells occurred at a much slower rate: leakage of inorganic phosphate and adenine nucleotides from mucosal cells was also minor. The surface ATPase may reflect the operation of a mechanism at the nutrient surface involved in acid secretion.


Acetylcholine-binding substance extracted by using organic solvent and acetylcholine receptor of electric organ of Narke japonica.

1. Proteolipid was extracted from the electric organ of Narke japonica by using chloroform/methanol (2:1, v/v). This extract was separated into acetylcholine-binding and non-binding substances by column chromatography. However, acetylcholine-binding substances did not show the characteristic properties of protein. 2. The membrane fragments of the electric organ were separated into three main parts by sucrose density gradient centrifugation. From the heaviest, the fractions were acetylcholine receptor rich, ATPase rich, and acetylcholinesterase rich. 3. The membrane fraction having acetylcholine receptor showed the excitability, the increase of Na+ permeability by the application of cholinergic agonists. However, the acetylcholine binding substance extracted by the organic solvent was richer in the lighter fraction. This substance differed from the true acetylcholine receptor.


Mechanisms by which Li+ stimulates the (Na+ and K+)-dependent ATPase.

The addition of LiCl stimulated the (Na+ + K+)-dependent ATPase activity of a rat brain enzyme preparation. Stimulation was greatest in high Na+/low K+ media and at low Mg-ATP concentrations. Apparent affinities for Li+ were estimated at the alpha-sites (moderate-affinity sites for K+ demonstrable in terms of activation of the associated K+-dependent phosphatase reaction), at the beta-sites (high-affinity sites for K+ demonstrable in terms of activation of the overall ATPase reaction), and at the Na+ sites for activation. The relative efficacy of Li+ was estimated in terms of the apparent maximal velocity of the phosphatase and ATPase reactions when Li+ was substituted for K+, and also in terms of the relative effect of Li+ on the apparent Km for Mg-ATP. With these data, and previously determined values for the apparent affinities of K+ and Na+ at these same sites, quantitative kinetic models for the stimulation were examined. A composite model is required in which Li+ stimulates by relieving inhibition due to K+ and Na+ (i) by competing with K+ for the alpha-sites on the enzyme through which K+ decreases the apparent affinity for Mg-ATP and (ii) by competing with Na+ at low-affinity inhibitory sites, which may represent the external sites at which Na+ is discharged by the membrane Na+/K pump that this enzyme represents. Both these sites of action for Li+ would thus lie, in vivo, on the cell exterior.


XC-cell fusion induced by murine plasmocytoma cells. II. Cytological and ultrastructural study.

The MF2 strain, a mouse myeloma derived cell line, was found to induce the mixed culture cytopathogenicity test when cocultured with XC cells. Only one MF2 cell was present per syncytium, as shown by autoradiography. Pretreatment of cells with inhibitors of DNA, RNA or protein synthesis suggested that a normal RNA synthesis was required to obtain optimal polykaryon growth. Immunoelectron microscopy using a syngenic mouse MF2 cell antiserum and peroxydase labeling revealed a complete mixing and redistribution of the respective plasma membrane sites of MF2 and XC cells on polykaryon surface.


[Energy conversion of the phosphate bond in the actomyosin-ATP system, accompanied by acceleration of myosin adenosine triphospatase].

A possible mechanism of the active motion ("tilt") of the actin-bound myosin head in ATP hydrolysis reaction is considered. This motion is considered. This motion can be goverened by actinmyosin affinity changes at two sites located on the head. Such an affinity change is assumed to take place already in the macroergic myosin (M) -products (P) complex MP. Then at a contact with actin a tilting force is generated, and the M energy liberation, coupled with the head ratation, goes on with a concomitant potential barrier lowering and corresponding acceleration of the MP destruction. In this way a destruction. In this way a decrease of free energy with a change of the head orientation is equivalent to the energy liberated, which amounts to 9 kcal.mol-1.


Sandhoff disease: impaired catabolism of sulfated glycosaminoglycans in cultured fibroblasts.

Fibroblasts cultured from the skin of patients with Sandhoff disease accumulate excessive amounts of sulfated glycosaminoglycans because of degradative inadequacy. Only a slight such abnormality in the metabolism of sulfated glycosaminoglycans was seen in fibroblasts from patients with Tay-Sachs disease. The defective glycosaminoglycan catabolism in Sandhoff fibroblasts is specifically corrected by intracellular replacement of beta-N-acetyl-hexosaminidase. Both beta-N-acetyl-hexosaminidase A and B are effective in bringing about such correction, although there seem to be differences in specificity. Our findings suggest that in Sandhoff disease there is an impaired catabolism of glycosaminoglycans in addition to the defect in the degradation of glycosphingolipids.


Subacute myelo-optic neuropathy and clioquinol. An epidemiological case-history for diagnosis.

Between about 1955 and 1970, some 100,000 Japanese were diagnosed as having subacute myelooptic neuropathy (SMON), a new disease characterized by abdominal and neurological manifestations, the former nearly always preceding the latter. Circumstantial evidence obtained in 1969-70 suggested that SMON might have been caused by clioquinol (CQL), a gastrointestinal disinfectant, and led to the suspension of further sales of CQL in Japan. However, several inconsistencies for the CQL theory of SMON have now emerged; first, CQL had been widely used in Japan for nearly 20 years before SMON occurred. Secondly, the SMON epidemic began to subside several months before CQL sales were suspended. Thirdly, a large proportion of SMON patients--probably about one-third and possibly more--had not taken CQL within six months of the onset of the disease (the modal interval between first taking CQL and the onset of SMON being about three weeks, and more than 100 days in only 4% of SMON patients); of the remaining two-thirds or so, many had taken CQL as part of the treatment of the first (that is, abdominal) symptoms of SMON itself. Fourthly, there was no dose-response relationship. Finally, SMON rarely, if ever, occurred outside Japan. CQL could, however, have been involved in the causation of SMON as an optional enhancer of some other necessary cause; the history of post-war environmental pollution in Japan is compatible with this hypothesis. Over-readiness to accept postulated toxic effects of medicines and chemicals as proven is likely to do at least as much harm as good to individual and community health.


H-rosette formation in T-cell-proliferative diseases.

In health up to 6% of human lymphocytes will form rosettes with homologous group O rhesus-negative erythrocytes (H rosettes). Increased numbers of H rosettes were found in T-cell-proliferative diseases-- namely, infectious mononucleosis, Sézary's syndrome, and T-cell leukaemia. H-rosette formation is thus a marker for a subpopulation of T lymphocytes, and this easily performed test of T-cell activation may have clinical value in characterising changes in blood lymphocyte populations in disease.


Effect of spectinomycin on T. pallidum in incubating experimental syphilis.

Animal experiments were performed to determine if a single-dose treatment for acute gonorrhoea with 2 g. spectinomycin could cure a simultaneously acquired syphilis at a very early stage of incubation. 300 treponemes (Nichols stain T. pallidum) were inoculated intratesticularly and 3 days later spectinomycin was administered in a dose which produced spectinomycin serum levels similar to those in patients who had received a single oral dose of 2 g. This dosage of spectinomycin did not prevent the development of syphilitic orchitis or reactivity to the FTA-ABS test, but it prolonged the subclinical incubation period.


The isolation and characterization of the ATPase inhibitory protein (TN-I) from bovine cardiac muscle.

The inhibitory component of the troponin complex (TN-I) was purified from bovine cardiac muscle, using a combination of ion exchange and molecular exclusion chromatographies in the presence of urea. It has the ability to inhibit the Mg2+-activated APTase (EC 3.6.1.3) of a synthetic cardiac actomyosin preparation and this inhibition is reversed by the addition of cardiac calcium binding component of troponin (TN-C). Conventional sedimentation equilibrium experiments suggest a molecular weight for cardiac TN-I of 22 900 +/- 500. However, sodium dodecyl sulfate (SDS) gels indicate a molecular weight of 27 000 +/- 1000. The mobility of TN-I on SDS gels may be anomalous due to the high proportion of basic amino acid residues in the protein. Cardiac TN-I and TN-C interact to form a tight complex, even in the presence of 6 M urea. The results of this study invite direct comparison with results published for rabbit skeletal TN-I.


A clinical trial of chemotherapy and RAJI immunotherapy in advanced acute lymphatic leukemia.

Patients in remission with advanced acute lymphatic leukemia were randomly assigned to receive chemotherapy alone or chemotherapy plus immunotherapy with a Burkitt lymphoma tissue culture cell line (RAJI). Remission duration in both groups were identical. Complement-dependent cytotoxic antibody was seen in 5 of 8 immunized patients and 0 of 8 controls. This antibody reacted with RAJI and both allogeneic and autologous acute leukemia cells. Antibody titers began to rise after 2 months, peaked at 4 months, and then declined prior to relapse in all patients. The time course of the increase in mixed-leukocyte culture response to RAJI was similar to immunized patients. An increased in vitro response to phytohemagglutinin was seen during drug administration in all patients. Although no clinical benefit was seen in this small number of patients with the RAJI injections, these in vitro responses are encouraging and new immunization schedules will be investigated.


Effects of chronic graded ethanol consumption on the metabolism, ultrastructure, and mechanical function of the rat heart.

Three sequential sets of ethanolic rats (E) and their matched controls (C) were fed regular chow containing standard vitamins with the ethanol group in each series also receiving a progressively greater alcohol intake for 3 to 6 months: E1 5%, E2 10%, and E3 25% ethanol. Electron microscopy showed swelling of mitochondria, transverse tubules and sarcoplasmic reticulum, dehiscence of intercalated discs and disintegration of myofibrils scattered throughout the ventricular myocardium in E1 and E2 as early as 7 wk after beginning 5% ethanol; in addition, there were clumping of mitochondria and supercontraction of myofibrils in E3. Concomitant with substructural abnormalities in E3, there were slight but significant depressions of cardiac myofibrillar ATPase activity and mitochondrial function. Cardiac catecholamines, hydroxyproline, and total bound glycerol were unchanged. Alteration of isometric contraction of isolated, supported left ventricular papillary muscles occurred initially in E2 and was clearly evident in E3 by significant reduction of duration of systolic active state (time from onset to peak tension), while total tension generated and peak rate of tension rise were not yet disturbed. Extra vitamin supplementation in additional rats drinking 25% ethanol minimally lessened decline in myofibrillar ATPase activity, but otherwise provided no protection. Thus, chronic daily ingestion of graded quantities of ethanol representing 10 to 30% of total calories in well-nourished animals exerted toxic effects on microstructure, metabolism and mechanics of the ventricle. These alterations are postulated to be pertinent to early pathogenesis of clinical alcoholic cardiomyopathy.


Evaluation of the specificity of a lymphoid chalone.

An extract from calf spleens, injected into mice, was found to inhibit their lymphocyte proliferative response to PHA-M and PWM in vitro. Despite the ability of the spleen extract to inhibit the response of lymphocytes to stimuli in vitro, no effect was observed on the repopulation of lymphocytes in the peripheral blood of sublethally irradiated mice. The data suggest that the spleen extract acts as a specific inhibitor of the innume competent cells since neither the precursors of lymphocytes nor other haematopoietic cells were affected.


Studies on plasma membranes. XXIII. Hormone-like action of concanavalin A on liver plasma membranes: inhibition of (Na+-K+)ATPase.

Concanavalin A inhibits the (Na+-K+)-ATPase activity of isolated rat-liver plasma membranes, while leaving the Mg2+-ATPase unaffected. Glucagon and cyclic AMP act supplementary to the lectin in the inhibition. The lectin effect is counteracted by insulin and L-epinephrine, and is completely abolished by the beta-adrenergic blocking agent propranolol. Results are discussed on the basis of the known interactions of concanavalin A with plasma membrane components, including its hormone-like action.


Calcium-accumulating properties of subcellular fractions of bovine vascular smooth muscle.

This study examined the calcium transport properties of two subcellular fractions of bovine aorta obtained by differential centrifugation. The vesicular fraction had a high-affinity (Km = 1.05 X 10(-6)M) calcium transport mechanism which could be potentiated by using the calcium-precipitating anion, oxalate. The mitochondria-enriched fraction's calcium transport system had a lower affinity for calcium than did that of the vesicular fraction. The calcium capacity of the vesicular fraction was determined by comparing the steady-state calcium uptake in the presence of oxalate in the whole homogenate with the same uptake by the vesicular fraction alone. A calcium capacity of about 100 mumoles Ca2+/kg aorta was obtained. It is concluded that the vesicular fraction has  all of the properties of a major site of subcellular calcium regulation in vascular smooth muscle.


Body position, electrode level, and respiration effects on the Frank lead electrocardiogram.

Frank lead ECG/VCG changes with deep inspiration, expiration, and body position were investigated in 194 patients, 100 with an old myocardial infarction and the remaining 94 chosen as a representative sample of catheterization laboratory patients with a variety of cardiac conditions. In a subgroup of 144 of the patients, Frank lead records were made both at the fifth and the fourth intercostal space. Diagnostic interpretation was performed using the VA-Pipberger ECG Program. The results indicate that, in general, body position and electrode level influence on mean intervals and orientation angles is negligible. There was a highly significant decrease in the R and Q wave amplitudes in leads X and Z and in the maximum spatial magnitude of QRS when electrodes were shifted from the fifth to the fourth interspace. The most pronounced decrease in ECG/VCG amplitudes took place in deep inspiration while mean orientation angles changed little, with the exception of QRS elevation. However, while mean changes with body position and electrode level were rather small, substantial orientation and magnitude changes took place in many patients. Diagnostic interpretation of the records changed in 12.5% with electrode level change, in 11.9% with the transition of body position, and in 16.8% with deep inspiration. Strict standardization of electrode positions and recording procedure is suggested, particularly when serial comparison of Frank lead records is planned.


Echocardiography in congenital and acquired absence of the pericardium. An echocardiographic mimic of right ventricular volume overload.

The purpose of this study was to investigate the echocardiographic effects of absence of the pericardium. Five patients with congenital complete absence of the left pericardium were studied. All had typical chest X-rays, four had cardiac catheterization which excluded any intracardiac shunts, and one had diagnostic pneumothorax. All five had an enlarged right ventricular dimension (RVD): 1.9 +/- 0.1 cm/m2 (normal: less than 1.3 cm/m2) and abnormal interventricular septal (IVS) motion (three Type A, two Type B). Sixteen additional patients were studied after pericardial stripping for a variety of conditions. In none was cardiopulmonary bypass used. Eight of these patients had preoperative echocardiograms; all showed normal IVS motion. After surgery RVD was large in all 16 patients, increasing from 1.0 +/- 0.2 cm/m2 preoperatively to 1.7 +/- 0.1 cm/m2 postoperatively, P less than 0.01. Fourteen of the 16 patients had abnormal IVS motion, nine Type A, and five Type B. We conclude that absence of the pericardium results in echocardiographic abnormalities which mimic those seen in right ventricular volume overload. This may be due to altered cardiac position and motion within the thorax resulting from loss of normal pericardial restraint.


A radioimmunoassay for the estimation of serum dehydroepiandrosterone sulphate in normal and pathological sera.

A radioimmunoassay is described for the measurement of dehydroepiandrosterone sulphate in serum. The method requires 0.1 ml of serum that needs no purification other than a single ether extraction. It is both rapid and economical, procedural losses are small and precision and accuracy are acceptable for both normal and pathological sera. Normal values have been established for children and adults which show a relationship to chronological age, values rising steadily as puberty intevenes, peak values occurring between 20 and 27 years, with a decline thereafter. An empirical curve was fitted to the data and approximate normal limits were obtained. The mean value for pregnanat women in the age range 20-40 years, was statistically significantly lower than in non-pregnant, premenopausal women. The levels of dehydroepiandrosterone sulphate in cord and newborn blood, were higher than at any other time during childhood until signs of puberty were demonstrable. Elevated levels are reported in six patients with untreated congenital adrenal hyperplasia and in one patient with a tumour of the adrenal cortex. It is suggested that this assay provides a useful and precise index of adrenal androgen secretion.


Hepatitis-associated (Australia) antigen and Down's syndrome.

Hepatitis-Associated (Australia) Antigen (HAA) was detected in 13 (5.8%) of 223 patients with Down's syndrome and in 14 (3.7%) of 378 patients with other forms of mental retardation. The frequency of HAA was 2.4 per cent in 127 noninstitutionalized patients with Down's syndrome, and 10.4 per cent in 96 institutionalized patients. The frequency of HAA with Down's syndrome was lower on the average in Japan than in the United States or Germany. HAA was detected in one (1.3%) of 78 mothers of infants with Down's syndrome. Our study suggests that maternal exposure to HAA, as reflected by the presence of either HAA or anti-HAA, was not associated with the subsequent birth of an infant with Down's syndrome.


Scattered mitoses in mature joint cartilage in rabbits after local trauma. A chalone effect?

This is a demonstration of local traumatization of articular cartilage inducing mitoses in parts of a joint other than the site of inquiry. Parts of one of the patellar cartilages of the full-grown rabbit were scored while the other knee was subjected only to arthrotomy. Radioactive thymidine and autoradiographic technique demonstrated that labeled chondrocytes appeared not only around the score in the patella but also scattered in the tibia and femoral condyles. Whether the mechanism of regional mitotic stimulation is a reduction of the concentration of the normal cell specific inhibitors of mitosis, or so-called chalones is not known and requires further investigation.


Defective membrane systems in dystrophic skeletal muscle of the UM-X7.1 strain of genetically myopathic hamster.

1. The function of mitochondria, sarcotubular membranes (heavy microsomes), sarcolemma and myofibrils from the hind-leg skeletal muscle of about 60- and 150-day-old normal and myopathic (UM-X7.1) hamsters was examined. 2. The mitochondrial calcium uptake as well as mitochondrial phosphorylation and respiratory rates were lower in 60-day-old myopathic skeletal muscle, unlike 150-day-old myopathic animals, when pyruvate-malate and glutamate-malate were used as substrates. However, mitochondria from 150-day-old myopathic animals showed depressed glutamate-dependent respiratory and phosphorylation rates and succinate-supported initial rate of calcium uptake. 3. The microsomal calcium-uptake, but not calcium-binding, and Ca2+-stimulated adenosine triphosphatase (ATPase) activity of the 150-day-old myopathic skeletal muscle were lower than the control values. Although microsomal calcium-binding, calcium-uptake and ATPase activities of the 60-day-old myopathic muscle were not depressed significantly, the initial rate of calcium uptake was less than the control. 4. The sarcolemmal Ca2+-ATPase, but not Mg2+-ATPase or Na+ +K+-ATPase, activity was higher in 60-day-old myopathic muscle whereas the activities of all these enzymes from 150-day-old myopathic animals were higher than the control. On the other hand, the Na+ +K+-ATPase activities from 60- and 150-day-old myopathic animals were inhibited by ouabain to a lesser extent in comparison with the respective control values. 5. The myofibrillar Ca2+-ATPase and Mg2+-ATPase activities as well as inhibition of Mg2+-ATPase due to Na+ and K+ in myopathic muscle were no different from the control values. 6. The results reported here give further support to the view that different membrane systems of the dystrophic muscle are defective.


Cadmium-induced experimental Fanconi syndrome.

We have demonstrated that repetitive injections of cadmium into rats leads to the abrupt development of Fanconi syndrome after the renal cortical concentration of cadmium has increased approximately eighty-fold. The syndrome is temporally associated with a fall in both Na-K-ATPase activity and ATP levels, suggesting a possible causal relationship between altered proximal tubular transport and inhibition of the ATP-Na-K-ATPase transport system.


Substrate oxidation and inhibition of sodium transport in the isolated perfused rat kidney.

The relationship between the oxidative substrate-turnover-rate and the active Na+-transport was investigated. Our special interest was directed to the possible dependence of the oxidative turnover-rate of one selected substrate from the mode of reduction of the Na+-transport. The following methods were used: 1. Blocking the active Na+-transport by the addition of ouabain to the perfusion medium (0, 5 mg/ml); 2. Reduction of the sodium concentration in the perfusion medium to 100mM substituting NaCl by Tris/HCl; 3. Reduction of the perfusion pressure resulting in a low GFR and a low tubular Na+-load. The perfusion medium contained the following substrates in normal plasma concentrations: Na-palmitate, Na-oleate, Na-propionate, Na-acetate, D-glucose, Na-pyruvate, Na-L-lactate and Na-glutamate. The substrate in question was uniformly 14C-labelled; its amount oxidized per unit time was calculated on the basis of the 14CO2 formed. The following substrates were studied; Na-acetate, Na-pyruvate and Na-L-lactate. There were no significant differences between the results of the three methods described.


Hormonal effects on Na-K-ATPase of various parts of the rat nephron.

Aldosterone reactivates the impaired Na-K-ATPase very rapidly along the course of the distal tubule and in the proximal convolution. Surprisingly the latent period before enzyme induction parallels with the latent period before the restoration of active sodium reabsorption after a physiological dose of aldosterone [7]. Induction of Na-K-ATPase can be prevented by cycloheximide or actinomycin D, inhibitors of protein synthesis; this proves that protein synthesis is necessary for the induction of Na-K-ATPase. Although this kind of data can be interpreted as due to de-novo-synthesis for the induced enzyme this conclusion is not proved by such data. Another way of aldosterone action could exist in demasking inactive enzyme molecules which are already present in the membrane. Because of the very short latent period between aldosterone application and restoration of Na-K-ATPase we cannot exclude this possibility. The most important finding of our study is the rapid reactivation of the impaired Na-K-ATPase in the proximal and distal tubule after a single dose of aldosterone.


A group training program in behavior modification for mothers of children with birth defects: an exploratory study.

This paper explores the impact of a group approach to teaching behavior modification principles and techniques to mothers of physically handicapped children. Five sessions involving presentations, discussions, and application of operant reinforcement techniques were followed by five sessions focusing on the importance of communication about the handicap, and applying the previously learned operant tecniques to opening communication in this sensitive, secretive area. Clinical and statistical findings suggest that this approach can effectively train mothers to apply the techniques, and increase their attempts to communicate with their handicapped children. Moreover, mothers' participation appeared to facilitate their children's readiness to communicate, as well as improve their children's self-esteem, enthusiasm, and likability.


[Androgen metabolism and Leydig-cell function in acute and chronic liver disease (author's transl)].

Diurnal variations of plasma testosterone and urinary excretion rates of testosterone-, androsterone-, aetiocholanolone-, and DHA-glucuronide, as well as DHA-sulphate were measured before and after selective Leydig-cell stimulation (with 3 X 5000 IU human gonadotrophin) in eight patients with alcoholic liver cirrhosis, ten with acute hepatitis and four with haemochromatosis. The circadian variation of plasma testosterone and the maximal testosterone concentration after human gonadotrophin stimulation were decreased in patients with cirrhosis and haemochromatosis, while in those with acute hepatitis the diurnal variations were evened out, whereas individual values were within the normal range. Urinary excretion of free testosterone and testosterone glucuronide was significantly reduced in those with cirrhosis and haemochromatosis before and after stimulation. The urinary pattern of C19 steroid metabolites was markedly changed in favour of the 5 alpha-steroids in acute heaptitis, cirrhosis, and haemochromatosis.


[Oxyphenisatin-induced liver disease (author's transl)].

Over a period of 18 months the development of hepatitis after intake of oxyphenisatin, a laxative, was established in 14 patients by re-exposure to the drug. The characteristic feature was nonspecific upper abdominal pain up to colic-like pain, lact of appetite, nausea or vomiting, and pruritus. The biochemical changes were those of chronic hepatitis with varying severity of biliary stasis and abnormal immunofluorescence. On re-exposure there was a particularly remarkable rise in GLDH activity. The histological picture showed acute inflammatory changes in the biliary passages on re-exposure, while the liver cells were clearly involved only secondarily. At a latter point the histological picture became non-specific. At laparoscopy there were different stages of minor periportal hepatic fibrosis to marked postnecrotic liver scars with portal hypertension and decompensation. Early diagnosis is difficult but crucial to the patient's fate, because this form of hepatitis regresses completely after oxyphenisatin has been stopped. Laxatives containing this drug should be withdrawn from the market.


[Diagnostic value of cytological investigations in metastatic tumours of the liver: comparison of laparoscopy, histology, and cytology (author's transl)].

Fine needle aspiration biopsy is generally a harmless method if contraindications are considered and provides sufficient tissue for cytological evaluation. The diagnostic value is very high: in 27 cases of metastatic tumours of the liver diagnosed by laparoscopy the diagnosis was confirmed in 26 cases and revised in one case. In a control group of 200 patients with various diffuse liver diseases or morphologically normal findings no false positive results were found. In no case were post-biopsy haemorrhage or other complications observed. Needle biopsy and cytological evaluation should be used with an increased frequency when a malignant tumour of the liver is suspected clinically. After primary localization of suspicious areas by ultrasound or scanning the pick-up rate is high. Laparoscopy with precisely aimed fine needle biopsy is the method for verifying the diagnosis in cases with cytologically negative results which is up to 100% accurate.


Inhibition of renal gluconeogenesis by guinolinate and hydrazine in diabetic rats.

Renal as well as hepatic gluconeogenesis is inappropriately accelerated in the diabetic state when plasma glucose levels are elevated. Known regulatory mechanisms influence gluconeogenesis in both organs. However, under certain circumstances gluconeogenesis may be affected in one organ and not the other. Recent studies with the tryptophan metabolite, quinolinate, suggest that hepatic gluconeogenesis in the diabetic is unaffected by this agent, whereas gluconeogenesis is blocked in the normal liver. These experiments have been interpreted as providing evidence for the lack of a specific physiologic repressor for gluconeogenesis in diabetic liver. In the present study quinolinate and hydrazine are shown to be effective inhibitors of the accelerated gluconeogenesis in the renal cortex of diabetic rats. Thus, the renal gluconeogenic mechanism in diabetics retains the capacity to recognize quinolinate as an inhibitor, but may be influenced by the depressed conversion of tryptophan to quinolinate in the intact diabetic organism.


Differential changes in the 5-hydroxytryptamine and insulin content of guinea-pig B-cells.

In the guinea-pig pancreas, 5-hydroxy-tryptamine (5-HT) occurs in the B-cells as well as in enterochromaffin cells scattered in the exocrine parenchyma. In the present study we examined the effects on the pancreatic 5-HT and insulin content of drugs known to affect the B-cells. For this purpose a radioimmunoassay of guinea-pig insulin was developed. Streptozotocin reduced the number of detectable B-cells and partially degranulated those that remained. It also lowered the insulin content of the pancreas and the 5-HT content of the B-cells but did not affect the 5-HT content of the enterochromaffin cells. Reserpine lowered the 5-HT content of both B-cells and enterochromaffin cells. The number and ultrastructure of the B-cells and the pancreatic insulin content was not affected. Streptozotocin + reserpine seemed to have additive effects of B-cell 5-HT. The results with these 2 drugs indicate that roughly 50% of pancreatic 5-HT is contained in the B-cells. Diazoxide markedly increased the pancreatic insulin content without affecting pancreatic 5-HT. Despite the fact that 5-HT and insulin are stored together in the cytoplasmic granules of the B-cells, 5-HT was differentially depleted from this store by reserpine. A marked disproportionality between 5-HT and insulin content was also induced by diazoxide.


Steroid hormone formation in bovine ovarian follicles.

In an attempt to assess histophysiological implication of the follicular compartment of the bovine ovary in steroid hormone formation and the effect of human chorionic gonadotropin (hCG) in vitro on follicular steroidogenesis, minces of follicular tissues from non-gravid bovine ovaries were incubated with radioactive testosterone or acetate in the presence and absence of hCG. Significant amounts of estrone and estradiol-17beta were formed on incubation with testosterone-4-14C; hCG decreased the conversion approximately by 30%. The major radioactive products formed from acetate-l-14C were androstenedione and testosterone with lesser amounts of dehydroepiandrosterone and 17-hydroxyprogesterone. In addition, small amounts of progesterone, 17-hydroxypregnenolone, estrone and estradiol-17beta were formed. Histology of the dissected follicle specimens was characterized by dominant theca cells undergoing luteinization with small amounts of granulosa cells, which showed neither proliferation nor luteinization. The pattern of distribution of radioactivity among the steroids formed from acetate-14C was considered to represent steroidogenic profile of bovine atretic follicles. The addition of hCG in vitro increased the overall incorporation of radioactive acetate into the steroids approximately by 50%, although the range of increase was not uniform in the individual steroids under the exprimental conditions.


[Transfer characteristic of the motor system of the hand obtained by electrical stimulation and its bearing on force tremor (author's transl)].

Pulse duration modulated signals were used to stimulate the extensor muscles of the hand. From this and from the isometric tension responses the frequency response of the human motor system was obtained. From the transfer characteristics of the components of the motor system which were taken from literature the frequency response of a closed-loop circuit model could be calculated. The theoretical frequency response was compared to the observed data. With increasing loop gain the model would show the characteristics of a filter tuned to the frequency of force-tremors.


The fibrous structure of human chromosomes in relation to rearrangements and aberrations; a theoretical consideration.

Human chromosomes as a type-sample for mammalian chromosomes consist of 200-A fibers, folded to chromomeres, which are interconnected by about a dozen longitudinal fibers. The average fiber at both interphase and metaphase contains 28.3 lengths of one double helix of DNA per length of fiber. The orientation of DNA imparts polarity to the fiber and thus to the chromosome and is an important constraint in concepts of chromosomal aberrations and rearrangements, some of which are being interpreted on the basis of fiber-fiber exchanges. Chromosomal rearrangements discernible by light microscopy are not likely to be fully synonymous with change in gene sequence. Chromosomes are considered to possess a plane of symmetry originating from semiconservative replication. Implications for chromosomal structure, centromeric function, and chromatid cohesion are discussed. Fibers connecting one chromosome to others are discussed in light of the proposal that fiber regions of repeated nucleotide sequences exist that facilitate fiber-fiber exchanges. No free fiber or DNA ends are thought to occur at any time in the nucleus.


Initial change of glycosaminoglycans in systemic scleroderma.

7 clinically uninvolved as well as 8 involved (6 moderately, 2 markedly) back or forearm skin specimens from 12 patients with systemic scleroderma were subjected to quantitative evaluation and to qualitative analysis of glycosaminoglycans (GAG) by one-dimensional and two-dimensional cellulose acetate electrophoresis. Skin specimens from the back, clinically uninvolved but histologically demonstrating the initial change, revealed increased amounts of hyaluronidase, chondroitinase-resistant GAG of varying electrophoretic mobilities, and one of them was chemically confirmed to be heparan sulfate variant, whereas involved skin specimens showed hardly this increase.


Sex variation in the activities of mucosal hydrolytic enzymes in the small intestine of the rat.

Mucosal response of alkaline phosphatase, ATPase and disaccharidase (lactase, maltase and trehalase) activities to sex hormones were studied by comparing male and female rats and castrated males and by injecting testosterone into castrated males. Alkaline phosphatase showed a very steep gradient in the small intestine from the oral to the aboral end, whereas ATPase activity in the ileum was still about 50% of that in the duodenum. Both enzymes showed only minor sex variations and weal response to castration. Lactase and maltase had peak activities in the jejunum, but trehalase activity was nearly equally high in the duodenal mucosa as in the jejunum. Jejunal lactase activity was about 50% lower in female than in male rats and castration decreased activity in males to the same low level as found in females. The administration of testosterone to castrated male rats did not enhance activity. Maltase activity showed similar sex variation, although castration was not able to decrease activity during the test period. Trehalase activity was lower in female than in male rats. The administration of testosterone enhance activity in castrated males.


Treatment with glucagon-binding antibodies alters the intestinal response to starvation in the rat.

Everted intestinal rings from partially starved rats accumulate the nonmetabolized amino acid 1-amino-cyclopentane-5-carboxylic acid (ACPC) at an enhanced rate. Plasma glucagon concentrations were found to be markedly elevated in these partially starved rats as well as in rats with experimentally induced diabetes, a condition previously shown to be associated with augmented intestinal uptake of amino acid. Treatment of partially starved rats with repeated injections of glucagon-binding antiserum prevented increased ACPC uptake of intestinal rings. Chronically elevated plasma glucagon levels may participate in the mechanism of the functional changes in the intestine in partial starvation.


Transport properties of membrane vesicles from Acholeplasma laidlawii. I. Isolation and general characteristics.

A new procedure for the isolation of membrane vesicles from Acholeplasma laidlawii cells is described. The membrane vesicles are completely free from contaminations of whole cells and cell debris and represent a homogeneous fraction as shown by electron microscopy, Ficoll density-gradient centrifugation, and titration on agar plate. Absence of cytoplasmic contaminations was confirmed by double-labelling of membranes with 3H-oleic acid and 14C-uridine, as well as by distribution of specific marker enzymes of membranes and cytoplasm. On the basis of light-scattering and electron microscopy, the vesicular nature of these membranes was established. The vesicles had the same orientation as intact cells (absence on membrane vesicles of ATPase and NADH dehydrogenase activities, localized in the inner surface of membrane). The respiratory activity of the membrane vesicles was low and was not stimulated by exogenous substrates, the respiratory chain of the vesicles being reduced and terminated by flavoproteins. The ability of membrane vesicles to take up carbohydrates was shown.


[Influence of long-term swimming training on the structure and enzyme activity of myosin in the rat myocardium (author's transl)].

Intermittent hemodynamic loading of the rat myocardium due to swimming training for several weeks leads to a significant increase in the specific ATPase activity of myosin. This enzymatic alteration of the myosin molecule is accompanied by changes in the stoichiometry of its light chains which are of great significance for the ATPase activity. The maximum shortening velocity of the unloaded myocardium (Vmax), estimated on the basis of afterloaded contractions, shows a slight increase as a result of the physical training. Since, on the other hand, the increase is not significant using the quick release technique, a close relationship between the specific ATPase activity and the augmented cross-sectional contractile capability cannot be proved in our experiments.


A membrane permeability test for the detection of cell surface antigens.

A convenient microtest is described which utilizes antibody-mediated release of [14C]nicotinamide ([14C]NA) from target cells for the detection of cell surface antigens. This test is considerably more sensitive and faster than the widely used 51Cr release test because most of the [14C]NA is rapidly released from the target cells in the initial phase of membrane permeability changes induced by activated complement, as distinct from the colloid osmotic phase of complement-mediated cytolysis.


In vitro proliferative response of BALB/c mouse spleen cells stimulated with trinitrophenylated syngeneic spleen cells.

Spleen cells from normal BALB/c mice showed in vitro proliferative response against hapten-conjugated syngeneic spleen cells. Trinitrophenylated (TNP) spleen cells were prepared by treating normal spleen cells with sodium 2,4,6-trinitrobenzenesulphonate (TNBS). Four-day cultures of TNP-labelled spleen cells incorporated 2.5-7.4 times more [3H]thymidine than similar cultures of untreated spleen cells. An obviously positive mixed lymphocyte reaction (MLR) by normal spleen cells against mitomycin C (MC) treated TNP-labeled syngeneic spleen cells was observed after 4 days of culture. The MLR to TNP-labelled syngeneic cells was inhibited in the presence of epsilon-TNP-L-lysine by 23-37%. The spleen cells from the mice injected intraperitoneally with TNP-labelled syngeneic spleen cells showed a higher MLR against TNP-labelled spleen cells than normal spleen cells. The sensitized spleen cells also showed an increased response to MC-treated spleen cells. These results suggest that normal spleen cells include cells which can recognize the hapten and new antigenic determinants introduced into syngeneic spleen by chemical modification.


Case detection rates for Central Burma (1962-1972).

A short description of the leprosy control program in Burmpa is given and the decrease of the case detection rates during thhe period 1962-1972 are presented to show the effects of control measures in the program emphasizing the importance of early case detection through annual examination of household contacts and school children, regular treatment and health education.


[Severe forms of acne with unusual clinical courses].

In two cases we could demonstrate the complications, which might appear in the clinical course of severe forms of juvenile acne. Our first case deals with a young male patient with leukaemic reaction in his peripheral blood and bone-marrow (sternal puncture) and excessively raised erythrocyte-sedimentation-rate, who developed bilateral ileo-sacral-fusion arthritis. The second case dealt with a young female patient with spondylitis and an enormously raised erythrocyte-sedimentation-rate.


[Cytochemical observations of hemocytes in decapod brachyuran crustacea (author's transl)].

Hemocytes from two species of crabs, Eriocheir sinensis and Carcinus maenas, have been submitted to different cytochemical reactions in order to bring out the nature of granules' content, either by specific coloration or be enzymatic digestion. Light as well as electron microscopy has been used. The granules appeared to be made essentially of basic proteins, rich in arginin and disulfide bridges but poor in tyrosin. These proteins are linked with nonglycogenic carbohydrates. Glycogen deposits of varied size are restricted to the cytoplasm in all the different cell-types but have never been detected inside the granules themselves. Lipids are poorly represented. The bulk of the granule material is made of neutral mucopolysaccharides although minute amounts of acid mucosubstances could be found in some instances.


[Applied phoniatry. III. Juvenile and hormonal dysphonias (author's transl)].

Juvenile and hormonal dysphonias are classified according to organic and functional etiologies. The clinical characteristics of each are described by emphasizing differential diagnostic and therapeutic aspects.


Physiological suppression of a transport defect in Escherichia coli mutants deficient in Ca2+, Mg2+-stimulated adenosine triphosphatase.

Transport properties of membrane vesicles isolated from two adenosine triphosphatase-deficient mutants of Escherichia coli, NR70 and DL54, were compared with those of vesicles prepared from the corresponding parental strains. As reported previously (Rosen, 1973; Altendorf et al., 1974), vesicles prepared from these mutants grown under aerobic conditions exhibited defective amino acid transport, and activity was restored after treatment with dicyclohexylcarbodiimide. In sharp contrast, however, vesicles isolated from the same mutants grown anaerobically in the presence of nitrate exhibited completely normal transport activity when assayed under either anaerobic or aerobic conditions. Suppression of the transport defect was not due to the manner by which the vesicles were prepared, and the adenosine triphosphatase deficiency was not ameliorated by anaerobic growth in the presence of nitrite. Finally, the transport activity of vesicles prepared from the mutants grown under aerobic conditions was relatively resistant to the effect of 1.0 M guanidine hydrochloride extraction, whereas the activity of vesicles prepared from mutants grown anaerobically was totally refractory to the effect of the chaotrope.


Anaerobic energy-yielding reaction associated with transhydrogenation from glycerol 3-phosphate to fumarate by an Escherichia coli system.

A particulate subcellular fraction from Escherichia coli K-12 induced in anaerobic sn-glycerol 3-phosphate (G3P) dehydrogenase and fumarate reductase can catalyze under anaerobic conditions the transfer of hydrogens from G3P to fumarate, with attendant generation of high-energy phosphate. The phsophorylation process is more sensitive than the transhydrogenation process to inhibition by the detergent Triton X-100. The same is true with respect to sensitivity to sodium azide, carbonyl cyanide m-chlorophenylhydrazone and N,N'-dicyclohexylcarbodiimide. Such a preparation derived from cells with beta-galactoside permease can accumulate thiomethyl beta-D-galactoside anaerobically, and the accumulation can be stimulated twofold by adding G3P and fumarate. Mutants lacking the membrane-associated Mg2+-dependent adenosine triphosphatase cannot grow anaerobically on glycerol with fumarate as the hydrogen acceptor, although they can grow aerobically on glycerol alone.


Electron transport chain from glycerol 3-phosphate to nitrate in Escherichia coli.

It is known that in Escherichia coli two dehydrogenases of the flavoprotein kind can participate in the transfer of hydrogens from sn-glycerol 3-phosphate (G3P) to nitrate and that possession of either enzyme is sufficient to permit anaerobic growth on glycerol as carbon source and nitrate as hydrogen acceptor. Results from this study show that under such a growth condition a protein with light-absorption characteristics of cytochrome b1 is induced. If G3P, nitrate, and adenosine diphosphate are added anaerobically to a particulate fraction prepared from these cells, four reactions can be detected: (i) the reduction of the cytochrome b1-like protein, (ii) the formation of dihydroxyacetone phosphate (DHAP), (iii) the formation of nitrite, and (iv) the generation of adenosine 5'-triphosphate (ATP). The anaerobic G3P dehydrogenase system can yield an ATP-DHAP (or ATP-nitrite) molar ratio of about 0.2, whereas the aerobic G3P dehydrogenase system can yield a corresponding ratio of about 0.3. The hydrogen transfer activity is sensitive to respiratory inhibitors such as cyanide, Rotenone, and 2-heptyl-4-hydroxyquinoline-N-oxide.


Linkage of six genes in Chlamydomonas reinhardtii and the construction of linkage test strains.

Linkage relationships were determined for six genes in Chlamydomonas reinhardtii: pyr, act, can-la, ey, y-1, and nic-7. Four of these were combined with five other genes to form linkage test strains.


Transport of sugars and amino acids in bacteria. XIV. Preferential inhibition of oxidase activities and active transport reactions for amino acids by azidebenzenes.

4-Methylazidebenzene and various azidebenzene derivatives were prepared, and the effects of these compounds on oxidase activities and active transport reactions for amino acids in Escherichia coli cells were studied. Azidebenzenes inhibited succinate oxidation by intact cells preferentially to  glycerol oxidation. However, the azidebenzenes could not inhibit succinate oxidation which was not coupled to phosphorylation. The compounds inhibited succinate driven proline uptake much more strongly than isoleucine uptake. Unlike sodium azide and diphenyl phosphorazidate, azidebenzenes did not inhibit membrane-bound, Mg2+-requiring ATPase [EC 3.6.1.3] of E. coli. Reactivities of various azide compounds in the mechanism of inhibition for energy transducing and energy transforming reactions were discussed briefly.


The amounts of adenosine di- and triphosphates bound to H-meromyosin and the adenosinetriphosphatase activity of the H-meromyosin-F-actin-relaxing protein system in the presence and absence of calcium ions. The physiological functions of the two routes of myosin adenosinetriphosphatase in muscle contraction.

The rates of the ATPase [EC 3.6.1.3] reaction of the H-meromyosin-F-actin-relaxing protein system were measured in 2 mM MgCl2, 50mM KC1, and 10mM Tris-HC1 at pH 7.8 and 20 degrees in the presence and absence of 0.05-0.1 mM Ca2+ ions. The concentrations of H-meromyosin (HMM) and the F-actin-relaxing protein (F-A-PR) complex were 3.4 and 3 mg/ml, respectively, and the ATPase reaction was coupled with 4 mg/ml of pyruvate kinase [EC 2.7.1.40] and 1 or 20 mM phosphoenolpyruvate to regenerate ATP. The amount of ADP bound to HMM during the ATPase reaction was determined by measuring the amount of ADP remaining in the reaction mixture. The amount of ATP bound to HMM was determined by subtracting the amount of bound ADP from the total amount of nucleotides bound to HMM, which was measured by a rapid flow-dialysis method. The following results were obtained. 1. The ATPase activity of the HMM-F-A-RP system increased linearly with increase in the amount of ATP added, and was independent of the presence of 0.05 mM Ca2+, when the amount of ATP added was less than 1 mole/mole of HMM. In the presence of 0.05 mM Ca2+, the ATPase activity reached a maximal level when 1.2-1.5 mole of ATP was added per mole of HMM, and maintained this level even at 3 moles of added ATP/mole of HMM. In the presence of 3mM EGTA, the ATPase activity decreased with increase in the amount of ATP added, from 1.5 to 3 moles of ATP/mole of HMM, and reached the level of the HMM ATPase reaction at 3 moles of added ATP/mole of HMM. Similar results were observed when the concentration of HMM was maintained at 3.4 mg/ml and the concentration of the F-A-RP complex was decreased from 3 to 1 or 0.5 mg/ml.


ATPase activity and filament formation of partially purified myosin from leucocytes.

Myosin was isolated from leucocytes in horse arterial blood by the same procedures used for the isolation of myosin from skeletal muscle. The Ca2+-, EDTA-, and Mg2+-ATPase [EC 3.6.1.3] activities of the protein was 0.148, 0.147, and 0.001 mumoles/min/mg, respectively, in 0.5 M KCl at pH 7.0 and 25 degrees. The Ca2+-ATPase activity decreased with decrease in the ionic strength. No difference was found between leucocyte myosin and skeletal myosin in the pH profiles of Ca2+- and EDTA-ATPases. The rate and amount of the initial burst of Pi liberation of leucocyte myosin were 0.002 mumoles/min/mg and 0.83 moles/4.8 X 10(5)g,  respectively. Leucocyte myosin aggregated into filaments of 0.3 mum length and 150 A diameter, which had a bare shaft and irregular projections. At high ionic strength, the protein bound to skeletal muscle F-actin to form a complex with the characteristic arrowhead structure.


Energy transduction in Escherichia coli. The role of the Mg2+ATPase.

Inverted membrane vesicles from strain 7, a wild type Escherichia coli K12 strain, actively transport calcium with energy supplied either by respiration or by ATP. These vesicles also have energy-linked quenching of quinacrine fluorescence. Membranes of strain 7, depleted of Mg2+ATPase by EDTA treatment, lack both activities. Membrane vesicles from strain NR70, a mutant lacking the Mg2+ATPase, show neither calcium transport nor energy-linked fluorescence quenching. Neither EDTA treatment nor genetic loss of the Mg2+atpase causes a reduction in respiration. Purified Mg2+ATPase from strain 7 can bind to EDTA-treated membrane vesicles from either strain 7 or NR70. This binding restored both calcium transport and fluorescence quenching, driven either by respiration or by ATP. Dicyclohexylcarbodiimide treatment mimics the effect of the Mg2+ATPase in the case of respiration-driven reactions. Treatment with EDTA, while not essential for the binding of the Mg2+ATPase to membrane vesicles of NR70, produced better restoration of both activities. The rate of restoration of fluorescence quenching showed a time lag which may indicate that binding of the Mg2+ATPase is a relatively slow process. Antiserum prepared against the Mg2+ATPase inhibited the quenching of quinacrine fluorescence when driven by ATP but not when driven by respiration. Addition of antiserum prior to addition of Mg2+ATPase prevented the restoration of fluorescence quenching, whether driven by respiration or ATP. These results clearly show that MG2+ATPase has an important role not only in catalyzing ATP synthesis and hydrolysis but also in maintaining the energized membrane state.


Regulation of mitochondrial biogenesis: enzymatic changes in cytochrome-deficient yeast mutants requiring delta-aminolevulinic acid.

Yeast cells almost completely deficient in all cytochromes were obtained by introducing two defective nuclear genes, cyd1 and cyc4, into the same haploid strain. The action of the two mutant genes is synergistic, since either gene acting singly results in only partial cytochrome deficiency. Normal synthesis of all cytochromes can be restored in the double mutant by adding delta-aminolevulinic acid to the growth medium. The optimum concentration of delta-aminolevulinate for restoration of cytochrome synthesis is about 40 muM; when higher concentrations are used, synthesis of cytochromes is partially suppressed, particularly that of cytochrome a.a3. Growth yield of the double mutant is stimulated by ergosterol and Tween 80, a source of unsaturated fatty acid. Methionine stimulates further. None of these nutrients is required for growth when sufficient delta-aminolevulinic acid is present in the growth medium. With respect to nutritional responses, the single-gene, cytochrome-deficient mutant, ole3, behaves like the double mutant. The frequency of the p-mutation in the double mutant grown in the absence of ergosterol, Tween 80, and delta-aminolevulinic acid is at least 15%. The frequency can be reduced to less than 1% by either delta-aminolevulinic acid or Tween 80. Ergosterol alone does not decrease the p- frequency. The ole3 mutant does not exhibit increased p-frequency under similar conditions of unsaturated fatty acid deficiency.


Studies of asymmetry in the three-dimensional structure of lobster D-glyceraldehyde-3-phosphate dehydrogenase.

An improved electron density map of lobster holo-D-glyceraldehyde-3-phosphate dehydrogenase has been computed to 2.9 A resolution based on two heavy atom isomorphous derivatives. This has been averaged only over the Q molecular 2-fold axis, which is known to be exact in the human holoenzyme. The map showed possible asymmetry between the subunits in which the active centers are closely related across the R axis (that is, between the red and green or between the yellow and blue subunits). A difference map between the electron density of citrate and sulfate-soaked crystals gave further evidence for possible asymmetry. The major differences of electron density between R axis-related subunits appear around the active center and suggest the following interpretations. 1. The conformation of the adenine about the glycosidic bond is the more frequently observed anti with a C-2' endo conformation for the ribose ring in the red and yellow subunits, but is probably syn with a C-3' endo conformation in the green and blue subunits.2. The adenine ribose has its 3'-hydroxyl group hydrogen-bonded to a main chain carbonyl group in the red and yellow subunits but not in the green and blue subunits, as a consequence of the differing ribose conformations. 3. Cysteine-149 is more closely associated with histidine-176 in the green and blue subunits, and appears nearer the nicotinamide in the red and yellow subunits.


Increase in dissociation rate constants of cardiotonic steroid-brain (Na+ + K+)-ATPase complexes by reduction of the unsaturated lactone.

Several cardiotonic steroids have been modified by reduction of the unsaturated lactone and their interactions with the sodium- and potassium-activated ATPase ((Na+ + K+)-ATPase) have been investigated. Reduction of the unsaturated lactone results in a decrease in binding affinity due primarily to an increase in the dissociation rate constant concomitant with a decrease in the activation free energy of dissociation. This decrease in activation free energy is about 2 to 4 kcal, which is approximately equal to the energy of one hydrogen bond. It is suggested that the increase in dissociation rate due to reduction of the unsaturated lactone may make possible the use of these compounds as affinity ligands for purification of the (Na+ + K+)-ATPase or an ouabain-binding fragment.


Interaction of inhibitors with muscle phosphofructokinase.

The interaction of several inhibitors with muscle phosphofructokinase has been studied by both equilibrium binding measurements and kinetic analysis. At low concentrations of citrate a maximum of 1 mol is bound per mol of enzyme protomer. Tight binding requires MgATP and very weak binding is observed in the absence of either magnesium ion or ATP. ITP at low concentrations cannot replace ATP. In the presence of MgATP and at pH 7.0, the dissociation constant for the enzyme-citrate complex is 20 muM. At 50 muM citrate and excess magnesium ion, the concentration of ATP required to give half-maximal binding of citrate is approximately 3 muM . Both P-enolpyruvate and 3-P-glycerate compete for the binding of citrate and the estimated Ki values are 480 and 52 muM, respectively. Creatine-P, another inhibitor of muscle phosphofructokinase, does not compete with the binding of citrate. Measurement of the equilibrium binding of ATP shows that citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P all increase the affinity of enzyme for MgATP with the concentration required to give an effect increasing in the order given. In kinetic studies, citrate, 3-P-glycerate and P-enolpyruvate each act synergistically with ATP to inhibit the phosphofructokinase reaction. This is indicated by the observation that the three metabolites do not inhibit the enzyme with ITP as the phosphoryl donor and that they inhibit at ATP concentrations that are not themselves inhibitory. Furthermore, the sensitivity to the inhibitors increases with increasing ATP concentrations. Striking differences in the extent of inhibition can be seen by varying the order of addition of assay components. Preincubation of the enzyme with ATP and citrate, 3-P-glycerate, or P-enolpyruvate results in greater inhibition than when the inhibitor is added after the reaction is started with fructose-6-P. Furthermore, the inhibition is reversed partially 10 to 15 min after the addition of fructose-6-P. This phenomenon is particularly striking with creatine-P as the inhibitor. Very high concentrations of this inhibitor are required to show any effect if the inhibitor is added after fructose-6-P. These effects are interpreted as reflecting slow conformational changes between an active form with high affinity for fructose-6-P and an inactive, or less active, conformation that binds the inhibitors. Citrate, 3-P-glycerate, P-enolpyruvate, and creatine-P increase the rate of the phosphofructokinase at subsaturating concentrations of MgITP. The results indicate a common binding site on the enzyme for citrate, 3-P-glycerate, and P-enolpyruvate that is distinct from the ATP inhibitory site. An additional site (or sites) for creatine-P is indicated. All four inhibitors act synergistically with ATP by increasing the affinity of the enzyme for MgATP at an inhibitory site. The inhibitors appear also to increase the affinity of the catalytic nucleoside triphosphate site for substrate.


Energy transduction in Escherichia coli. Genetic alteration of a membrane polypeptide of the (Ca2+,Mg2+)-ATPase.

Recent genetic analyses of the membrane components involved in energy transduction in Escherichia coli have concentrated on the (Ca2+, Mg2+)-ATPase complex (EC 3.6.1.3). Many mutants have been described with altered biochemical properties and defects in energy-requiring processes such as oxidative phosphorylation, transhydrogenase activity, and active transport of several solutes. This report describes the isolation of a mutant strain of E. coli that is defective in several energy-requiring processes. The strain BG-31 was obtained by "localized mutagenesis" using phage P1c1. The mutation maps at approximately 73.5 min on the E. coli chromosome. Reversion and suppression analyses indicate that the defect is the result of a single amber mutation. This strain is unable to utilize succinate, D-lactate, or malate for growth. Mutant cells are unable to couple the energy derived from the hydrolysis of ATP to the active transport of proline, although coupling of energy derived from electron transport to solute transport appears normal when examined in both cells and isolated membrane vesicles. Isolated membranes of the mutant are unable to couple the energy derived from the hydrolysis of ATP to transhydrogenase activity while they can utilize the energy generated from electron transport to drive transhydrogenase activity. Extracts of strain BG-31 have normal levels of (Ca2+, Mg2+)-ATPase activity. The ATPase portion of the complex, bacterial F1 (BF1), is poorly attached to the membrane portion of the complex. In vitro reconstitution of transhydrogenase activity with stripped membrane fractions and crude preparations of BF1 localize the defect in strain BG-31 to the membrane portion of the complex. Analysis of membranes of the strain BG-31 by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrate the absence of a single polypeptide of molecular weight about 54,000 and the appearance of a new polypeptide of lower molecular weight, about 25,000. Analysis of a spontaneous revertant of BG-31 shows complete restoration of the parental phenotype including the gel patterns. The characterization of this mutant provides the first demonstration of the consequences of a structural gene mutation on a polypeptide in the membrane portion of the complex and represents the initial stages in what we hope will be the biochemical definition and functional characterization of this important energy-transducing system.


Studies on the fatty acid inactivation of phosphofructokinase.

Investigation of phosphofructokinase in normal and regenerating livers led to the discovery of an inactivating factor in the extracts of these livers. The inactivating factor was found to be a mixture of free fatty acids. The fatty acid compositions of the normal and regenerating livers are the same, but the concentrations of most of the fatty acids are at least 3 to 4 times higher in the latter. Inactivation of phosphofructokinase by palmitate and oleate was investigated using purified rabbit muscle enzyme. Incubation of the enzyme with palmitate (250 muM) or oleate (50 muM) resulted in rapid inactivation of the enzyme with biphasic curves. The concentrations of oleate and palmitate required to produce 50% inactivation of the enzyme were 35 muM and 75 muM, respectively. Fructose-6-P (0.5 mM), MgATP, (1 mM), fructose-1,6-P2 (1 mM), AMP (1 mM), and cyclic adenosine 3':5'-monophosphate (20 muM) protected the enzyme against inactivation when these metabolites were incubated with the enzyme before the addition of fatty acid. Bovine serum albumin (100 muM) and beta-cyclodextrin (0.25 mM) also protected the enzyme against the inactivation. However, if the enzyme was inactivated by fatty acid, subsequent addition of the above metabolites or bovine serum albumin did not reactivate the enzyme. Binding studies with [3H]oleate revealed at least three types of binding sites. The first site binds 2 to 4 mol of oleate/mol of enzyme. Oleate binding to this site did not seem to affect the enzyme activity. The second binding site binds 5 to 15 mol of oleate/mol of enzyme resulting in complete loss of the activity. This is followed by an increase in oleate binding to the third site of the enzyme. Sucrose density gradient centrifugation of oleate-inactivated enzyme indicated that the enzyme dissociated to the dimeric form. Similarly, centrifugation of [3H]oleate-treated enzyme revealed that all polymeric forms of phosphofructokinase bound approximately 6 to 8 mol of oleate/mol of enzyme. In the presence of fructose-6-P, oleate is bound to the polymers to a lesser degree and therefore protects against the fatty acid inactivation. Various polymers which are cross-linked with dimethylsuberimidate are also inhibited by oleate.


Synthesis of myosin heavy and light chains in muscle cultures.

The weight ratio of myosin/actin, the myosin heavy chain content as the percentage of total protein (wt/wt), and the kinds of myosin light chains were determined in (a) standard muscle cultures, (b) pure myotube cultures, and (c) fibroblast cultures. Cells for these cultures were obtained from the breast of 11-day chick embryos. Standard cultures contain, in addition to myotubes, large numbers of replicating mononucleated cells. By killing these replicating cells with cytosine arabinoside, pure myotube cultures were obtained. The myosin/actin ratio (wt/wt) for pure myotube, standard muscle, and fibroblast cultures average 3.1, 1.9, and 1.1 respectively. By day 7, myosin in myotube cultures represents a minimum of 7% of the total protein, but about 3% in standard cultures and less than 1.5% in fibroblasts cultures. Myosin from standard cultures contains light chain LC1, LC2, and LC3, with a relative stoichiometry of the molarity of 1.0:1.9:0.5 and mol wt of 25,000, 18,000 and 16,000 daltons, identical to those in adult fast muscle. Myosin from pure myotubes exhibits light chains LC1 and LC2, with a molar ratio of 1.5:1.6. Myosin from fibroblast cultures possesses two light chains with a stoichiometry of 1.8:1.8 and mol wt of 20,000 and 16,000 daltons. Clearly, the faster migrating light chain, LC3, found in standard cultures is synthesized not by the myotubes but ty the mononucleated cells. In myotubes, both the assembly of the sarcomeres and the interaction between thick and thin filaments required for spontaneous contraction occur in the absence of light chain LC3. One set of structural genes for the myosin light and heavy chains appears to be active in mononucleated cells, whereas another set appears to be active in multinucleated myotubes.


Uninfected anastomotic false aneurysms following arterial reconstruction with prosthetic grafts.

Thirteen men, aged 49 to 76 years, average 60, were treated at the Little Rock, Arkansas Veterans Administration Hospital between January 1, 1969 and January 1, 1974 for 1-3 false aneurysms, a total of 18, following prosthetic arterial grafting. Most (ten patients, 15 aneurysms) had aorto-femoral bifurcation grafts for Leriche syndrome. Dacron prostheses were employed in ten, Teflon in three. The interval between the initial operation and the appearance of the aneurysms averaged six years. The end-to-side femoral anastomosis broke down in all ten patients with Leriche syndrome. Six of the eight unilateral aneurysms were on the right side, two had bilateral aneurysms and one patient had three. Five patients died some months or years after repair from vascular accidents. Three other patients required amputations sometime later because their grafts clotted. Silk sutures were employed in only two patients. Plastic sutures in the others were found to have pulled out of the recipient vessels. The aneurysms were all uninfected and were repaired by local stitching in four and the addition of further plastic grafting in nine. We conclude that false aneurysms occur with plastic sutures and they relate primarily to disruption of the end-to-side femoral artery attachment from the stresses of movement at the hip joint.


Proteoglycans in primate arteries. II. Synthesis and secretion of glycosaminoglycans by arterial smooth muscle cells in culture.

Glycosaminoglycan synthesis and secretion by primate arterial smooth muscle have been examined in cell culture. Mass cultures of diploid primate arterial smooth muscle cells were either double labeled with [35S]sulfate and [3H]acetate or single labeled with [3H]glucosamine for 24 h and glycosaminoglycans were extracted and isolated from the culture medium. Incorporation of labeled precursors into glycosaminoglycan was maximal during stationary phase of smooth muscle cell growth in culture and reduced, but not eliminated during logarithmic growth. The glycosaminoglycans synthesized and secreted into the culture medium were characterized by differential susceptibility to glycosaminoglycan-degradative enzymes and by cellulose acetate electrophoresis. Both assay procedures indicate that cultured primate arterial smooth muscle cells synthesize principally dermatan sulfate (60%-80% of total), chondroitin sulfate A and/or C (10%-20%of total) and little or no hyaluronic acid (0%-5% of total). This pattern of glycosaminoglycan formation differed significantly from that exhibited by isologous skin fibroblasts cultured under identical conditions. Dermal fibroblasts synthesize and secrete primarily hyaluronic acid (50%-60% of total) with lesser amounts of dermatan sulfate (10%-20% of total) and chondroitin sulfate A and/or C (10%-20% of total). These results indicate that differences exist in proteoglycan metabolism between these two connective tissue-producing cells in vitro, and suggest that the observed pattern of in vitro glycosaminoglycan synthesis by primate arterial smooth muscle cells may be characteristic for this cell type and not a general response to conditions of cell culture.


Incorporation of thymidine and iodeoxyuridine in mammalian cells in vitro.

The incorporation of labelled thymidine (dT) and iododeoxyuridine (IdU) into DNA was studied with tissue culture cells and with normal mouse cells in vitro. The rates of incorporation and the ratio dT/IdU incorporation both varied from one type of cell to another and from one suspending medium to another. Despite the known complexity of the regulation of DNA synthesis, the data for incorporation of exogenous dT and IdU could be fitted reasonably well by a model for single-step enzymic process. Deviations from the theoretical predictions were minimal in the presence of fluorodeoxyuridine.


The diagnosis of aorto-iliac stenosis: a comparison of thigh pressure measurement and femoral artery flow velocity profile.

Patients with vascular disease of the legs were studied by indirectly measuring thigh and ankle systolic pressures and recording the femoral artery flow profile before and after exercise with the aim of identifying those patients in whom significant aortoiliac stenosis may not have been diagnosed by angiography. A group of patients with aortoiliac stenosis or occlusion has been compared with a group with superficial femoral artery (SFA) occlusion and apparently normal controls. Resting thigh pressures were reduced in the group of patients with aortoiliac disease and with SFA occlusion. There was no significant change in thigh pressure with exercise in any group but ankle pressure fell with exercise in the patients with aortoiliac disease and with SFA occlusion. Femoral artery flow profiles provided better discrimination between the groups but six of the forty-two patients with SFA occlusions had abnormal tracings and a low thigh pressure suggesting they may have significant proximal disease.


Flaujeac trait. Deficiency of human plasma kininogen.

Flaujeac trait plasma resembled Hageman trait or Fletcher trait, in that the intrinsic coagulation pathway, plasma fibinolytic pathway, kinin-forming system, permeability factor of dilution (PF/dil) phenomenon were abnormal. The defect in each assay was reconstituted by afactor separable from Hageman factor or Fletcher factor. This substance was an alpha-globulin with an approximate mol wt of 170,000. Flaujeac plasma did not release a kinin upon incubation with kallikrein and was deficient in total kininogen antigen. Antiserum to kininogen inhibited the activity of the factor in solution. Flaufeac factor was identified as a kininogen of high molecular weight (HMW-kininogen). The mean total kininogen antigen in four children of the proposita was 51% (range 34-62%) of normal. A functional coagulation assay of HMW-kininogen in the children was 34% (range 23-55%). The results were consistent with autosomal recessive inheritance. The plasma pathways of intrinsic coagulation, fibrinolysis, kinin formation, and PF/dil generation are dependent upon HMW-kininogen. We believe this is the first demonstration of biological function for a kininogen apart from its role as a substrate for kallikreins.


Model phenylketonuria (PKU) in the albino rat: behaviroal, biochemical, and neuroanatomical effects.

Model phenylketonuria was induced in albino rats by injecting (at 1-20 days of age) and feeding (at 21-80 days) L-phenylalanine and D-L-para-chloro-phenylalanine. Behavioral testing on an eight-item battery occurred twice: first, whil the animals were either receiving the excess phenylalanines or not (original); and second, following a 90-day drug-free recovery period (retention). Results indicated drug-dependent deficits in learning and activity on both original and retention tests. Serum phenylalanine, serum tryptophan, and liver phenylalanine hydroxylase activity levels were positively correlated with the behavioral deficit. Clumped dense material in some myelin sheaths and associated degeneration of axons were found in experimental subjects.


Enzymes of carbohydrate metabolism in human epidermal tumors. 3. Enzymes between glucose and triose-phosphate.

The activities of five enzymes were determined by fluorometric micromethods in epidermal tumors (basal and squamous cell epithelioma, verruca seborrhoica and keratocanthoma) and compared to the activities in the adjoining, apparently normal epidermis. Most activities were higher in the tumoral tissues.


Denervation and reinnervation of fast and slow muscles. A histochemical study in rats.

A histochemical study, using myosin-adenosine triphosphatase activity at pH 9.4, was conducted in soleus and plantaris muscles of adult rats, after bilateral crushing of the sciatic nerve at the sciatic notch. The changes in fiber diameter and per cent composition of type I and type II fibers plus muscle weights were evaluated along the course of denervation-reinnervation curve at 1, 2, 3, 4 and 6 weeks postnerve crush. The study revealed that in the early denervation phase (up to 2 weeks postcrush) both the slow and fast muscles, soleus and plantaris, resepctively, atrophied similarly in muscle mass. Soleus increased in the number of type II fibers, which may be attributed to "disuse" effect. During the same period, the type I fibers of soleus atrophied as much or slightly more than the type II fibers; whereas the type II fibers of plantaris atrophied significantly more than the type I fibers, reflecting that the process of denervation, in its early stages, may affect the two fiber types differentially in the slow and fast muscles. It was deduced that the type I fibers of plantaris may be essentially different in the slow (soleus) and fast (plantaris) muscles under study. The onset of reinnervation, as determined by the increase in muscle weight and fiber diameter of the major fiber type, occurred in soleus and plantaris at 2 and 3 weeks postcrush, respectively, which confirms the earlier hypotheses that the slow muscles are reinnervated sooner than the fast muscles. It is suggested that the reinnervation of muscle after crush injury may be specific to the muscle type or its predominant fiber type.


Renal sodium-potassium adenosine triphosphatase. Optical localization and x-ray microanalysis.

The distribution of sodium-potassium adenosine triposphatase (Na-K-ATPase) activity in kidney sections has been studied by a method based on the hydrolysis of p-nitrophenyl phosphate in alkaline medium containing dimethyl sulfoxide. The products at each stage in the reaction sequence have been subjected to electron probe microanalysis. The initial product was identified as a mixture of KMgPO4 and Mg(PO4)2, and sequential analysis demonstrated the linearity of conversion of this product to a visible form. In human, rabbit and rat kidneys the distribution of activity was found to be essentially identical, with highest levels located in thick ascending limbs and distal convoluted tubules. The initial reaction was completely potassium dependent and was inhibited by ouabain in concentrations reflecting the relative sensitivity of microsomal Na-K-ATPase in each species. Measurement of initial product phosphorus by means of the electron probe is presented as a practical technique for direct quantitation of Na-K-ATPase activity in identified tubule segments.


Light microscopic localization of cytochemical reactions in epoxy-embedded material for electron microscopy.

Tissues from mice were fixed in 1.5% glutaraldehyde, treated for the ultrastructural localization of alkaline phosphatase or Mg++-dependent adenosine triphosphatase, post-fixed in osmium tetroxide, dehydrated and embedded in plastic for electron microscopy. The sites of reaction were visualized in 1-mu plastic sections counterstained with toluidine blue, using a phase contrast microscope. The data show a close correlation between the sites of reaction observed with the phase contrast microscope and the sites studied with the electron microscope. The use of this technique for the study of these phosphatases in normal and pathologic tissues is recommended in order to achieve a high degree of accuracy in selecting a portion of the tissue sample for electron microscopy and to obtain greater resolution in the localization of these enzymes with the light microscope.


Evaluation of the lymphocyte blastogenic response by rapid flow analysis.

The data presented show that the blastogenic response of human peripheral lymphocytes to mitogens and specific antigens can be easily evaluated with rapid flow analysis of mithramycin stained cells. The precision, reproducibility and rapidity of the method make it highly suitable to study lymphocyte response to antigens in patient populations.


Lentigo senilis and its evolutions.

Histologic studies have shown the evolution of lentigo senilis and have established the relationship between lentigo senilis, solitary lichen planus-like keratosis, and the reticulated form of seborrheic verruca. Histologic differentiation can be made between lentigo senilis and the early light-brown stage of lentigo maligna.


Quantification of bacteria in isolated pilosebaceous follicles in normal skin.

A technique for quantitating bacteria in isolated pilosebaceous follicles is described. This involves microdissection of the follicles from biopsies of skin, using the method of chemical pretreatment of skin to facilitate the separation of the epidermis and epidermal appendages from the dermis. The aerobic cocci and anaerobic diphtheroids in pilosebaceous follicles in 66 biopsies of scalp and 48 biopsies of skin of the upper back were quantitated using this technique. On the back, aerobic staphylococci were very sparse in normal follicles, indicating that their primary habitat on the skin must be on the skin surface rather than within follicles. Of 138 isolated follicles from skin of the upper back, 94 contained no aerobic cocci. Anaerobic organisms were present in high numbers within normal follicles. The geometric mean density of anaerobes in 138 isolated follicles from skin of the upper back was 3.8 X 10(4) diphtheroids per follicle. Eighty-eight follicles contained more than 10(4) anaerobic diphtheroids. Using data from scalp biopsies we found that there was a correlation between the weight of sebaceous glands and the density of anaerobes within the follicles attached to these glands (coefficient of correlation = 0.6).


Comparison of trimethoprim-sulfamethoxazole and amoxicillin in therapy of chloramphenicol-resistant and chloramphenicol-sensitive typhoid fever.

The efficacy of orally administered trimethoprim-sulfamethoxazole was compared with that of oral amoxicillin in therapy of typhoid fever due to both epidemic chloramphenicol-resistant and endemic chloramphenicol-sensitive Salmonella typhi. Both drug regimens were effective and of comparable value in treatment of chloramphenicol-resistant infections, as measured by duration of fever (124 hr and 115 hr, respectively) and duration of bacteremia (1.0 and 0.4 days, respectively). Trimethoprim-sulfamethoxazole therapy of infections due to chloramphenicol-sensitive S. typhi resulted in more rapid lysis of fever than did amoxicillin therapy. Trimethoprim and sulfamethoxazole were not synergistic in vitro against the chloramphenicol-resistant strain of S. typhi, and the role of sulfamethoxazole in treatment of such infections appears to be minimal. Oral administration of trimethoprim-sulfamethoxazole is effective therapy of chloramphenicol-resistant, and probably of ampicillin-amoxicillin-resistant, typhoid fever.


Effects on cancer patients of leukapheresis with the continuous-flow blood cell separator. II. Immunologic parameters in vitro.

The immunologic effects of leukapheresis on cancer patients and three other groups of donors using the continuous-flow blood cell separator are presented according to the protocol described in the preceding article. Transient declines were noted in per cent T-lymphocytes of some, but not all, leukapheresed normal donors and cancer patients. These declines were comparable to the small declines observed in sham donors and the fluctuations in per cent T-lymphocytes noted in individuals who did not undergo leukapheresis. There were no changes in lymphocyte-mediated cytotoxicity to a melanoma cell line in 2 of 4 melanoma patients, while values in 2 patients fell by about one-half within 4 hours and returned to preleukapheresis levels by 24 hours. Release of macrophage migration inhibition factor by lymphocytes from all 4 cancer patients and 4 normal donors, in whom this parameter was studied, fell transiently and returned to preleukaphresis levels within 24 hours. Blastogenic responses of lymphocytes from cancer patients and normal donors to phytohemagglutinin (PHA) and in one way mixed lymphocyte reactions increased in several individuals and decreased slightly in others. There appears to be no significant immunosuppressive effects (within the parameters studied) of a single leukapheresis for lymphocytes on the blood cell separator, since changes in lymphocyte parameters were relatively minor, transient, and variable and, where studied, paralleled fluctuations observed in sham donors and individuals not undergoing leukapheresis.


Dehydroepiandrosterone-induced thyrotrophin release during heat stress in rats.

Six groups each of 12 male albino rats were reared from day 21 of life at temperatures of 23, 34 or 37 degrees C. While the rats survived for unlimited periods at 23 and 34 degrees C, the animals reared at 37 degrees C succumbed within 5 days to heat stress. The latter group, when injected s.c. or i.p. with 50 mg dehydroepiandrosterone (DHA)/kg/day were no longer affected by the heat. During this treatment thyroid epithelial cell height doubled, colloid decreased by 20%, connective tissue did not change, the basic metabolic rate decreased by 10% and rectal temperatures of the treated rats increased with the ambient temperatures. Body weight increased only slightly, pituitary TSH decreased by 25%, serum TSH increased by over 50%, thyroxine and 125I uptake increased by 200%. The survival of the DHA-treated rats was apparently secured by blocking the hypothalamic thermoreceptors.


'Immobile' (im), a recessive lethal mutation of Xenopus laevis tadpoles.

'Immobile' (im) is a recessive lethal mutation discovered in the F3 of a Xenopus (Xenopus laevis laevis) originating from a mesodermal nucleus of a neurula transplanted into an enucleated egg. The im embryos do not contract after mechanical stimulation nor do they present any spontaneous contraction from the neurula stage onwards. Development proceeds normally during the first days after which deformation of the lower jaw and tail are observed. The im tadpoles die when normal controls are at the feeding stage. Nevous and muscular tissues are histologically normal in the mutant tadpoles; at advanced stages, however, an irregularity in the path of the myofibrils is observed which is especially conspicuous in the electron microscope. Cholinesterases and ATPase are present in the mutant muscles. Parabiosis and chimerae experiments have shown that parabionts and grafts behave according to their own genotype. Cultures of presumptive axial systems with or without ectoderm lead to the conclusion that, first of all, the abnormality is situated in the mesodermal cells and secondly that the first muscular contractions in normal Xenopus laevis are of myogenic origin. The banding pattern of the myofibrils is normal as was shown by obtaining contractions of glycerol extracted in myoblasts with ATP. It seems therefore that in this mutation, the abnormality is situated in the membraneous system of the muscular cell, sarcoplasmic reticulum and/or tubular system as is probably the case in the mdg mutation of the mouse.


Cell-mediated immunity: differential maturation of mixed leukocyte reaction and cell-mediated lympholysis.

While spleen cells from neonatal B10 (H-2b) are reactive (proliferate) in one-way mixed leukocyte culture, cell-mediated lympholysis reactivity does not arise until 7 days of age. When B10 cells are sensitized to B10.D2 (H-2d), cross-killing of third-party B10.BR (H-2k) target is always lower than the specific killing of B10.D2 targets and is not demonstrable until 27 days after birth.


Autologous stimulation of human lymphocyte subpopulation.

The background stimulation universally seen when lymphocytes are cultured in vitro has been shown to be markedly lowered by reducing the proportion of B lymphocytes. B-rich fractions of lymphocytes had extremely high background stimulation. It is concluded that stimulation of T cells, probably by autologous B cells, provides the most probable explanation for the findings described.