Role of phospholipid in the intermediate steps of the sodium-plus-potassium ion-dependent adenosine triphosphatase reaction.

The phosphorylation and dephosphorylation steps of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) reaction have been compared in 'normal', lipid-depleted and 'restored' membrane ATPase preparations. Partial lipid depletion was achieved by a single extraction with Lubrol W, and 'restoration' by adding pure phosphatidylserine. Gamma-32-P-labelled ATP was used for phosphorylation. The main findings were as follows. (1) Partial lipid depletion decreased but did not prevent Na-+-dependent phosphorylation, although it virtually abolished both Na-+-dependent and (Na-++K-+)-dependent ATPase activities. (2) 'Restoration' with phosphatidylserine produced an increment in phosphorylation that was the same in the presence and absence of added Na-+. (3) K-+ decreased the extent of Na-+-dependent phosphorylation of the depleted enzyme without producing a corresponding release of Pi. (4) K-+ rapidly decreased the extent of phosphorylation of the 'restored' enzyme to near-background value, with a concomitant release of Pi. (5) Na-+-dependent ATP hydrolysis was not restored. (6) The turnover of the 'restored' enzyme seemed to be higher than that of the 'normal' enzyme. The reaction sequence is discussed in relation to these results and the fact that the depleted enzyme retained about 50% of K-+-dependent phosphatase activity.


The effects of silymarin on experimental phalloidine poisoning.

The hepatoprotective action of silymarin, the active principle extracted from the fruit of Silybum marianum (L.) Gaertn., in animals (dogs, rabbits, rats, mice) intoxicated with phalloidine is evident, both after protective and curative treatment. A dose of 15 mg/kg of silymarin protects every animal when given 60 min before the toxin. When injected 10 mim after phalloidine, a dose of 100 mg/kg of silymarin again provides total protection. However, as the time span between administration of the toxic substance and start of treatment increases, so the efficacy of silymarin decreases; after 30 min its curative effect is negligible. The histochemical and histoenzymological studies show that during intoxication of the mice by phalloidine, silymarin inhibits the effect of the toxic substance and regulates the functions of the hepatocyte, when given either 60 min before or 10 min after phalloidine.


Langerhans cells in tissue cultures of guinea-pig epidermal cells.

Dispersed cell cultures of guinea-pig epidermis have been maintained for up to 36 days. The progress of these cultures has been studied by phase contrast microscopy, the adenosine triphosphatase reaction, and cinephotomicrography. Langerhans cells have been identified and their behaviour studied, particularly in relation to keratinocytes.


Contact dermatitis in animal feed mill workers.

Three cases of contact dermatitis due to additives in animal feed substances are described occurring in animal feed workers. There were two cases of sensitivity to ethoxyquin and one to halquinol.


Trichophyton rubrum infections according to age, anatomical distribution and sex.

Trichophyton rubrum caused 72.9% of the cases of dermatophytosis observed in a sample of caucasoids from Philadelphia. Tinea pedis was found in 84% of the female and male patients with dermatophytosis. Tinea manuum occurred with equal frequency in both sexes whereas tinea unguium was more prevalent in females. Tinea cruris occurred almost exclusively in males. Infections of two or more anatomical sites were observed more frequently in males. The data are compared with those reported by Rosman (1966) from a similar study done in Copenhagen.


Effects of intermittent and continuous hypoxia on the aortic wall in rabbits.

Male albino rabbits were exposed to intermittent nitrogen breathing every 30 sec for 5 sec, 15 min daily over a period of 3 weeks, and every 30 sec for 5 sec over a period of 10 hr. A third group of animals was exposed continuously to 8% oxygen breathing for 2 weeks. Neither intermittent not continuous hypoxia induced gross or microscopic alteration in the aorta. The effects of hypoxia upg which hypoxia was distributed than upon the total period or the degree of hypoxia. Exposure to hypoxia over a short period stimulated the synthesis of glycosaminoglycans, whereas distribution of the hypoxia over a longer period resulted in a reduction in the amount of glycosaminoglycans, probably secondary to an inhibition of the synthesis. Similarly, continuous exposure to 8% oxygen for a longer period decreased the aortic content of collagen. The alterations in the glycosaminoglycans and collagen induced by hypoxia may cause changes in the passage of macromolecules through the aortic wall. The changes may also influence the mechanical properties of the aorta and lead to impaired healing of vascular injury.


A fixed drug eruption due to paracetamol.

A fixed drug eruption due to paracetamol is reported. Over 10,000,000 prescriptions (EC10) for paracetamol are issued annually and rashes occurring as possible adverse reactions to this drug are reported fairly frequently to the Committee on Safety of Medicines (Committee on Safety of Medicines-personal communication). These include urticaria, angioneurotic oedema, purpura, morbilliform and scarlatiniform rashes, erythema nodosum, eczema, alopecia and nail changes but in many of these cases the cause-effect relationship is unproven. One strongly suspected case (Savin, 1970) was reported from St John's Hospital. This was a patient who developed a fixed drug rash after taking a chlormezanone-paracetamol combination. However, no other reports of a fixed drug rash which was proved to be due to parcetamol have been found.


Tylosis following post-corrosive stricture of the oesophagus.

A case of tylosis following corrosive stricture of the oesophagus in a male of 26 years is recorded. It is suggested that there is probably a connection between the state of the oesophagus and the state of palms and soles and that an oesophageal abnormality may precede tylosis of the late onset type.


Psoriasiform napkin dermatitis-a follow-up study.

One hundred and twenty-three children who had napkin dermatitis, with or without a secondary sensitization eruption, in infancy were reviewed 5-13 years later. Of the seventy-one who had a predominantly psoriasiform secondary eruption, twelve (17%) had psoriasis at review-three (4%) had atopic eczema. None of the forty treated for a predominantly seborrhoeic secondary eruption had psoriasis at review-15 (37%) had atopic eczema. The psoriasiform group had the highest incidence of psoriasis and the lowest incidence of atopy among first degree relatives. The converse incidence was found in the seborrhoeic group. It is suggested that infants who develop psoriasiform napkin dermatitis have a psoriatic diathesis.


Nickel dermatitis hazards from prostheses. In vivo and in vitro solubilization studies.

This report reviews data relating to the nickel hazard from implanted prostheses. It is shown that nickel is released from stainless steel prostheses by the action of sweat, blood and physiological saline solution. In laboratory animals, solubilized nickel isn the tissue near the implant.


Free energy and the kinetics of biochemical diagrams, including active transport.

In earlier papers on muscle contraction it was found very useful to relate the actual (not standard) free energy levels of the different states in the biochemical diagram of the myosin cross-bridge to the first-order rate constants governing transitions between these states and to the details of the conversion of ATP free energy into mechanical work. This same approach is applied here to other macromolecular biochemical systems, for example, carriers in active transport, and simple enzyme reactions. With the definition of free energy changes between states of diagram used here (and in the muscle papers), the rate constants of the diagram are firat order, the macromolecular transitions are effectively isomeric, the equilibrium constants are dimensionless, the free energy changes are directly related to first-order rate constant ratios, and the ratio of products of forward and backward rate constants around any cycle of the diagram is related to operational free energy changes (e.g. the in vivo free energy of ADP HYDROLYSIS). These general points are illustrated by means of particular arbitrary models, especially transport models. In contrast to the muscle case, the free energy conversion question in other biochemical systems can be handled at the less detailed, complete-cycle level rather than at the elementary transition level. There is a corresponding complete-cycle kinetics, with composite first-order rate constants for the different possible cycles (in both directions). An introductory stochastic treatment of cycle kinetics is included.


Correlation between the inhibition of the acto-heavy meromyosin ATPase and the binding of tropomyosin to F-actin: effects of Mg2+, KCl, troponin I, and troponin C.

When stoichiometric amounts of tropomyosin (TM) are bound to F-actin in the presence of 2 mM ATP, the MG2+-activated acto-heavy meromyosin (HMM) ATPase is inhibited by about 60% in 5 mM MgCl2-30 mM KCl. If the concentration of MgCl2 is reduced to 1 mM, the inhibition disappears because TM no longer binds to F-actin. Increasing the concentration of KCl to 100 mM restores both the binding and the inhibition. Thus, the binding of TM alone to F-actin causes significant inhibition of the ATPase provided that the HMM is saturated with ATP. (When the HMM is not saturated, TM activates the ATPase). When TM alone can bind stoichiometrically to F-actin, addition of troponin I (TN-I) increases the inhibition from 60% to about 85%, but the TM binding to F-actin is not affected. Under conditions such that TM alone neither inhibits the acto-HMM ATPase nor binds to F-actin, the inhibition caused by TN-I plus TM still approaches 100%. Direct binding studies under these conditions show that TN-I induces binding between TM and F-actin. A dual role for TN-I is proposed: first, TN-I can induce TM to bind to F-actin, causing inhibition of the ATPase; and second, TN-I can itself enhance the inhibition of the ATPase in a cooperative manner. The addition of TN-C in the absence of CA2+ has only a limited effect on the first role, but seems to be able to block completely the cooperative inhibition caused by TN-I such that the residual inhibition is a function only of the TM which remains bound.


ATP reversible Pi exchange and membrane phosphorylation in sarcoplasmic reticulum vesicles: activation by silver in the absence of a Ca2+ concentration gradient.

The activation of ATP reversible Pi exchange, normally associated with a Ca2+ concentration gradient in sarcoplasmic reticulum vesicles, can be obtained in "leaky" vesicles in 4-10 mM CaCl2. In the micromolar range, Ag+ activates the ATP reversible Pi exchange two- to fourfold. Similar concentrations of Ag+ promote a parallel inhibition of Ca2+- activated ATP hydrolysis and Ca2+ uptake in intact vesicles. Maximal inhibition of these activities by Ag+ leaves the Mg2+-dependent ATPase unaffected. No net synthesis of ATP was demonstrated in leaky vesicles. The effects of Ag+ depends on the protein concentration and persist after removal of Ag+ from the medium. Membrane phosphorylation from Pi or from ATP is respectively activated or inhibited by Ag+ in reciprocal fashion.


Studies on the interaction of adenine and nicotinamide ring systems in aqueous solution by high resolution nuclear magnetic resonance.

A Model system for NAD+ has been investigated using a paramagnetic transition metal ion as a probe. The well-known complexation of Mn2+ by adenine nucleotides was utilized to "label" adenosine 5'-diphosphate. A broadening effect on the 100-MHz proton nuclear magnetic resonance spectrum of N1-methylnicotinamide due to the adenine-metal ion complex was observed. It was found that the nicotinamide species showed no evidence for interaction with Mn2+ in the absence of the adenine nucleotide. These observations have led to the proposal that N1-methylnicotinamide associates with the adenine moiety of the adenine nucleotide-metal complex. This suggests a tendency of adenine and nicotinamide rings to interact in aqueous solution implying some tendency of the coenzyme NAD+ to occur in a folded or stacked conformation.


Bovine brain Na+,K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. K+-stimulated liberation of [32P] orthophosphate from [32P] phosphoenzyme and resolution of the dephosphorylation into two phases.

Dephosphorylation of [32P]phosphoenzyme of bovine brain Na+,K+-stimulated ATP phosphohydrolase (EC 3.6.1.3), labelled by [gamma-32P]ATP, was investigated at 21 degrees C by means of a rapid-mixing technique. On addition of a high concentration of KCl (10 mM) to [32P]phosphoenzyme at steady state in the presence of Mg2+ and Na+, very rapid dephosphorylation was obtained. Simultaneously, the amount of [32P]orthophosphate increased at about the same rate. It was concluded that this K+-stimulated dephosphorylation and liberation of [32P]orthophosphate from the [32P]phosphoenzyme was rapid enough to participate in the Na+,K+-stimulated hydrolysis of ATP. In order to study the dephosphorylation in absence of continuing 32P-labelling, excess unlabelled ATP or a chelator of Mg2+ was added. Simultaneous addition of a high concentration of KCl to the [32P]phosphoenzyme formed in the presence of Mg2+ and Na+ but in the absence of K+, resulted in an initial very rapid phase and a subsequent slower phase of dephosphorylation. With KCl also initially present in the incubation medium, only the slow phase was observed. The slow phase of dephosphorylation also seemed to be sufficiently rapid to participate in the Na+, K+-stimulated ATPase reaction. On addition of a high concentration of ADP (5 mM) to [32P]phosphoenzyme formed in the presence of Mg2+ and Na+, an initial comparatively rapid, and later slow phase of dephosphorylation were detected. This gave further support for different forms of phosphoenzyme. Approximate concentrations of these forms, in the absence and presence of KCl, were estimated by extrapolation and the turnover of these forms was calculated. The nature of the kinetically different components of phosphoenzyme and their role in the Na+, K+-stimulated ATPase reaction is discussed.


Bovine brain Na+, K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. Detection of a transient [32P]phosphoenzyme formed in the presence of potassium ions.

1. Conditions for binding of [gamma-32P]ATP to bovine brain Na+,K+-stimulated ATPase were investigated by the indirect technique of measuring the initial rate of 32P-labelling of the active site of the enzyme. 2. At 100 muM [gamma-32P]ATP in the presence of 3 mM MgCl2, approximately the same very high rate of formation of [32P]phosphoenzyme was obtained irrespective of whether [gamma-32P]ATP was added to the enzyme simultaneously with, or 70 ms in advance of the addition of NaCl. A comparatively slow rate of phosphorylation was obtained at 5 muM[gamma-32P]ATP without preincubation. However, on preincubation of the enzyme with 5 muM[gamma-32P]ATP a rate of formation of [32P]phosphoenzyme almost as rapid as at 100 muM[gamma-32P]ATP was observed. 3. A transient [32P]phosphoenzyme was discovered. It appeared in the presence of K+, under conditions which allowed extensive binding of [gamma-32P]-ATP. The amount of [gamma-32P]ATP that could be bound to the enzyme seemed to equal the amount of [32P] phosphorylatable sites. 4. The formation of the transient [32P] phosphoenzyme was inhibited by ADP. The transient [32P] phosphoenzyme was concluded mainly to represent the K+-insensitive and ADP-sensitive E1-32P. 5. When KCl was present in the enzyme solution before the addition of NaCl only a comparatively slow rate of phosphorylation was observed. On preincubation of the enzyme with [gamma-32]ATP an increase in the rate of formation of [32P] phosphoenzyme was obtained, but there was no transient [32P]-phosphoenzyme. The transient [32P]phosphoenzyme was, however, detected when the enzyme solution contained NaCl in addition to KCl and the phosphorylation was started by the addition of [gamma-32P]ATP.


Interaction of C1s and C4. A binding phenomenon.

Addition of enzymatically active 125-I-labeled C1s (the esterase which is part of the activated complex protein of serum designated as the first component of complement or C1) to purified C4 (the naturally occurring fourth component of human serum complement) results in binding of a portion of the C1s to C4 as indicated by sucrose density gradient ultracentrifugation. Demonstration of binding requires hemolytically active C4, but not enzymatically active C1s. The latter was demonstrated by using DFP inactivated C1s as well as fragments of C1s produced by prior protease treatment of the C1s. While treatment of C1s with proteases (human leukocyte lysosomal enzymes, trypsin or plasmin) resulted in progressive inactivation of the enzymatic activity, the decline in esteratic activity occurred at a much slower rate than the decline in functional activity (inactivation of C4 in free solution). The data lead to the probable conclusion that C1s contains an enzymatic (or esteratic) site in addition to a binding site. The latter might be important for positioning a large molecule, such as C4, in order to effect proteolytic cleavage at the proper bond and hence prepare C4 to participate in the complement sequence.


Thallium inhibition of ouabain-sensitive sodium transport and of the (Na+ plus K+)-ATPase in human erythrocytes.

The influence of Tl+ on Na+ transport and on the ATPase activity in human erythrocytes was studied. 0.1-1.0 mM Tl+ added to a K+-free medium inhibited the ouabain-sensitive self-exchange of Na+ and activated both the ouabain-sensitive 22Na outward transport and the transport related ATPase. 5-10mM external Tl+ caused inhibition of the ouabain-sensitive 22Na efflux as well as the (Na+ plus Tl+)-ATPase. Competition between the internal Na+ and rapidly penetrating thallous ions at the inner Na+-specific binding sites of the erythrocyte membrane could account for the inhibitory effect of Tl+. An increase of the internal Na+ concentration in erythrocytes or in ghosts protected the system against the inhibitory effect of high concentration of Tl+. A protective effect of Na+ was also demonstrated on the (Na+ plus Tl+)-ATPase of fragmented erythrocyte membranes studied at various Na+ and Tl+ concentrations.


Control over acid mucopolysaccharide production of cells in vitro.

Serum addition to stationary chich embryo fibroblasts in vitro causes a 300% increase in acid mucopolysaccharide production, this increase being linear both with time and concentration of serum added. At the same time, serum-stimulated production is reduced in cells which are actively dividing. The increase in acid mucopolysaccharide production caused by serum is in some manner dependent upon RNA synthesis, since the addition of actinomycin D reduces its effect by 50% within 4 h. A correlation between acid mucopolysaccharide production and chondroitin sulphotransferase activity (EC 2.8 2.5,3'-phosphoadenyl 5'-phosphosulphate: chondroitin sulphotransferase) is seen. Both increase to over 400% of control values 24 h after serum addition, and both are sensitive to the presence of actinomycin D. Serum control over production of these extracellular molecules is demonstrated and may be linked to the mitotic activity of the cell. It is proposed that this control by serum over acid mucopolysaccharide production can provide one mechanism for the cell to alter its external environment and thus affect events such as cell to cell interactions and hormonal signals necessary to cellular functions.


Plasmic degradation of human fibrinogen. IV. Identification of subunit chain remnants in fragment Y.

A method is presented for detection of cross-linking acceptor sites on fibrinogen chains, using monodansyl-cadaverine labeling in the presence of activated fibrin stabilizing factor, and polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Fluorescent gamma-chain monomers and dimers were produced at a considerably faster rate than the labeled alpha-chain derivative. Purified fragments X, Y and D were prepared all from the same plasmic digest of fibrinogen. Following incubation with fibrin stabilizing factor, thrombin and monodansyl-cadaverine, they were reduced with beta-mercaptoethanol and examined by sodium dodecyl sulfate/acrylamide electrophoresis. Three gamma-chains (mol. wts 49 000, 42 000 and 39 000) had reacted with dansyl-cadaverine while no alpha-chain remnant took up the label. Additional protein and carbohydrate staining further facilitated identification of the individual subunit chains. At least three critical peptide bonds, located on alpha, beta- and gamma-chain remnants, must be broken during conversion of fragment Y into D and E. Sequential cleavage results in heterogeneous appearance of reduced subunit chains. As a consequence, there exist several molecular entities of fragment Y, all of which may have the same molecular weight though they represent various products of progressive plasmic digestion. Our results are compatible with the model of asymmetric degradation of fibrinogen, according to which fragment X produces 1 mol of fragment E e and 2 mol of the monomeric fragment D.


Plasmic degradation of human fibrinogen. III. Molecular model of the plasmin-resistant disulfide knot in monomeric fragment D.

A mixture of fragments D, derived from fibrinogen by plasmic degradation, was S-reduced and carboxymethylated. Individual chains were separated by gel filtration on Sephadex G-100 and characterized by peptide mapping, N-terminal amino acid analysis, polyacrylamide electrophoresis in sodium dodecyl sulfate, and amino acid composition. It was demonstrated that all D species contain the same alpha- and beta-chain remnants, having mol. wts of 10 000 and 45 000, respectively. Their heterogeneity was shown to be caused by the gradual degradation of the gamma-chain at its C-terminal end. Denatured fragment D was further degraded with plasmin in the presence of 2 M urea. One beta- (mol. wt 17 000) and two gamma-fragments (mol. wts 5000 and 6000) were split from fragment D, in addition to non-characterized small peptides, leaving behind a plasmin-resistant core, designated as fragment d. Fragment d was in turn reduced and carboxymethylated, and the resulting constituent chains were isolated by chromatography on carboxymethyl-cellulose and Sephadex G-100. The reduced alpha-, beta- and gamma-chain remnants of fragment d were found to have been derived from the N-terminal portion of fragment D and have estimated mol. wts of 9000, 24 000 and 13 000, respectively. A tentative scheme for the conversion of an early fragment D into the core fragment d is proposed. Our results conclusively support the model of asymmetric degradation of fibrinogen, according to which 2 mol of monomeric fragment D are produced from 1 mol of fibrinogen.


Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells.

The K+ content and the K+ flux were measured in the cell lines ME2 and MF2 isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the seem K+ content and the same K+ steady state flux per unit of surface area. In ME2 cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K+ net movement. The (Na+ plus K+)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K+ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells. The K+ net flux is considerable increased when the intracellular K+ level is reduced after preincubation of the cells in a K+ -free medium. Thus, internal K+ seems to regulate the K+ influx.


Extraction and localization of a (Ca2+ and Mg2+)-stimulated ATPase in human erythrocyte spectrin.

(1) A water soluble  (Ca2+ plus Mg2+)-activated APTase has been extracted with 0.1 mM EDTA and 0.1 mM ATP from human erythrocyte membranes. (2) The specific activity of the extracted protein is increased 4- to 6-fold in comparison with untreated ghosts. (3) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [gamma-32P]ATP-labeled erythrocyte membranes shows that the (Ca2+ plus Mg2+)-activated ATPase is located in the "spectrin" region (Mr 220 000-240 000). The radioactivity of these high molecular peptide bands is decreased markedly after the extraction of the ATPase at low ionic strength.


Action of halothane on human erythrocytes. Mechanisms of cell lysis and production of sealed ghosts.

1. Suspension of human erythrocytes in halothane-saturated physiological media induces major cell deformation. 2. Release of halothane from erythrocyte suspensions after equilibrium with the anaesthetic produces complete hemolysis. 3. The membrane fragments isolated after halothane release are in the form of biconcave ghosts, impermeable to macromolecules (lactoperoxidase) and small molecules (ATP). 4. The protein composition of the membranes differs from that of ghosts produced by hypotonic lysis in the lack of components previously shown to be adsorbed at low ionic strength. 5. A hypothesis is presented explaining the action of halothane in terms of both its action on membranes and its capacity to modify water structure. 6. Halothane-induced hemolysis constitutes a simple method for the large-scale production of hemoglobin-depleted, sealed erythrocyte ghosts under physiological ionic conditions.


Biochemical effects of induced phenylketonuria in rats.

Phenylketonuria (PKU) was induced in rats by the combined feeding of 3 per cent excess phenylalanine and 0.12 per cent of p-chlorophenylalanine, an inhibitor of phenylalanine and tryptophan hydroxylases. Increased concentrations of phenylalanine and increased ratio of phenylalanine to tyrosine were demonstrated in blood from pregnant rats fed the experimental PKU diet from day 10 to 20 of pregnancy, in fetal blood and amniotic fluid of fetal animals from mothers fed the PKU diet, and in blood of rats fed the PKU diet for 28-30 days  beginning at 20-21 days of age. Both phenylpyruvic acid and orthohydroxyphenylacetic acid were excreted by rats fed the PKU diet, but neither were detected in urine in animals fed either excess phenylalanine or excess inhibitor alone. Reduced serotonin concentrations were found in brains of rats fed p-chlorophenylalanine, either alone or in combination with excess phenylalanine in the PKU diet. These biochemical changes in rats with induced PKU and the behavioral changes described earlier are similar to those of the human condition. The animal model should prove useful in searching for the mechanism of the disease.


Bone marrow and thymus transplantation in ataxia-telangiectasia.

Serum IgA concentrations were found to be normal in a boy with ataxia-telangiectasia and selective IgA deficiency several months after an infusion of bone marrow cells from an HL-A and MLR compatible normal sib. In addition, transient improvements were noted in both his in vivo and in vitro cellular immune responsiveness through the third month posttransplant. Despite this, no evidence of chimerism could be detected in karyotypic studies of his peripheral blood lymphocytes and bone marrow cells. No clinical or significant immunologic improvement was noted in his affected female sib following implantation of a fetal thymus or injection of transfer factor.


[Double-frequency oscillation in the  glycolytic system. Mathematical model].

A mathematical model describing the generation mechanism of double-frequency oscillations in the glycolytic system is proposed. Interaction of two connected glycolytic oscillation generators are put in the basis of this mechanism. It is assumed in the model that the first oscillation generator is formed due to product activation of phosphofructokinase (PFK) with adenosine diphosphate (ADP), while the second one is based on substrate inhibition of glyceraldehydephosphatededhydrogenase (GAPDH) with glyceraldehydephosphate (GAP).


Transfer factor therapy: evidence for nonspecificity.

Four patients were treated with multiple doses of dialyzed transfer factor in which one Wiskott-Aldrich patient with abnormal IgG monocyte receptors obtained benefit. All patients converted skin reactivity, and had improved in vitro lymphocyte responses to varying degrees. Three patients developed positive reactivity to allogeneic cells in MLC, and one patient developed DNCB reactivity. These latter two findings suggest transfer factor may act by inducing a nonspecific maturation of lymphocyte function. A possible mechanism for the action of transfer factor is discussed.


Ontogeny of cellular immunity in man.

The purpose of this study was to determine the precise developmental sequence of the acquisition of phytohemagglutinin (PHA) and allogenic cell responsiveness (MLR) in the human fetus. Reactivity was determined quantitatively by 3H-thymidine incorporation in various fetal lymphoid tissues. MLR reactivity was the earliest detectable response occurring first in liver lymphoid cells at 5 weeks fetal age. Reactivity to PHA then appeared in thymocytes at 10.5 weeks and at 14 weeks in the spleen and blood. MLR reactivity first appeared in the thymus at 12.5 weeks and in the spleen and blood at 14.5 weeks. Marrow lymphocytes failed to react up to 18 weeks. These results suggest that fetal thymus is immunocompetent by about 10.5 weeks and MLR responsiveness may be under nonthymic (hepatic) control during development.


Congenital and experimentally induced complement deficiency in rabbits.

Deficiency of the sixth component of complement in rabbits is an autosomal recessive defect which also renders C7-9 functionally unavailable. The function of C6-9 appears to be that of amplification of immunologic processes. Defective animals are associated with a subtle disadvantage. Normal rabbits injected with cobra-venom-anticomplementary-factor show primary C3-9 and secondary polymorphonuclear leukocyte and platelet functional deficiency. Animals treated with cobra venom showed striking deficits in manifestations of some immunologic functions. Thus the complement system plays a vital role in maintaining integrity of immunologic mechanisms.


Medical school libraries in the United States and Canada built between 1961 and 1971.

Twenty-four medical school libraries in the United States and Canada build between 1961 and 1971 were surveyed by means of questionnaires and visits. Results indicated that half of these libraries will have reached maximum functional capacity approximately six years after they were moved into their new quarters. Space for technical processing is generally much less than is required. Special features and examples of effective planning are described, and problems in arrangement, traffic patterns for people and materials, and the lack of logical expansion space are discussed. Comparisons are made with a similar survey of twenty medical school libraries made in 1961.


The failure of p-chlorophenylalanine to affect voluntary alcohol consumption in rats.

1 The effects of p-chlorophenylalanine (PCPA) administered orally and intraperitoneally on rat brain 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content were compared. The depletion of brain 5-HT and 5-HIAA following PCPA (316 mg/kg) injected intraperitoneally every third day was not significantly different from that following the administration of PCPA (316 mg/kg) by stomach tube on eight consecutive days. 2 Rats tested for alcohol preference before, during and after treatment with PCPA on two intraperitoneal dose regimens (either 316 mg/kg then 100 mg/kg four days later or 316 mg/kg three times at intervals of three days) showed no reduction in voluntary alcohol consumption. 3 The results indicated that depletion of brain 5-HT and 5-HIAA is not responsible for the reduction of voluntary alcohol intake which has been reported to follow chronic oral administration of PCPA to the rat (Myers & Veale, 1968). The possibility of a learned aversion to alcohol due to an association with PCPA administration is discussed.


Anabolic steroids in athelics:  crossover double-blind trial on weightlifters.

Thirteen experienced male weightlifters taking high-protein diets and regular exercise took part in a double-blind crossover trial of methandienone 10 or 25 mg/day to seeif the drug improved athletic performance.  Their improvemments were significantly greater on methandienone than on placebo; their body weights rose (though this seemed to be associated with water retention); and systolic blood pressure rose significantly.  Methandienone caused many side effects, and three men had to withdraw because of them.  All side effects disappeared after the drug was stopped.  Anabolic steroids are effective only when given combination with exercise and high-protein diet. We deprecate their use in athletics but can suggest no way of stopping it.


5-hydroxytryptamine in the central nervous system and sexual receptivity of female rhesus monkeys.

The role of 5-hydroxytryptamine (5-HT) in the control of sexual receptivity in female rhesus monkeys has been studied in 24 adult females paired with 6 adult males. p-Chlorophenylalanine (PCPA, 75 mg/kg or 100 mg/kg, every fourth day), a selective inhibitor of 5-HT, was found to reverse unreceptivity induced by adrenalectomy in ovariectomised, oestrogen-treated females. PCPA-treated females presented more frequently and initiated more sexual behaviour, or else they refused fewer of the male's attempts to mount. These effects were in turn reversed by 5-hydroxytryptophan (5-HTP, 20 mg/kg every second day), when this was given to PCPA-treated animals. In addition, 5-HTP given alone to ovariectomised oestrogen-treated females reduced their receptivity. Parallel biochemical experiments showed that PCPA in the doses used lowered the levels of 5-HT in the brain as measured by the levels of 5-hydroxyindole-3-acetic acid (5-HIAA) in the CSF, and that these were restored by 5-HTP. Both oestradiol benzoate (15 mug/day for 10 days) and testosterone propionate (250 mug/day or 400 mug/day for 10 days) lowered the turn-over rates of 5-HT in the brain (as measured by the probenecid test) in ovariectomised female monkeys. These effects of oestradiol on turnover were antagonised by progesterone (15 mg/day for 10 days, given with oestradiol). A substance other than an adrenal androgen has thus been found to reverse the effects of adrenalectomy on sexual receptivity in female monkeys. It is therefore possible that androgens regulate receptivity in female monkeys by modifying the activity of 5-HT-containing neural systems.


Hippocampal mediation of raphe lesion- and PCPA-induced hyperactivity in the rat.

These experiments provide a direct test of the hypothesis that median raphe lesion- or PCPA-induced hyperactivity in the rat is mediated specifically by the hippocampus. Previous studies had shown that a marked depletion of hippocampal serotonin accompanied median lesion-induced hyperactivity. In the present studies, aspiration of the anterodorsal hippocampus of adult male rats prior to median raphe lesions or PCPA administration abolished the ability of both of these treatments to produce locomotor hyperactivity in animals chronically housed in tilt cages. Control lesions of the overlying dorsal cortex and corpus callosum were ineffective in blocking the hyperactivity produced by these two treatments. The possibility that serotonin depletion-induced hyperactivity was dependent on the pituitary was excluded by the fact that PCPA effectively elevated the activity of hypophysectomized rats. These data indicate that serotonin depletion-induced hyperactivity in the rat is mediated by the hippocampus.


Monoaminergic mechanisms of stimulation-produced analgesia.

The roles played by the cerebral monoamines (dopamine, noradrenaline and serotonin) in stimulation-produced analgesia (SPA) have been investigated in the rat employing the tail flick test.  SPA was elicited through bipolar electrodes chronically implanted in the mesencephalic periaqeductal gray matter, an area previously shown to yield potent and reliable analgesic effects.  Four approaches were used to alter transmission in monoamine pathways.  (1) Depletion of monoamines by administration of tetrabenazine (TBZ), p-chlorophenylalanine (PCPA), alpha-methyl-para-tyrosine (AMPT), or disulfiram.  (2) Replacement of depleted monoamine stores by appropiate precursors (5-HTP or L-DOPA) in combination with a peripheral decarboxylase inhibitor.  (3) Potentiation of monoamine systems by administration of precursors to previously untreated animals or by administration of a dopamine receptor stimulator, apomorphine.  (4) Blockade of catecholamine receptors by haloperidol or of dopamine receptors by pimozide.  These four approaches yielded internally consistent results.  Depletion of all 3 monoamines (TBZ) led to a powerful inhibition of SPA.  Original levels of SPA were restored by injection of either 5-HTP or L-DOPA.  Specific depletion of serotonin (PCPA) caused a reduction in SPA, whereas elevation of serotonin levels (5-HTP) caused an increase in SPA.  Dopamine receptor blockade (pimozide) decreased SPA, whereas the precursor (L-DOPA) and a dopamine receptor stimulator (apomorphine) increased SPA.  On the other hand, selective depletion of noradrenaline (disulfiram) caused an increase in SPA; and at a time when noradrenaline levels are depressed and dopamine levels are elevated (AMPT + L-DOPA), SPA was seen to be particularly enhanced.  thus, dopamine and serotonin appear to facilitate SPA, whereas noradrenaline appears to inhibit it.  When a general catecholamine receptor blocker (haloperidol) was employed, SPA was diminished, suggesting that the influence of dopamine in SPA is greater than that of noradrenaline.  Most of the drugs used in this study significantly altered SPA at doses which left baseline tail flick latency unaffected.  It would appear, therefore, that SPA has a neural substrate at least partly independent of that underlying baseline pain responsiveness.  Consideration is given to various ascending and descending monoamine system as possible component paths in this neural substrate of SPA.  Finally, the present results are discussed in relation to studies by others on the site and mechanism of morphine's analgesic action.  Some striking parallels between SPA and morphine analgesia are noted.  These suggest the existence of a common pain-inhibitory system in the brain activated by morphine and by focal electrical stimulation.


Vitamin D -- induced changes in the renal membrane ATPase system.

Ca-dependent and Mg-dependent ATPase activity was found to be depressed significantly in renal membrane preparations from vitamin D-deficient as compared to normal rats. Administratiin of 2000 i.u. vitamin D2 to deficient rats 30 hours before sacrifice restored the membrane enzyme activity to normal. ATP binding by enzymes was also reduced in vitamin D deficiency and raised by repletion. Vitamin D may therefore be required for normal Ca-and Mg-dependent ATPase activity.


Characterization of developing antler cartilage matrix. I. Selected histochemical and enzymatic assessment.

Biopsy samples of the main beams and tines were obtained from the antlers of mature Rocky Mountain mule deer (Odocoileus hemionus hemionus) during the rapid phase of the antler grow-th cycle. The samples were studied using histochemical and enzymatic techniques for the demonstration of mucosubstances. The reserve mesenchyme contained chondroitin 4- and 6-sulfates, hyaluronic acid and sialic acid. During prechondroblastic and chondroblastic differentiation, some strongly sulfated mucopolysaccharides were complemented by low molecular weight or incompletely sulfated chondroitin 4- and 6-sulfates. During maturation of the cartilage strongly anionic mucopolysaccharides (possibly heparin, keratan and/or dermatan sulfates) were present in the general matrix and especially prominent along the capsular margins. Calcification foci, beginning initially in the mid-trabecular regions and eventually extending to the trabecular margins, were highly reactive for mucosubstances and were enzyme-resistant. This histochemical study establishes the cartilaginous nature of the developing deer antler, defines some of the mucosubstance constituents involved in the chondrogenic and calcification process, and confirms the utility of the antler as a model for chondrogenic and calcification studies.


Characterization of developing antler cartilage matrix, II, An ultrastructural study.

Cartilage from the main beams and tines of deer antler was examined with the electron microscope. The material studied included prechondroblastic, chondroblastic and chondrocytic matrices. Exdysial microfibrils (5-10 nm in diameter) were observed in the matrix of the prechondroblastic zone. These microfibrils and associated amorphous material were continuous with electron-dense material that probably represented extracellular units of collagen polymers. Matrix (proteoglycan) granules were first observed in the chondroblastic zone. They stained positively with colloidal iron and therefore probably represented proteinpolysaccharides. The matrix granules of the chondroxytic (unmineralized and mineralized) zone were twice the diameter of those in the chondroblastic zone. Matrix vesicles were present in all three stages of development. They were in contact with cellular extensions and also arose directly from cell membranes in the immature zones. As in somatic mineralizing cartilage, these vesicles served as the foci for early mineralization. The initial mineralization process was associated with the membrane of the vesicles.


Renal transport of organic acids and bases in genetically obese mice.

Accumulation of p-aminohippurate (PAH) and N-methylnicotinamide (NMN) by renal cortical slices was used to estimate transport capacity for organic anions and cations, respectively. In a previous study, renal organic anion transport appeared to be selectively depressed in animals rendered obese by high fat feeding. However, the effects of obesity could not be discretely separated from the effects of the 30% fat diet used to produce the obesity. Genetically obese hyperglycemic mice provided a model to determine the effect of obesity on renal transport systems without the complication due to diet. Accumulation of both PAH and NMN was depressed in renal cortical slices from genetically obese mice. Addition of plasma from thin or obese animals increased PAH accumulation by slices from thin animals. Accumulation by slices from obese animals was unaffected by addition of plasma. Oxygen consumption with acetate in the medium was less in kidneys from obese mice than kidneys from thin mice. Thus, in addition to inhibition of transport capacity, renal cortex of genetically obese mice has a biochemical defect that prevents response to stimulators. It is concluded that several renal functions are depressed in the genetically obese hyperglycemic mouse. Whether the depressed function results from the obesity or is concomitant with the gene for obesity is as yet undertermined.


Characteristics of Staphylococcus aureus associated with lysogenic conversion to loss of beta-hemolysin production.

Staphylococcus aureus strains 7-8 and 57 that produce beta-hemolysin but not staphylokinase (beta + K-) were lysogenically converted by certain serological group F bacteriophages to the loss of beta-hemolysin production and the gain in staphylokinase production (beta-K+). Serological group A phage 42E was found to convert S. aureus strains 7-8(beta-K-) and 57 (beta + K-) to beta - K-. Conversion of beta-hemolysin by lysogenization of a serological group A phage has not previously been reported. Phage 42E conversions differed from the group F conversions since staphylokinase was not affected. This indicates that conversion to beta-K+ involves separate loci on the phage chromosome. Several characteristics associated with virulence of staphylococci of human or animal origin other than staju;plomase production (coagulase, DNase, lipase, gelatinase, mannitol fermentation, and phage-sensitivity patterns) were not correlated with lysogenic conversions to loss of beta-hemolysin.


Effects of cancer chemotherapeutic agents on endocrine organs and serum levels of estrogens, progesterone, prolactin, and luteinizing hormone.

The effects of the following cancer chemotherapeutic agents on serum hormonal levels, estrous cycles, and endocrine organs were studied in mature, normal Sprague-Dawley rats by radioimmunoassay, vaginal smear examination, and organ weight end point: estradiol mustard (NSC 112259), testosterone mustard (NSC 112260), phenoestrin (NSC 104469), methotrexate (NSC 740), 5-fluorouracil (NSC 19893), vinblastine (NSC 49842), vincristine (NSC 67574), nitrogen mustard (NSC 762), and 1,3-bis(2-chloroethyl)-1-nitrosourea (NSC 409962). Following 2 weeks of treatment, estradiol-17beta levels were markedly elevated by all compounds except testosterone mustard and nitrogen mustard, which caused a decrease. Estrone levels were elevated by methotrexate, 5-fluorouracil, vinblastine, vincristine, nitrogen mustard, and 1,3-bis(2-chloroethyl)-1-nitrosourea, but were lowered by estradiol mustard. Progesterone levels were elevated only by estradiol mustard and testosterone mustard and were not affected by other compounds. Prolactin surge during proestrus was suppressed by phenesterin and methotrexate. Luteinizing hormone levels were lowered by methotrexate and nitrogen mustard. Estrous cycles of rats treated with estradiol mustard were arrested at proestrus, and the uterine and pituitary weights of these rats markedly increased. Uterine weight loss was significant following treatment with testosterone mustard, 5-fluorouracil, and nitrogen mustard. Thyroid weight was reduced by all compounds except methotrexate and vinblastine. Significant increases in pituitary weights occurred following treatment with all compounds except 1,3-bis(2-chloroethyl)-1-nitrosourea. The effects on ovarian and adrenal weights were minimal although significant by some compounds. Thus, in addition to their direct antitumor effects, these agents also produced changes in endocrine system which may be synergistic or antagonistic to the chemotherapy of endocrine-responsive neoplasms.


Protective effect of dehydroepiandrosterone against aflatoxin B1-and 7,12-dimethylbenz(a)anthracene-induced cytotoxicity and transformation in cultured cells.

Dehydroepiandrosterone, an adrenal steroid that has been reported to be produced in subnormal amounts and found in lower plasma concentrations in women with benign and malignant breast tumors, protects cultured rat liver epithelial-like cells against aflatoxin B1-induced cytotoxicity and protects hamster embryonic fibroblasts against aflatoxin B1- and 7,12-dimethylbenz(a)anthracene-induced transformation. Related androgenic steroids show significantly less protective effect. Dehydroepiandrosterone also inhibits the rate of conversion of [3H]-7,12-dimethylbenz(a)anthracene to water-soluble metabolites by rat liver epithelial-like cells, suggesting that the steroid probably exerts its protective effect by inhibiting carcinogen activation.


Phase II trials with procarbazine (NSC-77213), streptozotocin (NSC-85998), 6-THIOGUANINE (NSC-752), and CCNU (NSC-79037) in patients with metastatic cancer of the large bowel.

One hundred and ninety-seven patients with measurable metastatic cancer of the colon have been treated with one of four anticancer drugs which have had little prior trial in this disease. Objective tumor responses lasting a median of 9 weeks occurred with 0.5 g/m of streptozotocin given intravenously every week (10 percent), 130 mg/m of CCNU given orally every 6 weeks (10%), 1.0 mg/kg/day of 6-thioguanine given orally (8%), and 3 mg/kg/day of procarbazine given orally (3%). Performance status declined more rapidly with streptozotocin and 6-thioguanine and the median survival time was less (12 and 16 weeks respectively) than with procarbazine and CCNU (23 and 20 weeks respectively). This study suggests that procarbazine given in this way is ineffective but trials of streptozotocin or 6-thioguanine combined with other agents active against colon cancer should ensue as well as further exploration of the usefulness of other nitrosoureas.


The role of myocardial biopsy in cardiac diagnosis.

With the advent of cardiac catheterization, cardioangiography, and selective coronary arteriography, specific types of cardiac disease can be recognized and clearly defined. This is appropriate because myocardial biopsy alone rarely plays a major role in cardiac diagnosis. Excluding Aschoff's nodules in patients with rheumatic valve disease, the light microscopic findings in patients with rheumatic heart disease, congenital heart disease, pericardial disease, hypertensive and arteriosclerotic heart disease are similar and nonspecific. In these, interstitial fibrosis and/or myocardial hypertrophy is the dominant tissue diagnosis. Occasionally a pericardial and myocardial specimen is helpful to distinguish constrictive pericarditis and restrictive cardiomyopathy. Myocardial biopsy has provided the only method for diagnosis in a small number of patients with normal hemodynamics, normal coronary arteriograms and normal ventriculograms. The patients were studied because of chest pain and/or cardiac arrhythmias. Supraventricular and/or ventricular arrhythmias were encountered. In these patients the tissue diagnosis was interstital fibrosis and/or myocardial hypertrophy. These findings are consistent with primary myocardial disease which was not recognized clinically or by angiographic studies. The procedure seems to play a major role in the diagnosis of specific types of primary myocardial disease. It is valuable in the recognition of glycogen storage disease, amyloidosis, hemochromatosis, and myocarditis. On the basis of current experience, the indications for myocardial biopsies depend on the need for a tissue diagnosis in determining the management of the patient and the availability of adequately trained personnel to perform the procedure and manage the complications.


Hydrolase and serum treatment of normal chick embryo cells: effects on hexose transport.

We have asked whether treatment of normal cultured cells with proteases, other hydrolytic enzymes, or serum can convert them into transient phenocopies of transformed cells with respect to the very high rate of hexose transport characteristic of transformed cells. Treatment of density-inhibited cultures of normal chick embryo fibroblasts with trypsin, plasmin, neuraminidase, or hyaluronidase stimulated their rate of 2-deoxyglucose uptake to a level only marginally higher than that seen in normal exponentially growing cultures, and only 35-45% of that seen in transformed cultures. Addition of the hydrolytic enzymes to growing cell cultures had little effect on 2-deoxyglucose uptake. Serum, however, could stimulate 2-deoxyglucose uptake all the way up to the transformed level. Even though the hydrolases and serum differed in their ability to stimulate 2-deoxyglucose uptake, both reagents were capable of stimulating cell division equally well. Evidence is presented suggesting that the hexose transport rate is controlled by serum factors, and that proteolysis can affect the response of the cells of these factors.


[Symbiotic efficiency in spontaneous mutants of Rhizobium legumino-sarum resistant to streptomyocin, spectinomycin, or kanamycin].

Symbiotic effectiveness of 45 mutant strains selected from four wild effective strains of Rhizobium leguminosarum for resistance to streptomycin, spectinomycin or kanamycin was determined on Vicia faba.  Loss of effectiveness occurred in twenty of these mutants; distribution of ineffective mutants was uniform among the three types of antibiotic resistant mutants but varied with the parent strain from which mutants have been derived.


[Cathepsin D and the catabolic degradation of proteoglycans in granulomatous tissue].

Incubated in vitro at 37 degrees C, granulation tissues release glycosaminoglycans into the incubation medium.  Such release is inhibited if pepstatine is present in the medium.  From this result, it can be inferred that the protien moiety of the proteioglycans is degraded by cathepsin D.  Therefore a role of this enzyme in granulation tissues appears, especially in the late reparative phase.


Electrophysiologic and pathologic correlations in two cases of chronic second degree atrioventricular block with left bundle branch block.

This study concerns two cases of chronic 2 degrees atrioventricular (A-V) block with left bundle branch block (LBBB). Pathological studies included serial section of the conduction systems. Case 1 had type I 2 degrees block with LBBB. Electrophysiological studies revealed type I 2 degrees block proximal to the His bundle recording site and a prolonged H-V interval (60 msec). Pathologically there was a moderate to marked fibrosis of the approaches to the A-V node and of the A-V node, marked fibrosis of the left bundle branch, and moderate involvement of the right bundle branch. The changes proximal to the His bundle were more marked than the changes distal to this bundle. Case 2 had type II and 2:1 2 degrees A-V block with LBBB. Electrophysiologically the site of block was distal to the His bundle recording site, and there was a prolonged A-V node and severe involvement of both bundle branches. The changes distal to the His bundle were more severe than the changes proximal to the His bundle. This study reveals that the electrophysiologic data more closely approximated the pathologic findings than did surface electrocardiographic data alone. It also emphasizes that there may be multiple sites of disease in chronic 2 degrees block with bundle branch block.


Arteriovenous fistulae secondary to renal cell carcinoma. Clinical and cardiovascular manifestations: report of a case.

A 63-year-old woman presented with progressive congestive heart failure and unexplained cardiomegaly. Diagnostic workup revealed large arteriovenous fistulae in the lower pole of the left kidney. A total left nephrectomy was performed and microscopic exam revealed renal cell carcinoma. Following surgery, the congestive heart failure cleared and the patient has been asymptomatic for one year. The pertinent findings of the 22 patients who have been reported previously in the literature with arteriovenous fistulae complicating renal cell carcinoma are reviewed. Thirty percent of the patients presented with cardiovascular complaints, and 60% had significant cardiovascular findings during the course of evaluation. An abdominal bruit was the most discriminating finding on physical exam, and it occurred in 72% of the reported cases. The diagnosis was unexpectedly established by surgery in 13%, and by angiography in 87% -- usually in the course of a workup for hypertension, abdominal pain, hematuria, or during search for an occult malignancy. An extensive evaluation is required for early diagnosis of this correctible cause of hypertension and heart failure.


Analysis of urinary mucopolysaccharides using small ion exchange columns.

Methods are presented for the small scale column chromatography of urinary mucopolysaccharides. After isolation by cety pyridinium chloride mucopolysaccharides are applied to a small column of ion exchange resin and eluted by 2 M-sodium chloride. Colorimetric assays on this elutate allow clinically significant disease states to be detected.


Erythrocyte phosphofructokinase deficiency associated with an unstable variant of muscle phosphofructokinase.

A case of chronic non-spherocytic hemolytic anemia due to partial erythrocyte phosphofructokinase deficiency (61% of normal) is reported. Immunological studies in hemolystates, using anti-muscle and anti-leukocyte phosphofructokinase antisera, seemed to indicate that an isozyme of the muscle type was deficient in the patient. This hypothesis was confirmed by the studies of muscle phosphofructokinase; this enzyme was an unstable and fast variant. There was no deficiency in muscle because of the active synthesis of proteins by this tissue, but the deficiency could be detected in erythrocytes, old cells which are no longer able to synthesize proteins.


Micromethod for the determination of glycosaminoglycans in the serum. Results from the serum of healthy and varicose subjects.

Serum glycosaminoglycans (GAG) were isolated from 1 ml of serum by a modification of the method of Y. Emura and T. Mukuda (J. Jap. Biochem. Soc., 45 (1973) 30). The extraction was carried out as quantitatively as possible and the hexuronic acids in the GAG were measured with m-hydroxydiphenyl. This method for determination of the GAG is relatively easy, easily reproducible and requires no more than two days. Its application in clinical chemistry may prove to be useful in the detection of diseases involving connective tissue. A study was carried out with serum samples from 34 patients suffering from serious varicose conditions, compared with samples fro 30 healthy subjects. The mean concentration of GAG in the serum of healthy persons was 0.602 +/- 0.108 (p smaller than 0.0005). There were not, however, any significant variations between patients suffering from serious varices without trophic changes and those with comparable varices accompanied by trophic changes, such as phlebitis, thrombosis and ulcers.


Indices of gonadal function in the human male. I. Plasma levels of unconjugated steroids and gonadotrophins under normal and pathological conditions.

A radioimmunoassay technique for the simultaneous measurement of eight unconjugated steroids (progesterone, pregnenolone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, oestrone and oestradiol) in the peripheral plasma of human males is described. Determinations of these steroids and of immunoreactive FSH and LH were carried out on the plasma of twenty-one normal individuals and the levels were compared to those of eleven and ten males exhibiting oligospermia and azoospermia, respectively. Mean values and tolerance limits for each hormone, based on a lognormal distribution of individual values, are presented for all groups. Oligospermia was associated with a significant reduction in plasma dihydrotestosterone and testosterone levels. Azoospermic subjects also exhibited decreased dihydrotestosterone levels but a normal range of testosterone concentrations. Mean peripheral plasma levels of FSH were significantly elevated in both pathological groups and this was paralleled in the azoospermic men by increased concentrations of plasma LH.


Indices of gonadal function in the human male. II. Seminal plasma levels of steroids in normal and pathological conditions.

A radioimmunoassay method developed previously for the measurement of unconjugated pregnenolone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone and oestradiol in peripheral plasma was applied to the assay of these steroids in seminal plasma of normal, oligospermic and azoospermic males. It was not possible to use the plasma assay method for the determination of progesterone and oestrone in seminal plasma, because some of the reliability criteria were not fulfilled. A detailed analysis of these steroids in the peripheral plasma of the same subjects has been described previously. The levels of all steroids in seminal plasma were significantly lower than the corresponding blood levels. The ratios of blood plasma/seminal plasma levels of the various steroids varied from 37 (testosterone) to 1.7 (dihydrotestosterone). There was a positive correlation between the testosterone and dihydrotestosterone levels of the seminal plasma of normal and azoospermic subjects. The concentrations of dihydrotestosterone, pregnenolone and oestradiol were significantly lower in azoospermic subjects than in normals. The only pathological finding in the seminal plasma of oligospermic subjects was a diminished level of dihydrotestosterone. Enzymic hydrolysis of a seminal plasma pool resulted in a 3- to 8-fold increase in the concentration of pregnenolone, dehydroepiandrosterone, testosterone, dihydrotestosterone and oestradiol, indicating that human seminal plasma contains large amounts of steroid conjugates. It is suggested that the analysis of steroids in the seminal plasma in combination with determinations in peripheral plasma may be a valuable aid to the assessment of testicular function.


Chronic treatment regimens for hirsutism in women: effect on blood production rates of testosterone and on hair growth.

Twenty-five women with hirsutism were studied before and during treatment aimed empirically at suppresing testosterone production by adrenals, ovaries or both. Mean basal plasma testosterone was 70 plus or minus 30 ng/dl, significantly (P less than 0.01) higher than the mean of twenty-three normal women; basal metabolic clearance rate (MCR) of testosterone was also higher (P less than 0.01) than that reported for normal women. Production rate (PR) of testosterone exceeded 417 mug/24 h (1 SD above the mean reported for normal women), in nineteen of the twenty-five patients. After 4 months, small dose betamethasone therapy (0.5 mg at bed time) had reduced the mean PR of testosterone in thirteen patients from 509 mug/24 h to 356 mug/24 h (P = 0.05); anovulatory steroids reduced mean PR of testosterone in nine from 612 mug/24 h to 345 mug/24 h (P less than 0.05, greater than 0.025), and the combination of both therapies in ten reduced PR of testosterone from 528 mug/24 h to 148 mug/24 h (P less than 0.001). The latter regimen had moderate success in reducing hair growth (in six out of ten). Fourteen of the twenty-five claimed benefit in hirsutism and all thirteen with acne were improved. In individual cases, clinical benefit did not correlate well with reduction in PR of testosterone. Freedom from undesirable side effects allows these well-accepted forms of treatment to be given even longer trials.


Hepatitis associated antigen and the ABO locus in Down's syndrome.

The recent observation by Arndt-Hansen et al. (1974) of increased frequency of blood group A over group O in blood donors positive for the hepatitis associated antigen has been investigated in Down's syndrome, in order to establish if this could account for the increased frequency of the antigen in that syndrome. Seventy-one of 227 subjects with Down's syndrome (31.3%) were found to be positive for the antigen by haemagglutination, and comparison of these with the HAA-subjects failed to reveal any differences in the ABO blood groups.


Down's syndrome with additional XYY aneuploidy.

A further case of Down's syndrome with additional XYY aneuploidy is reported. The child has been followed for 5 years. It has not been possible to recognise any phenotypical feature which distinguishes the patient from others with Down's syndrome.


The "labial index" in Down's syndrome.

In newborn females with Down's syndrome the labia majora are of a typical appearance, being shorter and wider than in normals. This observation can be expressed numerically by the "labial index", which in mongoloids was found significantly lower.


Loin pain as a presenting symptom in idiopathic glomerulonephritis.

Loin pain may be a major presenting symptom in patients with glomerulonephritis. Most of these patients show an underlying focal and segmental proliferative glomeruloneyphritis and there may be associated deposits of IgA and Igg in the mesangium. In this group of patients, vascular lesions are often prominent in the absence of hypertension. Episodes of recurrent macroscopic hematuria also occur, but the pain cannot be attributed to colic due to blood clots in the ureter.


The dermolipectomy approach for augmentation mammaplasty.

Breast augmentation with silicone prosthesis through a dermolipectomy approach, its advantages (geometric preoperative planning, abdominal reduction in all three dimensions, providing an improved waistline and a navel of normal aspect, short bikini covered scar of good quality, breast augmentation without regional scars), and limitations (exclusive of major abdominal adiposites and moderately or severely ptotic, hypoplastic breasts) are discussed, and the technique is described.  The results obtained with the procedure are considered satisfactory for both patient and surgeon, and are recommended for cases of abdominal adiposity combined with unilateral or bilateral breast hypoplasias.


Potentiation of antinociceptive action of morphine by prostaglandin E1 in albino rats.

1. Prostaglandin E1 (0.4 ml/kg, i.p.) significantly potentiated the antinociceptive action of morphine in albino rats. 2. This potentiation was significantly inhibited by pretreatment with reserpine, p-chlorophenylalanine or methysergide but not by alpha-methyl-p-tyrosine, phentolamine or propranolol. 3. The results suggest that the potentiation of morphine by prostaglandin E1 is mediated by serotonin, since it is inhibited by pharmacological agents known to reduce central turnover of serotonin receptors.


Soluble high-molecular-weight E fragments in the plasmin-induced degradation products of cross-linked human fibrin.

1. The factor XIII-mediated cross-linked alpha chains in fibrin have no effect on the nature of the fragments released during the solubilization of fibrin by plasmin. 2. Besides the known D dimer and E fragments solubilized during the lysis of cross-linked fibrin, other fragments have been observed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which have a molecular weight of about 135 000. After prolonged plasmin digestion, these fragments (U fragments) were no longer evident on the gels and the high-molecular-weight E antigen was absent. It is assumed that the E antigen was associated with the U fragments. These fragments also cross-reacted with an anti-D serum. 3. The U fragments have been tentatively presumed to be a factor XIII-mediated cross-linked D-E complex since they degrade only after prolonged degradation with plasmin. Whereas it is known that the fibrin D dimer fragment contains the cross-linked gamma chain residues of the originating fibrin, the presumed covalent cross-linking of the D-E fragments has not been proved. 4. The presence of these high-molecular-weight fragments, containing the E antigen, in cross-linked human fibrin digests should be taken into account in the development of D dimer assays to monitor fibrin lysis in vivo.


Prevention of chronic experimental heart insufficiency by inosine.

The administration of inosine dosed 25 mg/kg daily during 10 1/2 months prevented the development of excentric cardiac hypertrophy on the background of experimental aortic stenosis in rats, reduced the lowering of the working capacity of the animals, and partially inhibited the full development of functional and morphological myocardial changes, detected by electrocardiographic, vectorcardiographic, microscopic, and electronmicroscopic examinations. Differences were found in the relative weights of the heart, thymus, liver and other organs; these findings attested to an anabolic activity of inosine. The results obtained justify the application of inosine for the prevention of excentric hypertrophy and decompensation of the heart in cardiac failures varying in etiology; any efficient preventive measure is of importance, because the degree of excentric hypertrophy is a factor influencing the survival of the patients.


Measurement of myocardial blood flow.

This paper represents a review of the recent literature on techniques to measure myocardial blood flow in man. A short discussion on flow meters in followed by a more detailed discussion of the radionuclide techniques used to measure myocardial blood flow. The radionuclide techniques are discussed in two groups: (1) qualitative measurement of blood flow using static images; and (2) quantitative measurements of blood flow using diffusible substances that actively enter the cell, radioactive labeled particles, and radioactive diffusible gases.


Myocardial coronary hemangiomatous tumors in children.

Persistent and severe ST segment and T wave changes associated with an apical diastolic murmur were observed in an asymptomatic five-year-old girl for ten years. Recent development of exertional dyspnea and chest pain prompted a detailed cardiovascular re-evaluation. The symptomatology and the electrocardiographic changes are attributed to an angiomatous tumor involving the anterolateral myocardium demonstrated by selective coronary arteriography. The literature regarding primary cardiac vascular tumors was reviewed and the clinical and therapeutic implications were discussed.


Electrocardiographic abnormalities in alcoholic cardiomyopathy. A study of 65 patients.

Electrocardiograms of 65 patients with alcoholic cardiomyopathy seen over a five-year period were reviewed. ST segment and T wave abnormalities, left ventricular hypertrophy, biatrial enlargement, left atrial enlargement, premature ventricular contractions, prolonged PR interval, and left anterior hemiblock are the most frequently encountered abnormalities. A combination of left ventricular hypertrophy and biatrial enlargement with or without left anterior hemiblock is most specific, Atrial flutter or fibrillation, pathologic Q waves, and bifascicular block are not uncommon findings, while isolated right atrial or right ventricular abnormalities, and isolated posterior hemiblock or right bundle branch block are rare. Electrocardiographic changes are in general similar to those seen in any diffuse cardiomyopathy and reflect biventricular involvement.


Cor pulmonale secondary to upper airway obstruction. Cardiac catheterization, immunologic, and psychometric evaluation in nine patients.

Previous reports of cor pulmonale due to upper airway obstruction have not noted that chronic hypoxemia due to alveolar hypoventilation can lead to left as well as right ventricular failure in these patients. We found elevated left ventricular end-diastolic pressure in four of five patients. Although these patients have frequent respiratory infections, no investigation of their immune status has previously been reported. We performed such investigations but could not demonstrate an immunologic deficiency. Although nine of 44 patients previously reported were mentally retarded, no psychometric data were available. We studied five patients but could not determine the role of mental subnormality in this syndrome. Diagnosis of this syndrome should be suspected when clinical and roentgenographic findings of biventricular heart failure are associated with right ventricular hypertrophy on electrocardiogram. The diagnosis of alveolar hypoventilation is confirmed by blood gas findings and examination of the upper airways. Surgical removal of obstructive tissue reverses the cardiac involvement.


DNA replication in the amphibia.

Autoradiographic techniques were used to measure rate of replication and length of the replication unit in cultured cells of Scaphiopus couchi, Bufo cognatus, Rana clamitans, and Triturus viridescens, having nuclear DNA amounts in the ratio 1:4:7:39 respectively. The autoradiographic experiments were designed to show whether the larger amounts of nuclear DNA are correlated with more rapid rates of synthesis and/or with longer replication units. -- The DNA replication rate was 2.5 mu/minute (corrected for two growing points) with 10 minutes 3H-thymidine label at 22 degrees C, but decreased with longer labelling durations. The length of the replication unit (estimated by the distance from the center of one autoradiograph to the center of the next in sequence) was most commonly in the 10-25 mu range with a 30 minute label, in all four species. The average center-to-center distance was 8 mu at 10 minutes and increased with label duration, to over 45 mu with 24 hours label. Replication was predominantly but not exclusively bidirectional. Neither rate of replication nor length of the replication unit was proportional to the amount of DNA in these species.


Different approaches to the mechanism of the sodium pump.

The way in which the sodium pump uses energy from the hydrolysis of ATP to perform osmotic and electrical work is not yet understood. We attempt to bring together the results of a number of different approaches to this problem. One approach has been to correlate biochemical changes and ionic fluxes, both when the pump operates normally and when it operates in various abnormal 'modes' in particular unphysiological conditions. A second approach has been to expose fragments of cell membrane to (gamma-32P)ATP and to study the properties of components of the membrane that become labelled. It is now clear that 32P can be transferred to the beta-carboxy group of an aspartyl residue in a pump polypeptide, but there is controversy about the interrelations of different forms of this polypeptide and its role, if any, in the normal functioning of the pump. A third approach has been to attempt to purify the pump and to determine the properties of the pure enzyme. It seems that the pump contains a polypeptide (molecular weight about 100,000), which bears the phosphorylation site, and a smaller glycopeptide, but there is disagreement about the molecular ratios. The results of these and other approaches cannot yet be fitted into a satisfactory model for the sodium pump, but we shall consider some of the problems involved in this task.


The roots of bioenergetics.

Understanding metabolic energy transformation began with the realization of an 'intrusion' of phosphate into the mechanism of alcoholic fermentation. The discovery of an analogous participation of phosphate in muscle glycolysis connected the metabolic generation of energy-rich phosphate bonds fed into a common transmitter, adenosine triphosphate (ATP), with the production of mechanical energy through the finding that the phosphoryl group of creatine phosphate transferred to ATP could supply the energy for muscle contraction. In this way, a functional applicability of the energy of the phosphate bond was first shown. This observation was soon followed by the recognition that the phosphoanhydride bond of ATP provided the driving force in biosynthetic reactions; in this type of bond, metabolic energy apparently collects before it is transmitted for functional and biosynthetic use. The storage of energy in ATP was first detected in anaerobic energy-yielding reactions but soon was also found in respiratory and photosynthetic energy production. However, the mechanism by which energy derived from metabolites was converted into phosphate-bond energy in the latter processes appeared to differ from that of anaerobic energy transmission. Whereas phosphorylated compounds mediate the latter in homogeneous solutions, aerobic phosphorylation and photophosphorylation in prokaryotes seem to require special submembranous structures; and in eukaryotes, energy conversion is a function of special organelles, the mitochondria and chloroplasts. The evolutionary aspects of the transition from prokaryotes to eukaryotes are of considerable interest. In conclusion, the relevance of an apparent prokaryotic origin of the energy-transforming organelles in the eukaryotes will be commented on.


Energy changes during the formation and interconversion of enzyme-substrate complexes.

The rate constants and equilibrium constants of the individual steps of several enzyme reactions may be determined by the application of rapid reaction methods and isotope techniques. This makes it possible to complement the formalism of the Haldane relation with details of the reaction mechanism. It has been shown that, in several enzyme reactions, steps involving chemical catalysis are fast and have small free-energy changes compared with those of the substrate binding and product dissociation processes. Data are presented in this paper for three enzyme reactions for which different methods have been used to elucidate the kinetic parameters of the elementary steps. For cardiac lactate dehydrogenase (EC 1.1.1.27), absorption and fluorescence spectroscopy have been used to distinguish the step involved in the chemical process from those involved in the formation of the substrate complex and the release of the product. The rate of interconversion between enzyme-bound substrates and products is fast compared with other steps and the equilibrium constant for the process is near unity. Consequently, the difference of standard free energy changes for the formation of the two ternary complexes correspons approximately to the overall free-energy change of the hydrogen transfer reaction. Isotope kinetic techniques can be used to study the reactions of triosephosphate isomerase (EC 5.3.1.1). With this enzyme, the interconversion of enzyme-bound substrate into product is comparable in rate to product dissociation. The reactions of myosin subfragment 1 with ATP, studied by fluorescence spectroscopy and chemical quenching, follow a similar pattern in that the equilibrium constant of the chemical step in which water reacts with protein-bound ATP is 9. In this case, however, there is a remarkably large free-energy change associated with a first-order process involved in the binding of ATP. The possible significance of these results to energy transduction in muscle contraction as well as in the biosynthesis of ATP is discussed.


Gas chromatographic determination of unconjugated dehydroepiandrosterone, androstenedione, testosterone, pregnenolone and progesterone in human ovarian tissue.

A gas chromatographic method has been presented for the determination of unconjugated dehydroepiandrosterone, androstenedione, testosterone, pregnenolone and progesterone in human ovarian tissue. The procedure utilized radioactive tracers added to homogenate for correcting methodological loss, preliminary separation of steroids by thinlayer chromatography, acetylation, rechromatography on chromatoplate and gas chromatography on 3% SE-30 or 1% XE-60 columns with flame ionisation detection of steroids by using internal standards. Results of control experiments and representative clinical findings on normal and polycystic ovaries are reported.


Concentration of unconjugated adrenogenic hormones and their precursors in normal and polycystic ovaries.

Dehydroepiandrosterone, androstenedione, testosterone, pregnenolone and progesterone concentration was determined by our sensitive gas-liquid chromatographic method in ovarian tissues obtained from surgery of patients without hirsutism and with Stein-Leventhal syndrome. The steroids, except testosterone, were detectable in all ovaries studied. Dehydroepiandrosterone and androstenedione, regarded as preandrogens, were present in an increased amount in almost all patients with polycycstic ovaries. Gas chromatographic evidence was obtained for the presence of testosterone in two of the cases. The delta4/3betaOH ratio reflecting 3beta-hydroxysteroid dehydrogenase activity was decreased only in same patients with the Stein-Leventhal syndrome suggesting that the impaired function of this enzyme is not an obligatory feature of polycystic ovaries. Concentration of pregnenolone and progesterone measured in a part of cases varied in a great range although the determination was caried out before luteal phase. Simultaneous determination of hormones in both ovarian tissues revealed an active and an inactive period of the gland in the given time, since a great difference of hormone concentration in bilateral ovarian tissues were observed. A comparison of hormone content in ovaries and the urinary excretion of metabolites showed poor correlation between the two parameters of hormone production.


[Steroid metabolism in primates. XVI. Secretion of corticosteroids in the baboon Papio hamadryas during chronic administration of sodium chloride].

The adrenal steroid secretion was investigated in male baboons (Papio hamadryas) treated for a long time with sodium chloride, in comparison to an untreated control group. In animals treated with NaCl, the secretion of progesterone, 17alpha-hydroxyprogesterone, 11-deoxycortisol, aldosterone and corticosterone was decreased, while cortisone, pregnenolone, 7-keto-cholesterol, 7-keto-DHEA, DHEA and adrenosterone were increased.


The importance of adrenal glands in the improved adaptation of trained animals to physical exertion.

The importance of adrenal glands in the adaptation of rats to physical exertion was studied. Exercise training, significantly increased the working ability and prevented the water accumulation in myocardial cells during acute exertion. The latter shift was characteristic of untrained animals. However, adrenalectomy abolished the benefical effect of training on working ability and myocardial adaptation  to exertion. The results obtained supported the view that the effect of training is partly mediated through the improved adrenocortical function while adrenal hormones, by regulating the heart metabolism and function, significantly influenced the adaptation of rats to physical exertion.


Effect of acute hypoxia on the enzymes involved in the metabolic and nervous functioning of rat brain.

Rats were exposed to a simulated altitude of 25,000 ft for 4 h in a decompression chamber, and the activity of some tissue enzymes estimated. Succinate dehydrogenase activity was significantly decreased and cholinesterase activity significantly elevated in the brain homogenates of the hypoxic rats, succinic dehydrogenase activity was significantly increased. There was no change in the activity of Mg+2-ATPase and Na+-K+-ATPase in the microsomal fractions of liver or brain homogenates of the hypoxic animals.


Biochemical effects of di-2-ethylhexyl phthalate.

Di-2-ethylhexyl phthalate (DEHP) is a widely used plasticizer in the plastics industry. Biochemical changes in rat blood and liver were studied at 21 days after 3 intraperitoneal injections of DEHP on days 1, 5 and 10. In the liver, a decrease in the activity of succinic dehyrogenase (SDH) and adenosine triphosphate and an increase in alkaline phosphatase activity was observed. In serum the activity of acid and alkaline phosphatases and glutamic oxaloacetic and glutamic pyruvic transaminases remained unaltered. Under in vitro conditions, only the activity of SDH was inhibited when homogenates or mitochondrial preparations from normal rat liver were incubated with DEHP and the degree of inhibition was related to the concentration of the plasticizer.


Effect of coumarins on mitochondrial function.

Several derivatives of coumarin inhibited mitochondrial respiration and ATPase activity.  The extent of inhibition depended on the concentration of the coumarins as well as on the substituents of the coumarin ring.  Some of the coumarins stimulated ATPase activity, but all of them inhibited uncoupler-stimulated ATPase activity.  Coumarins with free or substituted phenolic groups were found to exert profound effects on respiration and ATPase activity.


Studies on energy-linked reactions: isolation, characterisation and genetic analysis of trialkyl-tin-resistant mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae resistant to triethyl tin sulphate have been isolated and are cross-resistant to other trialkyl tin salts. Triethyl-tin-resistant mutants fall into two general phenotypic classes: class 1 and class 2. Class 1 mutants are cross-resistant to a variety of inhibitors and uncoupling agents which affect mitochondrial membranes (oligomycin, ossamycin, valinomycin, antimycin, erythromycin, chloramphenicol, '1799', tetrachlorotrifluoromethyl benzimidazole carbonylcyanide-m-chlorophenylhydrazone and cycloheximide). Class 2 mutants are specifically resistant to trithyl tin and the uncoupling agent "1799' [bis-(hexafluoroacetonyl)-acetone]. Triethyl tin at neutral pH values is a specific inhibitor of mitochondrial energy conservation reactions and prevents growth on oxidisable substrates such as glycerol and ethanol. Triethyl-tin-resistant mutants grow normally on glucose and ethanol in the presence of triethyl tin (10 muM). Biochemical studies indicate that the mutation involves a modification of the triethyl tin binding site on the mitochondrial inner membrane, probably the ATP-synthetase complex. Triethyl tin resistance/sensitivity in yeast is determined by cytoplasmic (mitochondrial) and nuclear genes. The mutants fall into a nuclear and a cytoplasmic (mitochondrial) class corresponding to the phenotypic cross-resistance classes 1 and 2. In the cytoplasmic mutants the triethyl tin resistance segregates mitotically and the resistance determinat is deleted by the action of ethidium bromide during petite induction. Recombination studies indicate that the triethyl tin mutations are not allelic with the other mitochondrial mutations at the loci RI, RIII and OLI. This indicates that the binding or inhibitory sites of oligomycin and triethyl tin are not identical and that the triethyl tin binding site is located on a different mitochondrial gene product to those which are involved in oligomycin binding. Interaction and cooperative effects between different binding sites on the mitochondrial inner membrane have been demonstrated in studies of the effect of the insertion of the TETr phenotype into mitochondrial oligomycin-resistant mutants and provide an experimental basis for complementation studies at the ATP-synthetase level.


Identification of two products of mitochondrial protein synthesis associated with mitochondrial adenosine triphosphatase from Neurospora crassa.

Soluble mitochondrial ATPase (F1) isolated from Neurospora crassa is resolved by dodecylsulfate-gel electrophoresis into five polypeptide bands with apparent molecular weights of 59000, 55000, 36000, 15000 and 12000. At least nine further polypeptides remain associated with ATPase after disintegration of mitochondria with Triton X-100 as shown by the analysis of an immunoprecipitate obtained with antiserum to F1 ATPase. Two of the associated polypeptides with apparent molecular weights of 19000 and 11000 are translated on mitochondrial ribosomes, as demonstrated by incorporation in vivo of radioactive leucine in the presence of specific inhibitors of mitochondrial (chloramphenicol) and extramitochondrial (cycloheximide) protein synthesis. The appearance of mitochondrial translation products in the immunoprecipitated ATPase complex is inhibited by cycloheximide.. The same applies for some of the extramitochondrial translation products in the presence of chloramphenicol. This suggests that both types of polypeptides are necessary for the assembly of the ATPase complex


Huntington's chorea associated with normal pressure hydrocephalus.

Two patients with Huntington's chorea (HC) developed a gait disturbance more suggestive of normal pressure hydrocephalus (NPH) than HC. The diagnosis of NPH was confirmed by pneumoencephalography and isotope cisternography. Both patients were shunted and both showed an improvement not only in their gait but, unexpectedly, a decrease in their abnormal involuntary movements (AIMs). The association of HC with NPH is discussed.


Isogeneic or allogeneic transplantation of duct-ligated pancreas in streptozotocin diabetic mice.

Recovery from hyperglycaemia was observed in three different mouse hybrids that were made diabetic with streptozotocin, and then transplanted with pancreas from isogeneic donors whose pancreatic ducts were ligated over 8 weeks earlier. Recovery did not occur in recipients of allografts indicating that islets in ligated pancreas cannot be successfully transplanted across a major histocompatibility barrier. However, in lethally irradiated mice injected with allogeneic bone marrow (allogeneic radiation chimeras), subsequent transplantation of ligated pancreas from either isogeneic donors or allogeneic donors of the same strain as the bone marrow donor decreased blood glucose of diabetic recipients. Thus, irradiation and allogeneic bone marrow therapy allow subsequent allogeneic transplantation of functional islet tissue in diabetic mice. Observations on diabetic mice transplanted isogeneically with ligated pancreas suggest that recovery from hyperglycaemia was more consistent when grafts were placed subcutaneously rather than intraperitoneally and when hosts received ligated pancreas from three donors rather than one. Thus, the site and amount of tissue transplanted are important considerations in the transplantation of ligated pancreas. Persistence of endocrine beta-cells in ligated pancreas was required for recovery in grafted mice since diabetic hosts failed to recover when grafted with ligated pancreas obtained from alloxan or streptozotocin-treated, diabetic donors.


Behavioral correlates of serotonin depletion.

Depletion of telencephalic serotonin (5-HT) content by medical forebrain bundle lesions, which interrupt the ascending serotonergic pathways or by DL-p-chlorophenylalanine produces an increased sensitivity to pain as measured by the flinch-jump, stabilimetric, or hot-plate methods. Examination of the effects of a number of other lesions and drugs indicated that dopamine, norepinephrine and acetylcholine are not involved in pain sensitivity. Dosages of 75 mg/kg DL-5-hydroxytryptophan(5-HTP), 37.5 mg/kg L-5-HTP or 50 mg/kg Ro 4-4602 (NI-(DL-seryl)-N2-(2,3,4-trihydroxybenzyl)hydrazine) plus 37.5 mg/kg L-5-HTP administered to medical forebrain bundle lesioned rats returned both the telencephalic content of 5-HT and the pain threshold to normal values. Injection of 37.5 mg/kg of D-5-HTP or an equimolar dose of L-dopa had no effect on pain threshold. Normal animals display increased sensitivity to pain and decreased 5-HT contents in frontal pole, hippocampus, and amygdala during dark as compared to light hours. All three of these telencephalic areas are innervated by the ascending serotonergic pathways, and cells in these areas show inhibition of firing following the iontophoretic application of 5-HT. Taken together these data suggest that the serotonergic system normally acts to inhibit the effects of painful stimuli. A review of a variety of behavioral effects of 5-HT depletion including an enhanced response to lysergic acid diethylamide and amphetamine suggests that the ascending serotonergic system may have a general role in the inhibition of arousal, rather than a specific role with respect to various categories of behavior.


Cassaine: mechanism of inhibition of Na+ +K+ -ATPase and relationship of this inhibition to cardiotonic actions.

The erythrophleum alkaloid  cassaine shares many of the pharmacological actions of the cardiac glycosides but lacks the structural characteristics typical of cardiac glycosides. To further investigate the relationship between Na+ +K+ -ATPase inhibition and the cardiotonic actions of these drugs we investigated the interaction of cassaine with the Na+ +K+ -ATPase. Cassaine inhibited rat brain Na+ +K+ -ATPase with about one quarter of the apparent affinity of ouabain for this enzyme. This inhibition was non-competitive with respect to K+. Cassaine also inhibited this enzyme in the presence of Mg2+ and this inhibition was enhanced by Pi and antagonized by Na+. In the presence of Na+, Mg2+ and (gamma-32P)-ATP cassaine acted to stabilize the phosphorylated intermediate of Na+ +K+ -ATPase. Cassaine also acted to displace specifically bound (3H)-ouabain from this enzyme. These observations suggested that cassaine inhibited the Na+ +K+ -ATPase by interacting at the cardiotonic steroid binding sites of Na+ +K+ -ATPase. Consistent with this hypothesis, dog, guinea pig and rat heart Na+ +K+ -ATPase showed differing sensitivities to cassaine paralleling their differing sensitivities to ouabain. The principal difference between the interaction of cassaine and ouabain with Na+ +K+ -ATPase appeared to be the more rapid dissociation of cassaine from the cardiotonic steroid binding site(s) of Na+ +K+ -ATPase. In keeping with this the rates of offset of cassaine-induced inotropy in Langendorff perfused dog and guinea pig hearts were several times faster than those of ouabain-induced inotropy.


Irreversible inhibition of 3H-ouabain binding to Na+ +K+ -ATPase by digoxigenin-3,12-dibromoacetate, an alkylating derivative of digoxigenin.

Digoxigenin-3,12-dibromoacetate (DDB), an alkylating derivation of digoxigenin, was synthesized and tested as a cardiotonic steroid (CS) site directed affinity label for Na+ +K+ -ATPase (ATP phosphohydrolase EC 3.6.1.3). DDB inhibited rat brain Na+ +K+ -ATPase with an I50 of 5 times 10(-6)M and readily displaced specifically bound 3H-ouabain from its binding sites on Na+ +K+ -ATPase. If the enzyme was exposed to DDB prior to the addition of 3H-ouabain its ability to bind 3H-ouabain was decreased, consistent with the concept that DDB interacted irreversibly with the cardiotonic steroid binding sites of Na+ +K+ -ATPase. However, DDB proved to be an even more effective inhibitor of 3H-ouabain binding under conditions where it was unlikely that it could interact with the CS binding sites of this enzyme, suggesting that DDB inhibited 3H-ouabain binding by non-cardiotonic site directed actions. Similarly, the presence of excess strophanthidin did not protect this enzyme against irreversible inhibition by DDB. The data suggest that the presence of a bromoacetate group at the 12 position on cardiotonic steroids does not confer CS binding site directed alkylating properties on these drugs.


The effect of harmaline on membrane potentials of rat atrial contractile fibers.

Rat atria either beating spontaneously or electrically driven were were perfused in vitro at 30 degrees C. When the preparations were exposed to harmaline 8.3 x 10(-5) or 16.6 x 10(-5) M the atrial rate showed down and the following changes in membrane potentials were observed: (1) a transient increase in the amplitude of the action potential (AAP), in the absence of any change in the magnitude of the resting potential (RP), followed by a progressive decrease of AaP; (2) a progressive decline of the slope of the fast ascending phase of the action potential, which was initially independent of any change in the magnitude of the rp; (3) a progressive increase of the duration of the action potential; (4) a fall in the magnitude of the resting potential. The conclusions proposed are that harmaline: (1) stimulates the slow current responsible for the last part of the upstroke and the beginning of the repolarization; (2) inhibits the sodium current responsible for the fast component of the upstroke; (3) slows doen the processes responsible for the repolarization; (4) inhibits the (Na+-K+) activated membrane ATPase.


Effect of duration of insulin deficiency on membrane-bound and free ribosomes from livers of diabetic rats.

In order to define the effect of duration of diabetes on hepatic protein sysntesis, membrane-bound and free ribosomes were isolated from livers of rats, 3, 7 and 28 days after administration of intravenous streptozotocin (75 mg/kg). Hepatocytes from the same rats were subjected to ultrastructural quantitative analysis. By day 3 there was a significant loss in the amount of rough endoplasmic reticulum (RER) per volume cytoplasm; however, the normal ratio of membrane-bound ribosomes per unit length of membrane was maintained. These hepatocyte ultrastructural changes continued over the ensuing four weeks. In spite of this decrease in amount of RER, in vitro protein synthetic activity of hepatic membrane-bound polyribosomes was unchanged from controls at three days, and by 28 days protein synthetic activity of bound hepatic ribosomes from diabetic rats was almost twice that of normal controls (p less than .01). In contrast to the effect of diabetes on bound ribosomes, there was no change in protein synthetic activity of free polyribosomes isolated from livers of rats, 3, 7 or 28 days after induction of diabetes. Thus, the effect of any given degree of diabetes on hepatic protein synthesis appears to vary with the population of hepatic ribosomes being studied, and with duration of insulin deficiency.


[Ultrastructural changes of hypertrophied myocardium in severe experimental hypoxia. Effects of diphosphothiamine and of an antagonist of the vitamin B1. (author's transl)].

The ultrastructural changes occurring in hypertrophic heart cases during severe experimental hypoxia, conducted intermittently for 140 and 250 hours, are reported in the present study. For this purpose, serial right myocardial specimens of control and treated groups of animals were obtained and examined by electron-microscopy. Most areas of the individual myocardial tissue taken during prolonged degrees of exposures to normobaric hypoxia, showed an increased number of mitochondria, their prominent destructive changes, enlargement of cisternae of sarcoplasmic reticulum, reduction and derangement of the cristae and an increase of glycogen and lipif droplets cellular content. Different responses were associated with the various experimental designs which include simple hypoxia, hypoxia with administration of Diphosphothiamine (DPT, 30 mg/hie/Kg body weight) and hypoxia with administration of an antagonist of the vitamin B1, neopyrithiamine (PyTh, 20 mg/die/Kg body weight). Alterations in the fine structure of myocardial cells and cellular organelles could complement the increased glycolitic activity and phospholipid biosynthesis for the compensatory mechanism of the heart to hypoxic stimulus. These changes were most evident in the hypoxic animals treated with PyTh because of a large disturbance of energy production caused by the antivitamin. Most of ultrastructural and glycogen content changes disappeared when DPT was administered to the animals in hypoxia. However, the mitochondria were larger in size and their matrices had higher electron density than in the normal control animals. The significance of the morphological and biochemical findings are discussed.