Studies on coagulation and fibrinolysis in blood from puerperal women with and without oestrogen treatment. Coagulation and fibrinolytic studies were made on 25 women who had a normal puerperium and breast fed their babies and on 32 otherwise healthy women who had lactation suppressed by diethylstilboestrol. In the untreated patients the fibrinogen, Factors VIII and IX, platelet count, and antifibrinolytic activity increased, and the levels of Factors  II, VII, and X and of plasminogen and plasminogen proactivator decreased. The incidence of cold activation of Factor VII decreased slowly from the 5th postpartum day onwards. The concentration of antithrombin III which was depressed in pregnancy, increased in the puerperium and was the only observed change that counteracted an increased coagulation potential. Oestrogen treatment produced further increases of Factors II, VII, and X, platelet count, and the cold activation of Factor VII while it decreased the concentration of antithrombin III. These changes favoured coagulation but were possibly to some degree counterbalanced by an increased concentration of plasminogen and decreased antifibrinolytic activity.


Regulatory proteins of lobster striated muscle. The regulatory proteins of lobster muscles consist of tropomyosin and of troponin. Troponin contains a 17,000 chain weight component, two closely related components of about 30,000 and a 52,000 chain weight component. In addition to troponin, tropomyosin is required for the inhibition of the magnesium activated actomyosin ATPase activity in the absence of calcium and for the reversal of this inhibition by calcium. Lobster tropomyosin interacts with rabbit actin and lobster troponin interacts with rabbit tropomyosin. The 30,000 doublet component corresponds to the troponin-I of rabbit and inhibits the ATPase activity of actomyosin both in the presence and in the absence of calcium. The 17,000 component corresponds to the troponin-C of rabbit; it binds calcium and reverses the inhibition of the ATPase activity by troponin-I in the presence of calcium. No more than 1 mol of calcium is bound by a mole of troponin-C or by troponin. The 52,000 component interacts with tropomyosin and has been tentatively identified as troponin-T; however, it has not been demonstrated as yet that this component had a role in the regulation of lobster actomyosin.


Amino acid sequence studies on plasmin-derived fragments of human fibrinogen: amino-terminal sequences of intermediate and terminal fragments. The progressive changes in amino-terminal sequence brought about by the digestion of human fibrinogen by plasmin have been studied. In addition, the limit products (fragments D and E) have been isolated and characterized in the same way. These studies have confirmed the generally accepted scheme of fibrinogen being changed into a large molecular weight fragment X, which in turn is converted into an intermediate fragment Y and a limit fragment D, followed by the breakdown of fragment Y into an additional fragment D and another core fragment E. Our data allow the precise identification of several of the junctions being attacked, including one in a region of the gamma-chain whose sequence has not previously been reported. The cleavages are not singular in any case, however, and, as suggested by others, intermediate species exist which correspond to "early D," "late D," etc. In addition to localizing the exact bonds split by plasmin, we have been able to sequentially position the core fragments relative to each other, since the gamma-chain amino terminus of fragment D has been found to be contiguous to the known carboxy-terminal sequence of fragment E.


Subunit dissociation and unfolding of rabbit muscle phosphofructokinase by guanidine by hydrochloride. The denaturation of rabbit skeletal muscle phosphofructokinase by guanidine hydrochloride has been studied using fluorescence, light scattering, and enzyme activity measurements. The transition from fully active tetramer (0.1 M potassium phosphate (pH 8.0) at 10 and 23 degrees) to unfolded polypeptide chains occurs in two phases as measured by changes in the fluorescence spectrum and light scattering of the protein: dissociation to monomers at low guanidine hydrochloride concentrations (similar to 0.8 M) followed by an unfolding of the polypeptide chains, which presumably results in a random coil state, at high concentrations of denaturant (greater than 3.5 M). The initial transition can be further divided into two distinct stages. The native enzyme is rapidly dissociated to inactive monomers which then undergo a much slower conformational change that alters the fluorescence spectrum of the protein. The dissociation is complete within 2 min and is reversible, but the conformational change requires about 2 hr for completion and is not reversible under a variety of conditions, including the presence of substrates and allosteric effectors. The conformationally altered protomer reaggregates to form a precipitate at 23 degrees, but is stable below 10 degrees. The second major phase of the denaturation is fully reversible. A simple mechanism is proposed to account for the results, and its implications for the corresponding renaturation process are discussed.


Stoichiometry of labeling of myosin's proteolytic fragments by a purine disulfide analog of adenosine triphosphate. A site-specific analog of ATP, 6,6'-dithiobis (inosinyl imidodiphosphate (S2P-PNP), inactivates the ATPase activities of myosin's proteolytic fragments, heavy meromyosin (HMM) and subfragment one (SF1), by formation of mixed disulfides between the 6 position of the purine ring and certain key cysteines. The stoichiometry of the reaction was determined by quantitatively displacing the thiopurine nucleotides from the labeled enzymes with sodium[14-C]cyanide. The thiocyanatoenzyme formed regained 25 percent of the original activity showing that the cysteines modified were not essential for catalysis. The rate of uptake of label paralleled the rate of inactivation. HMM was completely inactivated when 4 mol of thiopurine nucleotide was bound. SF1 made by a papain digestion of myosin incorporarted 2 mol of thiopurine nucleotide when completely inactivated. Having adenylyl imidodiphosphate, areversible competitive inhibitor of myosin's ATPase, present during the inactivation of HMM by S2P-PNP demonstrated that only one cysteine per head needed to be blocked to inactivate the enzyme. Moreover, SF1 made by a trypsin digest of HMM was completely inactivated when only 1.1 mol of the thiopurine nucleotide bound again indicating that blocking only a single cysteine per head was sufficient to cause inactivation. This sulfhydryl is thought to be at an ATP binding site distinct from the ATPase site. The properties of this second ATP binding site are consistent with it being an ATP regulatory site.


Subunit location of sulfhydryl groups of myosin labeled with a purine disulfide analog of adenosine triphosphate. A purine disulfide analog of ATP, 6,6'-dithiobis(inosinyl imidodiphosphate), forms mixed disulfides with cysteine residues at what are believed to be ATP regulatory sites of myosin. Blocking these sites causes inactivation of the ATPase activity at the active sites. Two cysteine residues per head are specifically modifed by this disulfide analog. The thiopurine nucleotides can be stoichiometrically displaced from myosin by [14-C]cyanide to give a more stable thiocyanato derivative of the enzyme. [14-C]Thiocyanatomyosin (3.7 14-CN/myosin) was dissociated in 4 M urea and the individual subunits were isolated. The heavy chains each had 0.78 14-CN bound per 200,000 molecular weight unit. The light chain with molecular weight of 20,700 had 1.00 14-CN bound and the 16,500 molecular weight light chain had 0.65 14-CN bound. The two 19,000 molecular weight light chains were not labeled. The two labeled light chains have only a single cysteine which is stoichiometrically modified. These two light chains show a high degree of homology and presumably perform identical functions in myosin. Their specific modification by the purine disulfide analog and their other known properties suggest that they contribute directly to the ATP regulatory sites and may, in fact, function as regulatory subunits.


Localization of sodium pump sites in frog urinary bladder. [3H]Ouabain binding in frog and toad urinary bladder was investigated by short-circuit current (SCC), scintillation counting and autoradiographic techniques. SCC data and analysis of tissue digests following serosal exposure to ouabain showed that ouabain binding and inhibition of Na+ transport was completely reversible in toad bladder whereas, in frog bladder, [3H]ouabain was tightly bound and Na+ transport remained suppressed even after a 60-min washout. Mucosal exposure of frog bladder to [3H]ouabain or serosal exposure after preincubation with unlabeled ouabain led to a marked reduction in binding. Specificity of binding was assessed further  by adjusting the concentration of certain (Na+ -K+)-ATPase ligands(K+, ATP) to levels known to reduce ouabain binding. High K+ concentrations and depletion of endogenous ATP by incubation under anoxic conditions resulted in a significant drop in [3H]ouabain binding. Autoradiographic analysis showed that grains are localized primarily to the basolateral plasma membranes of the granular cells, providing direct morphological evidence for the location of Na+ pumps at these sites. Although autoradiographs did not provide sufficient resolution to rule out unequivocally ouabain binding to the mitochondria-rich cell, morphological evidence suggests that grain densities are significantly higher between adjacent granular cells than between granular cell-mitochondria-rich cell interfaces.


Temperature-dependent transitions of the myosin-product intermediate at 10 degrees C in the Mn(II)-ATP hydrolysis. 1. While below 10 degrees C, the initial burst of Pi liberation in the hydrolysis of Mn(II)-ATP by heavy meromyosin or myosin subfragment 1 was inhibited by the pre-addition of ADP without any change in the steady-state activity, it was not inhibited above 10 degrees C. The burst size was about one mole per two moles of myosin active sites. 2. Above 10 degrees C, the ultraviolet absorption spectrum of heavy meromyosin induced by ATP in MnCl2 was similar to that induced in MgCl2 and the spectral decay to the ADP-induced level occurred only after all the ATP in the solution was depleted. In contrast, below 10 degrees C the spectrum induced by ATP in MnCl2 decayed to the ADP-induced level within a few seconds after the addition of ATP, although ATP was present in the solution. 3. These two results indicate that in Mn-ATP above 10 degrees C at the burst site there is a myosin*-ADP-Pi complex generated by ATP hydrolysis while below 10 degrees C there is a myosin-product complex identical with the one generated by adding ADP (and Pi) to myosin. 4. At tempertures both above and below 10 degrees C, the Mn-ATP hydrolysis of heavy meromyosin was activated by actin  and superprecipitation of actomyosin occurred. Characteristics of these phenomena showed a transition at around 10 degrees C.


Nucleotide-binding properties of native and cold-treated mitochondrial ATPase. 1. The bound nucleotides of the beef-heart mitochondrial ATPase (F1) are lost during cold inactivation followed by (NH4)2SO4 precipitation. The release of tightly bound ATP parallels the loss of ATPase activity during this process. 2. During cold inactivation, the sedimentation coefficient (s20, w) of the ATPase first declines from 12.1 S to 9 S, then to 3.5 S. (NH4)2SO4 precipitation of the 9-S component also leads to dissociation into subunits with s20, w of 3.5 S. 3. The 9-S component still contains the bound nucleotides, which are removed when it dissociated into smaller subunits. 4. Reactivation of cold-inactivated ATPase by incubation at 30 degrees C is increased by the presence of 25% glycerol. ATP, however, does not have any clearcut effect on the degree of reactivation in the presence of glycerol. 5. ADP is an inhibitor of the reactivation, probably because it exchanges during reactivation for bound ATP giving rise to an inactive 12-S component. 6. The exchange of tightly bound nucleotides with added adenine nucleotides is more extensive and faster with cold-inactivated ATPase than with the native enzyme. During reactivation up to 1.6 moles of ATP and 1.0 mole ADP can exchange per mole enzyme. 7. Incubation with GTP, CTP or inorganic pyrophosphate induces an increased activity of the ATPase, which, however, soon declines in the presence of ATP. It also disappears on precipitation of GTP-treated enzyme with (NH4)2SO4.


Energy transduction in photosynthetic bacteria. VII. Inhibition of the coupling ATPase by N-ethylmaleimide related to the energized state of the membrane. N-Ethylmaleimide, at millimolar concentrations, irreversibily inhibits photophosphorylation and ATPase activity of photosynthetic membranes from Rhodopseudomonas capsulata. The inhibitory effect of N-ethylmaleimide is evident only the membranes are preincubated with the inhibitor in the light and in the absence of phosphorylation substrates. ADP and orthophosphate (or arsenate) exert a protective effect against the inhibition if they are present during the preillumination stage. The energization of the membrane by ATP hydrolysis, measured as ATP-induced quenching of 9-aminoacridine fluorescence, also is inhibited irreversibly by N-ethylmaleimide. Uncouplers protect the ATPase from inhibition by N-ethylmaleimide at concentrations at which they inhibit photophosphorylation. The ATPase, as measured either in the dark or in the light, is also inhibited by carbonylcyanide p-trifluoromethoxypenylhydrazone in parallel with photophosphorylation. These results are interpreted as evidence that the high-energy state of the membrane induces a conformational change of the ATPase, making it sensitive to attack by N-ethylmaleimide; this conformational change might be related to the active state of the ATPase.


Energy transduction in photosynthetic bacteria. VIII. Activation of the energy-transducing ATPase by inorganic phosphate. ATPase activity and ATP-induced energization of photosynthetic membranes from Rhodopseudomonas capsulata are stimulated by phosphate; the maximum stimulatory effect occurs at a concentration between 1 and 2 mM. The sensitivity of the ATPase to oligomycin increases in the presence of phosphate since all the Pi-stimulated activity is inhibited by this antibiotic. Aurovertin, which has no effect on ATPase in the absence of phosphate, inhibits completely the activity elicited by this anion. The addition of Pi induces a substantial increase in the V of ATPase activity without changing the affinity of the enzyme for ATP or ADP. Arsenate, at the same concentrations, produces effects very similar to those of phosphate. The stimulation by arsenate of the transfer of energy from ATP to the membrane suggests a non-hydrolytic role of this anion as a modifier of the ATPase activity.


Further characterization of gradient-fractionated submitochondrial membrane fragments from beef heart mitochondria. We have recently reported that with a linear sucrose density gradient centrifugation two distinct types of membrane fragments, designated as X- and Y-fragments are obtained (Huang, C. H., Keyhani, E. and Lee, C. P. (1973) Biochim. Biophys. Acta 305, 455-473). Further characterization of these two membranes fragments is reported. (1) Potassium chloride at the concentration of 0.15 m extracts 7% and 30% of cytochrome c from the X- and Y-fragments, respectively. (2) When cytochrome c was added to the mitochondrial suspension prior to sonication, the cytochrome c content was increased by 6-8-fold in both X- and Y-fragments. Subsequently KC1 extraction resulted in loss of cytochrome c by 1/4 in the X- and by 2/3 in the Y-fragments. (3) With partially inhibitory concentrations of KCN, cytochrome c in either the X- or the KC1 extracted X-fragments showed uncoupler-sensitive, biphasic reduction kinetics upon the addition of NADH to the oligomycin-supplemented system. Under identical conditions rapid first order reduction kinetics were seen for cytochrome c in Y-fragments supplemented with either oligomycin or oligomycin + carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). (4) When cytochrome c was added to the mitochondrial suspension after sonication, a significant amount of cytochrome c was bound to both X- and Y-fragments, but was readily removed with a high ionic strength medium. (5) Lubrol had little effect on the ATPase activity of the X- and the Y-fragments, suggesting a lack of membrane-buried ATPase. (6) Partial depletion of ATPase in X-fragments did not induce an increase in reactivity towards externally added cytochrome c. (7) Both the X- and the Y-fragments showed an energy-linked fluorescence enhancement of 8-anilinonaphthalene-1-sulfonate and an energy-linked fluorescence decrease of quinacrine. (8) In the presence of K-+ nigericin alone or in combination with valinomycin exhibited a stimulating effect on the rate of NADH oxidase of the oligomycinsupplemented X- and Y-fragments.


Physiological and genetic modifications of the expression of the yeast mitochondrial adenosine triphosphatase inhibitor. 1. The oligomycin-sensitive ATPase activity of submitochondrial particles of the glycerol-grown "petite-negative" yeast: Schizosaccharomyces pombe is markedly stimulated by incubation at 40 degrees C and by trypsin activations are treatment. Both increased in Triton-X 100 extracts of the submitochondrial particles. 2. A trypsin-sensitive inhibitory factor of mitochondrial ATPase with properties similar to that of beef heart has been extracted and purified from glycerol-grown and glucose-grown S. pombe wild type, from the nuclear pleiotropic respiratory-deficient mutant S. pombe M126 and from Saccharomyces cerevisiae. 3. ATPase activation by heat is more pronounced in submitochondrial particles isolated from glycerol-grown than from glucose-grown S. pombe. An activation of lower extent is observed in rat liver mitochondrial particles but is barely detectable in the "petite-positive" yeast: S. cerevisiae. No activation but inhibition by heat is observed in the pleitotropic respiratory-deficient nuclear mutant S. pombe M126. 4. The inhibition of S. pombe ATPase activity by low concentrations of dicyclohexylcarbodiimide dissapears at inhibitor concentrations above 25 muM. In Triton-extract of submitochondrial particles net stimulation of ATPase activity is observed at 100 muM dicyclohexylcarbodiimide. The pattern of stimulation of ATPase activity by dicyclohexylcarbodiimide in different genetic and physiological conditions parallels that produced by heat and trypsin. A similar mode of action is therefore proposed for the three agents: dissociation or inactivation of an ATPase inhibitory factor. 5. We conclude that "petite-positive" and "petite-negative" yeasts contain an ATPase inhibitor factor with properties similar to those of the bovine mitochondrial ATPase inhibitor. The expression of the ATPase inhibitor, measured by ATPase activation by heat, trypsin or high concentrations of dicyclohexylcarbodiimide, is sensitive to alterations of the hydrophobic membrane environment and dependent on both physiological state and genetic conditions of the yeast cells.


Energy requirements for biosynthesis of DNA in Escherichia coli. Role of membrane-bound energy-transducing ATPase (coupling factor). A mutant of Escherichia coli missing energy-transducing ATPase and known to be defective in a variety of membrane functions from earlier studies (Yamamoto, T. H., Mével-Ninio, M. and Valentine, R. C. (1973) Biochim. Biophys. Acta 314, 267-275; Thipayathasana, P. and Valentine, R. C. (1974) Biochim. Biophys. Acta 347, 464-468; Mével-Ninio, M. and Yamamoto, T. (1974) Biochim. Biophys. Acta 357, 63-66) has been found to be blocked for anaerobic DNA synthesis. The rate of anaerobic DNA synthesis in the mutant, measured as radioactive adenine incorporation into the alkali-resistant fraction of whole cells, is about 1/6 the rate of DNA synthesis in the wild type culture under similar conditions. Addition of NO-3- or O-2 restores DNA biosynthesis in the mutant. The entry of radioactive adenine is not appreciably affected in the mutant by anaerobiosis. It is concluded that coupling factor plays a role in some step(s) of DNA biosynthesis.


The effects of phosphate and electron transport on the carbonyl cyanide m-chlorophenylhydrazone-induced ATPase of rat-liver mitochondria. 1. The effects of phosphate and electron transport on the ATPase induced in rat-liver mitochondria by the uncoupler carbonyl cyanide m-chlorophenylhydrazone have been measured at different uncoupler concentrations and compared with those of ATP, oligomycin and aurovertin. 2. The inhibitory action of respiratory-chain inhibitors on the ATPase activity, which is independent of the actual inhibitor used, is greatly delayed or prevented by the presence of uncoupler, and, in the case of rotenone, can be reversed completely by the subsequent addition of succinate (in the absence of uncoupler). These results can be explained on the basis of the proposal previously made by others that coupled electron transfer causes a structural change in the ATPase complex that results in a decreased affinity of the ATPase inhibitor for the mitochondrial ATPase. 3. Inorganic phosphate specifically stimulates the ATPase activity at high uncoupler concentrations (greater than 0.2 muM), but has no effect at low concentrations. The stimulation is prevented or abolished by sufficiently high concentrations of aurovertin. 4. Aurovertin prevents the inhibition of the uncoupler-induced ATPase by high uncoupler concentrations. 5. It is proposed that the steady-state concentration of endogenous P-i may be an important regulator of the turnover of the ATPase in intact mitochondria and that the inhibition of ATPase activity by high concentrations of uncoupler is at least partially mediated via changes in the concentration of endogenous P-i.


Solubilization of bacterial membrane proteins using alkyl glucosides and dioctanoyl phosphatidylcholine. The non-ionic detergent octyl glucoside solubilizes a substantial amount of Streptococcus faecalis membrane protein without loss of the monitored enzyme activities. A secondary detergent, dioctanoyl phophatidycholine, appears to increase the yield of solubilized material. In addition, the effect of ionic strength indicates that it may be possible to selectively extract groups of membrane proteins by their characteristic solubility at different ionic strengths. The solubilized membrane-associated enzymes, ATPase and NADH dehydrogenase, enter polyacrylamide gels as distict species. Electrophoretic studies suggest that there are two membrane-associated ATPase in the Streptococcus faecalis, one which dissociates from the membrane in the absence of Mg-2+ ions and the other which remains particulate until solubilized by detergents. Octyl glucoside can be easily removed from a solution containing solubilized proteins and lipid by dialysis.


Membrane ATPase of Proteus L-forms. Solubilization and molecular properties. The Mg2+ -dependent ATPase (EC 3.6.I.3) of Proteus L-form membrane has been solubilized according to various procedures (Tris - HCL shock-wash with or without MG2+, EDTA, Triton X-100). The best results were obtained by the same 33mM Tris-HCL (pH 7.5) shock-wash without MG2+ used for ATPase of protoplasts from Streptococcus faecalis. The solubilized enzyme after 105 000 times g centrifugation was purified on acrylamide/agarose. The molecular weight was established to be 360 000 by gel filtration and by sedimentation coefficient (12.5 S). Polyacrylamide disc-gel electrophoresis in sodium dodecylsulphate revealed two classes or subunit of mol. wt. 64 000 (alpha) and 58 000 (beta), associated in ratio 1:1. We propose a formula alpha-3beta-3 for the native ATPase of Proteus L-forms. Structural similarities to ATPase of various origins are discussed.


Hydrolysis of erythrocyte membrane phospholipids by a preparation of phospholipase C from Clostridium Welchii. Deactivation of (Ca-2+, Mg-2+)-ATPase and its reactivation by added lipids. 1. Haemoglobin-free erythrocyte ghosts were prepared in 40 imosM bicarbonate buffer, pH 7.4, containing 1 mM EDTA (40 imosM/l mM EDTA). The ghost preparation was highly permeable on preparation but partially resealed on incubation in media containing Ca-2+. 2. A partially purified preparation of phospholipase C from Clostridum welchii caused an increase in observed Mg-2+-ATPase activity, reflecting a change in the permeability of the ghost to substrate. The phospholipase did not decrease Mg-2+-ATPase even at the highest levels tested. Mg-2+-ATPase activity could therefore be used as a permeability indicatior in these experiments. 3. Both (Ca-2+, Mg-2+)-ATPase activities of the ghosts were progressively lost as a result of the phospholipid hydrolysis induced by phospholipase C. 4. When a haemolysin in the commercial preparation was destroyed by heat-treatment, deactivation of the (Ca-2+, Mg-2+)-ATPase and (Na+, K+, Mg-2+)-ATPases were still observed but permeability changes were greatly reduced. 5. The products of phospholipase action were not inhibitory to the Ca-2+, Mg-2+)-ATPase. 6. Lysolecithin brought about a reactivation of the (Ca-2+, Mg-2+)-ATPase which was superimposed upon permeability changes in the preparation. 7. Reactivation of the (Ca-2+, Mg-2+)-ATPase was brought about by a nonlytic, mixed lipid preparation without significant effect upon permeability. 8. Human erythrocyte (Ca-2+, Mg-2+)-ATPase therefore appears to be an enzyme which responds to perturbation of the lipid environment in the membrane and is a "lipid-dependant" enzyme.


Enzymic synthesis of steroid sulphates.  XI. Study of the oestrogen binding site of oestrogen sulphotransferase by affinity labelling with 4-mercuri-17beta-oestradiol. Oistrogen sulphotransferase (3"-phosphoadenylylsulphate: oestrone sulphotransferase, EC 2.8.2.4) contains asingle sulphydryl group thought to be at, or near, the oestrogen-binding site. 4-mercuri-17beta-oestradiol, the activity of the enzyme decreased with increasing concentration of the oestrogen derivative. However, some 40% of the activity remained when all the sulphydryl had reacted to form mercaptide. Formation of mercaptide was only marginally decreased in the presence of the substrate 17beta-oestradiol. Other steroids, such as 11-deoxycorticosterone and testosterone, which are non-substrates for the enzyme, were more effective than 17beta-oestradiol in inhibiting mercaptide formation. Bovine serum albumin also reacted with 4-mercure-17beta-oestradiol and the effects of various steroids on mercaptide formation by the affinity label closely paralleled those found for the enzyme. 2t is concluded that the single sulphydryl group in the enzyme is not directly involved in the binding of oestrogen at the active site but is perhaps in closer proximity to a second site capable of binding certain non-substrate steroids.


Enzymatic degradation of heparin-related mucopolysaccharides from the surface of endothelial cell cultures. When cultures of endothelial cells prelabeled with H2 -35-SO4 are exposed to a purified preparation from induced Flavobacterium heparinum containing heparinase and heparitinase activities, radioactivity accumulates in the supernatant medium.  After further treatment in vitro with crude enzyme this material migrates, in part, as glucosamine (N,O-disulfated glucosamine), a break-down product characteristic of heparin and heparin-related mucopolysaccharides.  After exposure of the cultures to the purified enzyme, the amount of acid-insoluble -3 5-S radioactivity that can be removed with EDTA is decreased compared to that that can be removed from control cultures.  Since the amount of radioactivity that is released as break-down products is much higher than the amount of radioactivity that is secreted into the supernatant medium as intact (non-dialysable) mucopolysaccharide chains in control plates, the action of the enzyme appears to be on the cell itself.  The data presented support previous studies suggesting that chains of heparitin sulfate that are accessible to the action of the enzyme are present at the surface of endothelial cells.


Dynamic affinity chromatography of heavy meromyosin subfragment-1. Heavy meromyosin subfragment-1 and its trinitrophenylated derivative have been chromatographed on immobilized ATP, ADP and adenosine 5'-(geta, gamma-imino) triphosphate affinity chromatography columns, in the presence and in the absence of Ng-2+ or Ca-2+.ma-32-P] ATP columns.  While the divalent cations had little effect on the chromatographic pattern in the case of the non-hydrolyzable ADP and adenosine 5' (beta, gamma-imino) triphosphate, they catalyzed splitting in the case of ATP and at the same time strongly increased the affinity of adsorption of the proteins.  The protein-elution and the Pi-release patterns were different for the native and the modified proteins.  These results have been interpreted in terms of protein binding to the various intermediates of the ATP hydrolysis reaction.


Apparent sulfation of glycosaminoglycans by ascorbic acid 2-[3 5-S] sulfate: an explanation. The sulfation of glycosaminoglycans by ascorbic acid 2-[35S]sulfate was studied in costal cartilage and chondrocytes in vitro. Negligable (if any) sulfation of glycosaminoglycans was detected with immediately isolated ascorbic acid 2-[35S]sulfate. However, formation of [35S]glycosaminoglycans was readily detected with ascorbic acid 2-[35S]sulfate which had been stored at minus 20 degrees C for several days. The [35S]glycosaminoglycans did not result from the direct transfer of 35S from ascorbic acid 2-sulfate but rather from a decomposition product of ascorbic acid 2-[35S]sulfate. Evidence is presented to show that the sulfation pathway with the decomposition product involves exchange with inorganic sulfate, and strongly suggests that sulfation proceeds via 3'-phosphoadenosine 5'-phosphosulfate. The decomposition product appears similar to inorganic sulfate in several test systems. In view of these observations, it is suggested that previous conclusions implicating as acid 2-sulfate as a biological sulfate donor, based on the use of ascorbic acid 2-[35S]sulfate be re-evaluated.


Structural studies of heparitin sulfates. Heparitin sulfate fractions with a large range in sulfate content were subjected to degradation by Flavobacterium heparinase and by nitrous acid. The products obtained were fractionated by chromatography, characterized, and used to arrive at tentative structures for these complex polysaccharides. The heparitin sulfate chains examined appear to be composed of: 1. uninterrupted blocks of N-acetylglucosamine containing disaccharides; 2. larger blocks with a molecular weight range of 5000 to 6000 which include the N-acetyl block but do not contain heparinase sensitive linkages; 3. segments containing mainly areas where N-acetyl, N-sulfate and some disulfated units alternate in the chain. The size and arrangement of these polymer segments seem to vary with the sulfate content of a particular heparitin sulfate. For instance, the polysaccharides with the highest degree of sulfation do not appear to contain N-acetyl blocks of significant size.


In vitro restoration of nitrate reductase: investigation of Aspergillus nidulans and Neurospora crassa nitrate reductase mutants. Extracts of Aspergillus nidulans wild type (bi-1) and the nitrate reductase mutant niaD-17 were active in the in vitro restoration of NADPH-dependent nitrate reductase when mixed with extracts of Neurospora crassa, nit-1. Among the A. nidulans cnx nitrate reductase mutants tested, only the molybdenum repair mutant, cnxE-14 grown in the presence of 10-minus 3 M Na2 MoO4 was active in the restoration assay. Aspergillus extracts contained an inhibitor(s) which was measured by the decrease in NADPH-dependent nitrate reductase formed when extracts of Rhodospirillum rubrum and N. crassa, nit-1 were incubated at room temperature. The inhibition by extracts of A. nidulans, bi-1, cnxE-14, cnxG-4 and cnxH-3 was a linear function of time and a logarithmic function of the protein concentration in the extract. The molybdenum content of N. crassa wild type and nit-1 mycelia were found to be similar, containing approx. 10 mu g molybdenum/mg dry mycelium. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The enzyme purified from wild-type N. crassa contained more than 1 mol of molybdenum per mol of enzyme, whereas the enzyme purified from nit-1 contained negligible amounts of molybdenum.


Glomerular protocollagen lysyl-hydroxylase activity in streptozotocin diabetes. Using [14-C]lysine protocollagen substrate prepared from chick embryo tibiae, lysyl hydroxylase activity was found in the 17 000 times g supernatant and particulate fractions obtained from homogenates of isolated rat renal glomeruli. Specific activities using the latter as an enzyme source were about 20-30% that of the supernatant. [14-C]Hydroxylysine formation was proportional to substrate and enzyme concentration, and to time for up to 120 min of incubation. Omission of alpha-ketoglutarate and ascorbate in the incubational assay markedly depressed activity. Hydroxylation of substrate by supernatant enzyme from streptozotocin diabetic rats was significantly increased over that of normal. In contrast, the activity of supernatant fractions from glomeruli of pancreatectomized, normoglycemic animals did not differ from that of non-operated controls. It is concluded that elevated glomerular lysine hydroxylase activity accompanies the increased glomerular collagen synthesis found in streptozotocin diabetes, and that chronic hyperglycemia may be implicated in these changes.


Characterization of the terminal degradation products of canine fibrinogen by plasmin. Fibrinogen, isolated from canine plasma by the successive procedures of (1) freezing and thawing, (2) fractional precipitation with 25% saturated (HN4)2SO4 and (3) Sepharose 6B gel-filtration, had a molecular weight of 282 000 by the rapid sedimentation equilibrium method. However, a molecular weight for canine fibrinogen of 332 000, which is closer to that reported for human and bovine fibrinogens (340 000 plus or minus 20 000), was obtained from the sum of the molecular weights of the Aalpha, Bbeta and gamma chains, determined from dodecylsulfate gel electrophoretic patterns of reduced fibrinogen. Canine fibrinogen, subjected to proteolysis by urokinase-activated plasminogen for 24 h, contained degradation fragments D and E which were isolated by starch block electrophoresis and Sephadex G-200 gel-filtration. The purified D and E fragments with sedimentation coefficients of 5.0 S and 2.5 S had weight average molecular weights of 89 000 and 42 000, respectively by the rapid sedimentation equilibrium method. The ratio of D to E was 2:1 per parent fibrinogen molecule. Antigenic analysis according to anti-fibrinogen antiserum showed that both D and E fragments were antigenically deficient to native fibrinogen and revealed a reaction of non-identity with each other. Upon immunoelectrophoresis at pH 8.2, D and E had different electrophoretic mobilities. Preliminary studies indicate that based on thrombin time alone, D has anticoagulant activity while E appears to be a coagulation potentiator. Canine fibrinogen apparently consist of two core fragments with dissimilar chemical characteristics in common with the fundamental structures of human and bovine fibrinogens.


The maintenance of the energized membrane state and its relation to active transport in Escherichia coli. 1. An ATPase mutant of Escherichia coli and two partial revertants of that mutant were examined for the ability to generate a high energy membrane state with D-lactate or ATP, as measured by the quenching of the fluorescent dye quinacrine. 2. All three strains showed reductions in the aerobically-driven quenching of fluorescence compared to the wild type, but the reduction could be reversed by the addition of eitherN,N'-dicyclohexylcarbodiimide or the crude soluble ATPase of the wild type. 3. The mutant exhibited a decreased ability to accumulate sugars and amino acids and showed an increased permeability to protons. 4. One partial revertant showed a slight increase in active transport and a slight decrease in proton permeability. 5. The other partial revertant showed a large increase in transport ability and a large decrease in proton permeability. 6. A model is proposed in which the conformation of the Mg-2+-ATPase is important in the utilization of energy derived from the electron transport chain and this function is independent of the catalytic activity of the Mg-2+-ATPase.


Micrococcus lysodeikticus ATPase.  Purification by preparative gel electrophoresis and subunit structure studied by urea and sodium dodecylsulfate gel electrophoresis. Micrococcus lysodeikticus ATPase was purified by preparative gel electrophoresis after its "shodk wash" release from the membrane.  The method afforded the highest yield of pure protein in the minimum time as compared with former purification procedures.  The pure protein had a specific activity of 7 mumol Pi-min- minus 1-mg- minus 1 with incubation times not longer than 3 min, 345 000 mol. wt and was not stimulated by trypsin.  By gel electrophoresis at alkaline pH (8.5) in 8 M urea or in sokium dodecylsulfate, the ATPase revealed a complex pattern with two major subunits (alpha and beta) and two minor ones (gamma and delta).  The non-identity between the major subunits was demonstrated.


ATPase inhibitor from yeast mitochondria.  Purification and properties. 1. Mitochondria from Candida utilis CBS 1516 and Sacchromyces cerevisiae JB 65 possess an ATPase-inhibitor activity.  The inhibitor activity depends on the growth conditions of the yeast cells.  It is markedly decreased when the cells are grown in the presence of a high concentration of glucose, which suggests that glucose represses the synthesis of the ATPase inhibitor or of a protein required for the insertion of the inhibitor into the inner mitochondrial membrane.  2. The ATPase inhibitor has been isolated from D. utilis mitochondria and purified to homogeneity.  The minimal molecular weight calculated from amino acid composition is close to 7500.  Dtermination of the molecular weight by sokium dodecylsulfate-polyacrylamide gel electrophoresis gives a value close to 6000.  3. The ATPas inhibitor of C. utilis mitochondria differs from the beef heart ATPase inhibitor by a number of properties.  It has a lower molecular weight (6000-7500 vs 10500), a different amino acid composition, and a more acidic isoelectric point 5, 6 vs 7, 6).  In spite of these differences, the C. utilis inhibitor cross-reacts with the ATPase of beef heart submitochondrial inhibitor-depleted particles.  4. The interaction of the C. utilis inhibitor with the ATPase of inhibitor-depleted particles requires the addition of Mg-2+-ATP or ATP in the incubation medium.  5. 14-C labelling of the C.utilis inhibitor has been achieved by growing C. utilis in a medium supplemented with [14-C]leucine.  It has been found by titration experiments that the C. utilis 14-C-labelled inhibitor binds to the homologous submitochondrial inhibitor-depleted particles with a KD of about 10- minus 7 M.  The number of binding sites is of the order of 0.1 nmol/mg protein.


Tightly bound nucleotides of the energy-transducing ATPase of chloroplasts and their role in photophosphorylation. 1. Like other energy-transducing membranes, chloroplast membranes bear a coupling ATPase with especially tight binding sites for adenine nucleotides.  Membranes washed several times still contain 2.5 nmol ATP and 1.3 nmol ADP bound per mg chlorophyll, which is equivalent to 1.9 ATP and 1.0 ADP per coupling ATPase.  2. In de-energized membranes, these nucleotides exchange to only a limited extent with added nucleotides.  In membranes illuminated in the presence of pyocyanine, however, complete exchange of the bound nucleotides occurs rapidly, irrespective of whether ATP or ADP is present in the medium.  3. Pi can exchange into these nucleotided at both the beta and gamma positions when the membranes are energized in the presence of Mg-2+.  Equilibrium with the beta and gamma groups of th ebound nucleotides is, however, not complete.  4. The inhibitors and uncouplers Dio-9, S13 and EDTA have different effects on the exchange of nucleotides, the exchange of inorganic phosphate and photophosphorylation.  5. The bound ATP level on the membrane is stable to a wide variety of conditions.  The ADP level, however, drops to near zero under conditions of maximal activation of the emmbrane ATPase.


Effect of concanavalin A on membrane-bound enzymes from mouse lymphocytes. The ionic influence and ouabain sensitivity of lymphocyte mg-2+-atpase and Mg-2+-(Na+ +K+)-activated ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5'-nucleotidase and Mg2+-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of (Na+ +K+)-ATPase was located inside the membrane. Concanavalin A induced an early stimulation of Mg2+-APTase and (Na+ +K+)-ATPase both on intact cells and purified plasma membranes. In contrast, 5'-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg2+-ATPase activity was 3-5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20%). (Na+ +K+)-ATPase activity was undectectable in thymocytes. However, in spleen lymphocytes (Na+ +K+)-ATPase activity can be detected and was 30% increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg2+-ATPase, in lymphocyte stimulation.


A comparative study on the effects of different bile salts on mucosal ATPase and transport in the rat jejunum in vivo. The effects of deoxycholate, taurocholate and cholate on transport and mucosal ATPase activity have been investigated in the rat jejunum in vivo using closed-loop and perfusion techniques. In the closed-loops, 5 mM deoxycholate selectively inactivated (Na+ + K+)-ATPase, and net secretion of Na+ induced by 2.5 mM deoxycholate was due to reduced lumen to plasma flux of the ion; deoxycholate (2.5 mM) produced marked inhibition of 3-0-methylglucose transport. Luminal disappearance rates of deoxycholate (60.5 plus or minus 2.9% per g wet st of gut) greatly exceeded those of taurocholate (4.3 plus or minus 1.0). In the perfusion studies 1 mM deoxycholate induced net secretion of water, Na+ and C1-, and inhibited active glucose transport; concomitantly "total" ATPase, (Na+ + K+)-ATPase, and Mg-2+-ATPase were inhibited. At higher concentrations (5 mM) deoxycholate stimulated Mg-2+-ATPase activity. Taurocholate and cholate at 1mM had no effect on transport of (Na+ + K+)-ATPase. Mucosal lactase, sucrase and maltase activities were not affected by 1 mM deoxycholate, taurocholate or cholate. These results suggest that deoxycholate inhibits sodium-coupled glucose transport by inhibition of (Na+ + K+)-ATPase at the lateral and basal membranes of the epithelial cell, rather than from an effect at the brush-border membrane level.


Absence of (Na+,K+)-ATPase involvement in lactose production by lactating guinea pig mammary gland. The role of the (Na+, K+)-ATPase system in lactose production by the lactating guinea pig mammary gland has been studied in vitro with slices of the gland. In this system there is an initial fast lactose release, mainly representing secretion of preformed lactose, followed by a continuous slow lactose release, representing mainly lactose synthesis. The latter process occurs at a rate of 1.6 to 2.4 g lactose/kg wet wr/h, which value is about half of the lactose production in vivo (3.9 g/kg set wt/h). Incubation of slices in the presence of 10-4 M ouabain does not influence the rate of overall lactose production. When determined separately, it does not change either the rate of secretion or that of synthesis. This pleads against a role of the (Na+, K+)-ATPase system in lactose secretion or synthesis, in particular it seems to rule out control of the rates of these processes by the intracellular potassium concentration. An explanation for the generally observed correlation between the lactose and potassium concentrations in milk, may be that both the maintenance of the intracellular potassium concentration and the lactose synthesis rate require the presence of ATP.


Clinical applications and theoretical analysis of NMR blood flowmeter. Nuclear magnetic resonance principles have been applied to measure biological blood flow. The method is non-intrusive and can be used to measure peripheral blood flow or blood flow from various organs. The mechanisms of NMR are described and studies of blood flow in limbs and the head are discussed.


The Departments of Medical Eelectronics, St. Bartholomew's Hospital--the first ten years. The organisation of an electronics department required to support the work of a large teaching hospital is described. The research and development programme involves the interaction of graduates from many disciplines since problems can emerge from any of the specialities, for example general surgery, obstetrics and gynaecology, clinical neurophysiology. Examples from the research and development programme of the department are described in order to illustrate the range of medical electronic techniques needed to provide adequate support for the clinical research programmes which have received support from the Joint Research Board of the Hospital and Medical College.


Measurement of venous blood flow and pressure using thermoelectric and pressure transducers. In order to simplify procedures for testing the circulatory function of a patient, changes in the venous blood flow as well as venous pressure following exercise were continuously observed using the thermoelectric flowmeter. The level of zero flow necessary for evaluation of the velocity of flow was successfully indicated without occlusion of blood vessels by means of a form of compensation for thermal diffusion devised by the authors.


[The influence of rheologic properties of the blood on adaptive processes in the myocardium]. Changes in rheological properties of the blood were produced by intravenous injection of a high-molecular weight dextran and lysin-vasopressin. The animals were decapitated in one hour. Oxygen absorption by mitochondria of the heart in oxidation of 2.5-10 mM of the succinate increased by 90-120%, as compared to control. Stimulation of respiration by ADP was decreased 1.5-2 times. Simultaneous administration of the succinate and glutamic acid normalized the respiration and phosphorylation. A possibility of inhibition of succinic-dehydrogenase by the oxalo-acetic acid was suggested. Switching of respiration to succinic acid and limiting of the SDG activity can be considered as adaptive factors under conditions of changes in rheological properties of the blood, and are directed to the maintenance of cardiac activity, this being evidenced by the absence of changes in the ATP-asic activity and in the myosin content of the heart.


MLC stimulatory capacity and production of blastogenic factor in patients with chronic lymphatic leukemia and Hodgkin's disease. The aim of the present study is to evaluate the stimulatory capacity of chronic lymphatic leukemia (CLL) and Hodgkin's disease (HD) lymphocytes in the one-way mixed lymphocyte reaction (MLR) and to investigate whether, in patients with these diseases, a correlation exists between the degree of stimulatory capacity and production of a blastogenic factor. These studies might help to resolve the controversy about the nature of the stimulating cells and to offer some insight into the mechanism of the stimulation in the MLR. The significant observations from this study are summarized as follows: (1) Stimulatory capacity of CLL lymphocytes was intact or increased, while their responding capacity was markedly depressed. (2) In our group of patients with advanced Hodgkin's disease, both stimulating and responding capacities were imparied. (3) In these patients a correlation existed between the degree of stimulatory capacity and production of a blastogenic factor. This observation would raise the possibility that the production of a blostogenic factor may in some way be involved in the stimulation of responding lymphocytes in mixed cultures. Our results, which show a dichotomy between MLC stimulatory and responding capacities of CLL lymphocyte, may suggest that different factors are involved in stimulation and response.


The effect of various amine-depleting drugs on the fever response exhibited by rabbits to bacterial or leucocyte pyrogen. 1 The concentration of noradrenaline, dopamine and 5-hydroxytryptamine (5-HT) in the rabbit brainstem was measured during fevers produced by either an injection of bacterial pyrogen (BP) or continuous infusion of leucocyte pyrogen (LP). 2 Both procedures had little effect on the concentration of noradrenaline in the preoptic/hypothalamic area but significantly (P smaller than 0.001) lowered the concentration of noradrenaline in the midbrain and pons/medulla. 3 BP significantly (P smaller than 0.01) lowered the concentration of 5-HT in the preoptic/hypothalamic area but had no effect in the midbrain or pons/medulla, whereas LP significantly (P smaller than 0.01) lowered the concentration of 5-HT in the midbrain and pons/medulla but had little effect in the hypothalamus. 4 The concentration of dopamine throughout the brainstem was little affected by either BP or LP fevers. However the concentration in the midbrain was significantly reduced by LP (P smaller than 0.001). 5 Alpha-Methyltyrosine (200 mg/kg) pretreatment diminished the pyrogenic response to both BP and LP whilst p-chlorophenylalanine (300 mg/kg) slightly enhanced the response to both forms of challenge. 6 Reserpine (1 mg/kg) diminished both types of fever whilst a combination of alpha-methyltyrosine and p-chlorophenylalanine slightly enhanced the fevers produced by either BP or LP. 7 The results obtained are discussed in relation to the mechanisms involved in the production of fever and to the possible function of noradrenaline and 5-HT as thermoregulatory transmitters.


Treatment of deep vein thrombosis with streptokinase. From September 1962 to May 1972 145 patients with acute or subacute deep vein thrombosis confirmed by phlebography were treated with streptokinase. During the same period 42 patients considered unfit for thrombolytic therapy were treated with herapin and oral anticoagulants. The results, assessed by repeat phlebography, in 93 of the patients treated with streptokinase were compared with those in 42 patients treated with heparin. The age, sex, and severity of occlusion were roughly similar in both groups. Streptokinase treatment was successful in 42 per cent, partially successful in 25 per cent, and unsuccessful in 32 per cent of the 93 patients compared with none, 10 per cent, and 88 percent respectively in the 42 patients treated with heparin. Streptokinase was more effective when the thrombus was in proximal rather than calf veins. Thrombi of more than six days old were readily lysed. Plasma fibrinogen levels were below 0-8 g/1 (80 mg/100 ml) in nearly all patients successfully treated. The incidence of pulmonary embolism was no greater with streptokinase than with heparin treatment. Only prolonged follow-up would show whether thrombolytic treatment would be effective in preventing late complications of deep vein thrombosis such as chronic venous insufficiency.


Radiofibrinogen catabolism in nephritis. A radiofibrinogen catabolism study performed on 40 patients with glomerulonephritis has shown increased fibrinogen catabolism in active immune-complex disorders.


Complications of laparoscopy.

Complications of laparoscopy. Five years' experience with the laparoscope raises doubts about its continued use for sterilization. As a diagnostic tool, however, it serves a useful function.


Manipulation in treatment of low back pain: a multicentre study. In a multicentre trial 456 selected patients with low back pain were randomly allocated to one of four treatments-manipulation, definitive physiotherapy, corset, or analgesic tablets. Patients were reassessed clinically after three weeks' treatment and again after a further three weeks. Questionnaires were used to find out the patients' condition three months and one year after admission to the trial. There were never any important differences among the four groups of patients. A few patients responded well and quickly to manipulation, but there was no way of identifying such patients in advance. The response to a corset was slow, but the long-tern effects were at least as good as those of the other treatments. Patients treated only with analgesics fared marginally worse than those on the other three treatments. There is no strong reason, however, for recommending manipulation over physiotherapy or corset.


Antibiotic susceptibility of Neisseria gonorrhoeae isolated in the Western Pacific in 1971. Isolates of Neisseria gonorrhoeae from 256 patients contracting acute gonococcal urethritis, primarily in the Republic of the Phillipine Islands, in 1971, were tested for the minimum inhibitory concentrations of five antibodies. The median is advocated as a measurement for comparison of ranges of MIC values; the median MIC of penicillin for 258 isolates was 0-23 mug/ml. A positive correlation coefficient was observed for the susceptibility of isolates to penicillin, spectinomycin, cephaloridine, and tetracycline.


Sectinomycin in the treatment of gonorrhoea in females and males. 126 female patients with proven gonococcal infection were treated with 4 g. spectinomycin bihydrochloride given intramuscularly in a single does. In 120 cases followed up there were four possible treatment failures, giving a cure rate of 97-6 per cent. 99 male patients with acute gonococcal urethritis were treated with a single dose of 2, g. sectinomycin bihydrochloride. Of 88 cases followed up, there were three possible treatment failures, giving a cure rate of 96-6 per cent. Spectinomycin is an important addition to gonorrhoea therapy but should be reserved for penicillin sensitive patients and penicillin insensitive infections.


Subunit structure and some properties of pyruvate kinase of Neurospora. Pyruvate kinase isolated from Neurospora and purified to homogeneity has been shown to be a tetramer of molecular weight around 242 000 by gel filtration studies and 239 000 daltons by sedimentation equilibrium measurements. The monomer produced by treatment with guanidine hydrochloride is found to be 51 000-52 000 daltons by sedimentation equilibrium studies; a molecular weight of 62 000 was determined for the monomer generated by SDS treatment by electrophoresis in SDS-polyacrylamide gels. The enzyme has an isoelectric point of 6.35-6.41; Substrate saturation kinetics of PEP show a variable extent of cooperativity depending upon the buffer ions employed in the assay. ADP is the most effective phosphoryl group acceptor, GDP and IDP being poor substitutes. A divalent cation, Mg-2+, is required for activity. At low concentrations, Ca-2+ acts as an activator of pyruvate kinase but it is inhibitory  at high concentrations. Fructose 1,6-diphosphate is the most potent allosteric activator, fructose 6-phosphate being next in order of effectiveness. Valine is a powerful inhibitor. Phenylalanine, tyrosine, and tryptophan are without any effect individually, but their simultaneous presence results in a considerable activation. Alanine does not affect this enzyme appreciably.


Rabbit cardiac myosin. I. Physical and chemical characterization of the native molecule. Rabbit cardiac myosin, isolated from frozen tissue, was effectively purified by batchwise treatment with DEAE-cellulose in addition to suing cilution-precipitation techniques. An extensive experimental program was subsequently carried out with respect to the enzymic amino acid, optical and physicochemical properties of native cardiac myosin. This program has included the following: examination of the effects of pH and varying concentrations of ATP, CaCl2, MgCl2, and PCMB on its ATPase activity; measurement of its circular dichroic spectrum in solvent buffers, at different pH or containing ATP in the absence or presence of Ca-2+ or Mg-2+ ions; study of the concentration dependence of its viscosity and sedimentation velocity at low temperatures; and investigation of its molecular weight by the Archibald method and low- and high-speed sedimentation equilibrium. The results of these studies were consistent with the interpretation that cardiac myosin is comprised of highly asymmetric, semi-rigid molecules with a molecular weight in the order of 4.7 times 10-5, which display non-ideality even in solvent buffers of high ionic strength at neurtal pH. In addition, computer analysis of the high-speed sedimentation equilibrium data has provided evidence for the presence of a self-association reaction at low protein concentration. Even though the specif ATPase activity of cardiac myosin was found to be approximately one-third that reported for skeletal myosin in all cases, it was concluded, on the the basis of the essentially analogous physical and chemical properties of rabbit cardiac and skeletal myosin, that the two proteins are very similar in terms of molecular size, shape, and secondary structure.


Dehydroepiandrosterone and dehydroepiandrosterone sulfate dynamics in obesity. Dehydroepiandrosterone (D) and dehydroepiandrosterone sulfate (DS) dynamics were studied in three obese female subjects following a single injection of [4-14-CA1D and [7 alpha-3-H]-DS tracers. Dynamic parameters were calculated simultaneously by both the urinary and blood method of compartmentalization; Estimates for the urinary secretion and production rates of D were found to be high, and those of DS varied within normal range. Calculation of the conversion factors, rho DDS and rho DSD, by the urinary method revealed a noraml extraglandular DS yields D conversion, while that for D yields DS appeared deficient in obese female subjects. Estimates of inner and outer pool distribution volumes were extremely increased for free D; in contrast to this, moderately increased inner and decreased outer pool volumes of DS were observed. The metabolic clearance rates of D were normal or decreased and those for DS were greater than normal. The blood production rates of both B and DS were higher in obese female subjects than those estimated for normal women in our previous study; These observations suggest a considerable uptake of unconjugated D by adipose tissue, an overall poor D yields DS conversion and an accelerated DS metabolism in obese female subjects.


Existence of two chalone-like substances in intestinal extract from the adult newt, inhibiting embryonic intestinal cell proliferation. The inhibiting effect of tissue extract from fully differentiated intestinal mucosa of adult animals on proliferation kinetics of exponentially growing embryonic epithelial gut cell populations was studied in the newt Pleurodeles waltlii. Crude extract was fractionated by G-200 Sephadex chromatography and the effect of fractions on cell proliferation was studied using both mitotic index and 3-H-thymidine incorporation methods. The inhibitions we obtained were then displayed by means of cytophotometric study of age distribution of intestinal gut cells around the cell cycle, measuring the Feulgen-DNA content. The results revealed the presence of two chalone-like substances in the intestine of adults. One (factor 1) is characterized by a molecular weight of between 120,000 and 150,000 and inhibits the cell cycle at the end of the G1 phase, the other (factor 2) is characterized by a molecular weight lower than 2000 and inhibits the cell cycle in the course of the G2 phase. The cells delayed in the G2 phase escape from inhibition but the cells delayed in the G1 phase do not, although availability time of both factor 1 and factor 2 is about 12 hr. It is thus thought that cells prevented from dividing in G1 phase are indefinitely delayed in this phase and possibly differentiate.


Chemical definition of the mucopolysaccharidoses. Glycosaminoglycans were isolated from the urines of 46 patients with mucopolysaccharidosis; 11 with Type I (Hurler), 8 with Type II (Hunter), 16 with Type III (Sanfilippo A and B), 9 with Type V (Scheie), one with Type VI (Marateaux-Lamy), and one unclassified. All 46 patients excreted in their urine excessive amounts of dermatan sulfate, heparan sulfate or both. In addition, patients of certain types excreted excessive amounts of chondroitin sulfates A and/or C. There is a trend in each type of the disease towards the same carbazole/orcinol ratio, glucosamine/galactosamine ratio and glycosaminoglycan composition. Molecular weight distribution of the urinary glycosaminoglycans by gel filtration from Sephadex G-200 is characteristic for each different type of mucopolysaccharidosis and is distinguished from normal controls and patients without mucopolysaccharidosis. Preparation of elution diagrams from Sephadex G-200 allows an estimation of the composition of the glycosamino-glycans. Practically all heparan sulfate and a sizable part of dermatan sulfate from the urinary glycosaminoglycans of all these patients have been highly degraded. In all the patients in which the specific enzyme defect was demonstrated, the assignment of the type of mucopolysaccharidosis, on the basis of the elution diagrams, was correct. Patients with mucopolysaccharidosis Type V displayed two conspicuously different types of elution patterns, suggesting heterogeneity. Indeed, only a portion of these patients showed alpha-L- iduronidase deficiency. Carriers had normal urinary glycosaminoglycan output and composition and exhibited normal elution diagrams.


[Study of soluble proteins from human amniotic fluid by fraction chromatography (author's transl]. The nature and origin of soluble proteins from human amniotic fluid have been investigated by gel filtration chromatography on Sepharose 6B and ion-exchange chromatography on Ecteola-cellulose. Each fraction has been studied by immunoelectrophoresis. Specific antisera against serum proteins, amniotic fluid proteins, fetal proteins and salivary proteins have been used. These various antisera showed that the amniotic fluid contains maternal proteins, but also more specific proteins from fetal origin such as beta2-microglobulin, urinary mucopolysaccharides, salivary proteins and carcinoembryonic antigen. These results not only confirm that amniotic proteins are essentially from maternal origin, but prove the important fetal contribution and emphasize the importance of the fetoplacental unit in the monitoring of high risk pregnancies.


Familial balanced (7;11;21) translocation and Down's syndrome in two siblings. Cytogenetic studies of a family with two children with Down's syndrome have revealed a balanced reciprocal translocation between chromosomes No. 7, 11 and 21 in the mother. One of three daughters has inherited this translocation. two mongoloid daughters have a supernumerary chromosome No. 21 in addition to the translocation.


Investigations on Huntington's disease in the Canadian Prairies. I. Prevalence. One hundred and sixty-two confirmed cases of Huntington's disease have been ascertained in the provinces of Saskatchewan and Manitoba in the Canadian Prairies. They were encountered in 97 sibships which were derived from 58 kindreds. The prevalence of the disorder was estimated to be 8.4 per 100,000 individuals. Taking into account the mean age of onset of the disorder, 23.3 per 100,000 individuals are expected to be heterozygous for the Huntington's disease allele, the frequency of which is thus computed to be 0.0001168.


Investigation on Huntington's disease in the Canadian Prairies. II. Fecundity and fitness. The reproductive performance of 157 patients with Huntington's disease, their 170 normal siblings and 150 matched unrelated controls is presented. Comparison with the general Canadian Prairie population in the appropriate age group is undertaken. Patients with Huntington's disease are not only more fecund than their unaffected sibs but also out-reproduce unrelated normal controls and members of the general population of corresponding age. Their fitness is thus estimated to be 1.14. Should the present reproductive pattern continue, the allele for Huntington's disease will double its frequency with consequent doubling of the prevalence of the disorder in six generations or approximately 150 years' time.


Investigation on Huntington's disease. III. Biochemical observations, a possibly predictive test? The possible biochemical basis underlying the aetiology and pathogenesis of Huntington's disease (H.D.) is discussed. The development of a predictive test for presymptomatic detection of the disorder, based on measurement of serum caeruloplasmin and serum dopamine beta-hydroxylase (DBH) is also described. Possible means of appraising the proposed test are suggested.


Cellular control of calcium movements in bone. Interrelationships of the bone membrane, parathyroid hormone and alkaline phosphatase. Bone ECF is separated from the general ECF by a functional membrane which has been shown to limit the mineralization of embryonic tibiae and to selectively pump Ca out of the bone ECF. Energy to run this pump may be derived from ATP hydrolyzed by Ca-2+-stimulated ATPase, an enzyme activity which bone alkaline phosphatase may possess. The data suggest that PTH rapidly and selectively increases Ca pumping possibly by increasing ATPase activity in the bone cell cytosol through increased Ca influx and by increasing the intracellular ATPase level through inhibited secretion or excretion of the enzyme.


Sutureless epicardial pacemaker lead: a satisfactory preliminary experience. The first pacemaker systems used the epicardial electrode. The transvenous endocardial electrode rapidly supplanted the epicardial electrode since it could be positioned with less morbidity and mortality and was associated with a lower incidence of wire breakage. The long term complication rate of the transvenous electrode had not been inconsequential. The sutureless epicardial electrode combines the greater reliability of the epicardial lead system with the ease of insertion and low morbidity of the endocardial lead system. We have used this electrode in 33 patients. The electrode was positioned using the subxiphoid approach and local anesthesia in most patients. There have been few complications and none that resulted in long term morbidity. There have been no deaths related either to the operative approach or to the pacing system. There has been no instance of lead failure during the follow-up period.


Vectorcardiograms in severe aortic insufficiency. Clockwise rotation of QRS loop in the horizontal plane. In La Paz Hospital, Madrid, recently, vectorcardiograms (VCGs) were performed on 74 patients with aortic insufficiency. These cases presenting electrocardiographic and radiologic signs of left ventricular hypertrophy, were divided into three groups. In 25 patients (Group 1) the QRS loop in the horizontal plane (HP) showed counterclockwise rotation; 43 patients (Group 2) showed clockwise rotation; and 6 patients (Group 3) showed a pattern of left bundle branch block (LBBB). In Group 2, 31 of the 43 patients (group 2-A) had a Qloop of normal orientation and generally increased voltage, and in the remaining 12 patients (group 2-B) the Q loop was absent. All of the patients in Group 2 had severe aortic insufficiency. Thus, that group's valuation of the left ventricular hypertrophy using maximum QRS vector voltage in the HP, was higher than in Group 1 mean 3.75mV in Group 2 versus mean 2.71mV in Group 1 (p less than 0.01). Postoperative VCGs were performed in 12 patients of group 2-A, showing in all of them a clockwise to counterclockwise rotation change of the QRS loop in the HP. On the other hand, this fact was observed in only two of the eight patients in group 2-B. The clockwise rotation of the QRS loop in the HP is an important datum in our report, particularly in the diagnosis of severe aortic insufficiency. Conversely, a clockwise to counterclockwise rotation change would be a positive sign of good post operative evolution. Finally, two reasons are given to explain this anomalous rotation in the horizontal plane: dilatation and fibrosis.


Pulmonary atresia with ventricular septal defect: report of the oldest known surviving case. The case of a 54-year-old housewife, oldest published survivor with pulmonary atresia and a ventricular septal defect (pseudotruncus arteriosus), is reported. Her remarkably favorable course is likely related to the absence of progressive hemodynamic changes, with moderate pulmonary flow adequate for nearly normal arterial oxygenation yet without increasing pulmonary vascular resistance. This case reemphasizes the relationship of longevity to pulmonary blood flow volume with this defect.


Results of replacement of cardiac valves by homologous dura mater valves. Homologous dura mater valve was employed in a series of 533 patients in the period between January 1971 and May 1974. The dura mater was sterilized and preserved in 98 percent glycerol solution at room temperature. Important data were the following (1) no significant pressure gradient through the vale at rest; (2) no bacterial endocarditis; (3) two cases of fungal endocarditis; (4) no degeneration or retraction of the leaflets; (5) no thrombus formation in the valve; and (6) no anticoagulants were used in this series.


[Androgens in the plasma of women with hirsutism (author's transl)]. 109 women with hirsutism were investigated (102 with idiopathic hirsutism, four with Stein-Leventhal syndrome, one each with adrenal adenoma, adrenal carcinoma and hilus-cell tumour). Levels of testosterone, dihydrotestosterone, androstenediol and dehydroepiandrosterone were measured, usually in the course of a combined adrenal-ovarian stimulation-suppression test. Measuring several plasma androgens made it possible to distinguish idiopathic hirsutism from other endocrine diseases with hirsutism. Mean plasma testosterone level in idiopathic hirsutism was not significantly different from that in healthy women. Elevated testosterone and(or) dihydrotestosterone levels were found in only 22 of 102 women. There was a marked increase in plasma testosterone after HCG injection in the patient with Stein-Leventhal syndrome. Androstenediol and dehydroepiandrosterone were both important in the diagnosis of autonomous adrenal steroid secretion.


Developmental study of androgen responsiveness in the submandibular gland of the mouse. The development of androgen responsiveness in the submandibular gland of normal and androgen-resistant (Tfm) mice of different ages was studied after varying hormonal treatments. Total esteroproteolytic (tamase) activity of submandibular gland homogenates was used as a marker for androgen action. Newborn mice of all four genotypes (normal male, normal female, carrier Tfm females, and Tfm males) were resistant to androgen. However, at 3 weeks of age the capacity to develop a tamase response appears in normal and carrier Tfm animals given androgen and rapidly rise to maximal levels. The level in the normal animal is regulated thereafter primarly by the level of circulating androgen. In contrast, the tamase response in the Tfm male of all ages and under all androgen regimens was minimal.


Adaptation of red cell enzymes and intermediates in metabolic disorders. The metabolic activity of the red cell glycolytic pathway hexose monophosphate shunt (HMP) with dependent glutathione system was studied in patients with hyperthyroidism (n = 10), hyperlipoproteinemia (n = 16), hypoglycemia (n = 25) and hyperglycemia (n = 23). In uncontrolled diabetics and patients with hyperthyroidism the mean value of glucose phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G-6-PD), glutathione reductase (GR) was increased, whereas these enzyme activities were reduced in patients with hypoglycemia. Apart from a few values of hexokinase (HK) which were lower than normal the results in hyperlipoproteinemia patients remained essentially unchanged, including the intermediates such as 2,3-diphosphoglycerate (2,3-DPG), adenosine triphosphate (ATP) and reduced glutathione (GSH). While increased rates of 2,3-DPG and ATP in hypoglycemia patients were obtained, these substrates were markedly reduced in diabetics.


ATP-sensitive and ATP-insensitive phosphofructokinase in Escherichia coli K-12. The purification and kinetic characteristics of two phosphofructokinases are described. Aerobic cultures of Escherichia coli exhibit two types of phosphofructokinase. Both types are dimers of mol. wt 150,000 (subunit mol. wt 73,000), whereas the anaerobic culture of E. coli revealed only one type, which is a tetramer of mol. wt 350,000 (subunit mol. wt 90,000). Type 1 of the aerobic enzyme, representing approximately 70% of the total enzyme activity, is ATP-insensitive, whereas type II and the anaerobic enzyme are ATP-sensitive. The addition of AMP stimulates the tetramer, relieving ATP inhibition, and also the type II dimer, which is, however, inhibited at concentrations higher than 0.5 mM AMP. No effect was observed on the type I dimer of the aerobic preparation. ADP stimulates the tetramer and inhibits type I more strongly than type II of the aerobic dimer. The kinetic characteristics together with the effect of metabolites on these phosphofructokinase types are described and discussed in the light of their importance for the regulatory mechanism of the Pasteur effect.


Precocity of pulmonary vascular obstruction of Down's syndrome. Studying the pulmonary vascular resistance in children with ventricular septal and endocardial cushion defects, the authors found a statistically significant earlier and more severe reaction in children with Down's syndrome as compared to normals. Cardiac investigations with regard to surgical intervention should be made early in life, if the parents wish their child with Down's syndrome to undergo surgery.


Snapshot diagnosis: syndromes, facies and heart disease. In some syndromes the facial appearance of the patients is so typical that a correct diagnosis can be made 'à vue'. The five cases presented here are examples of this statement and the additional clinical data make it possible to decide on the syndromes and the complicating cardiac anomalies.


Amantadin e tremor, a 5-hydroxytryptamine-mediated response? Amantadine-induced tremor has been investigated using mice. Experiments with, mebanazine, reserpine, diethyldithiocarbamate, and p-chlorophenylalanine suggest that the tremorgenic action of amantadine is influenced by a balance between three putative central nervous system (CNS) transmitters: noradrenaline, dopamine and 5-hydroxytryptamine (5-HT). Drugs which reduce the concentration of the catecholamines in brain increase amantadine induced tremor. p-Chlorophenylalanine, which specifically depletes brain 5-HT, antagonises amantadine-induced tremor. An ED50 (tremor) dose of amantadine decreases the concentration of 5-hydroxy-indoleacetic acid (5-HIAA) in rat brain, particularly when this elevated due to pretreatment with 5-hydroxytryptophan. Neither inhibition of monoamine oxidase nor reduction of 5-HT-reuptake appear to be responsible for this decrease. Experiments on rat fundus suggest that amantadine increased the sensitivity of receptors to 5-HT. A similar mechanism of action in the CNS could explain both the tremor and the decrease in brain 5-HIAA. The possible relevance of these findings is discussed with respect to the known anti-Parkinson action of amantadine.


Are muscarinic receptors in the central and peripheral nervous system different? The concentration-dependent binding of atropine-3 H to membrane fractions from bovine tracheal muscle, parotid gland and caudate nucleus, measured by equilibrium dialysis, revealed the presence of virtually identical high affinity binding sites in all three tissues. Sch 1000 and Sch 1178 geometrical isomers of N-isopropylatropine bromide with a large potency ratio as antimuscarinics, inhibited atropine binding identically in all three tissues. Differences in properties of muscarinic receptors in these tissues are either non-existent or too small to be detected by the applied techniques.


[Effects of caudate nucleus stimulations on the primary and secondary endings activity of soleus muscle spindle]. Effects of repetitive stimulation of the contralateral caudate nucleus on the static discharge and dynamic sensitivity of soleus muscle spindle primary and secondary endings were studied in cats anaesthetized with Halothane (Fluothane). By progressive increasing the depth of anaesthesia two different fusimotor effects could be observed. 1. A static facilitatory effect; under light anaesthesia the static discharge of primary and secondary endings were strongly increased. The response of the primary endings to phasic stretch of the muscle was generaly decreased. 2. A depressant effect; under deep anaesthesia the static discharge of primary and secondary endings were decreased. The dynamic sensitivity of the primary endings was lightly increased or remained unaltered. The ventro-lateral part of the caudate nucleus is mainly responsible for these effects; Caudate nucleus fusimotor effects were compared with those induced by gamma dynamic and gamma static fibers stimulations.


[Modulation of primary and secondary endings activity of tibialis anterior muscle spindles during contraction induced by caudate nucleus stimulations]. 1. Effects of repetitive stimulations of caudate nucleus were studied on static and dynamic sensitivities of contralateral flexor (tibialis anterior) muscle spindle primary and secondary endings in cats anaesthetized with Halothane (Fluothane). Fusimotor effects are interpreted in term of gamma dynamic and gamma static activation. 2. Threshold stimulation induce a gamma dynamic effect on the primary endings sensitivity. Secondary endings activity and muscle tension remainded unaltered. 3. A slight increase of the stimulus strength elicit a gamma dynamic-gamma static effect on the primary endings sensitivity. Simultaneously secondary endings activity was increased and muscle contractions induced. 4. Using graded voltage stimulation permit to increase primary and secondary endings activity without muscle contraction. 5. Two stimulations applied at sufficiently short time interval produce different fusimotor effects. The first stimulation induce the general motor effect previously obtained: gamma dynamic-gamma static modulation of primary endings sensitivity, increase of secondary endings activity, muscular contraction. The second stimulation elicit a gamma dynamic effect on primary endings sensitivity and a depressant effect on secondary endings activity during muscular contractions. 6. Tendon organs discharge during caudate nucleus stimulation was studied comparatively with the primary and secondary endings activity.


Perfluoro compounds as artificial erythrocytes. Some liquid perfluoro compounds dissolve relatively large amounts of oxygen and can be used in dispersed form as substitutes for erythrocytes. The commonly used perfluoro compounds contain about the same amount of oxygen as do equal volumes of erythrocytes when equilibrated with 100% oxygen. However, when equilibrated with alveolar air, the perfluoro compounds contain much less oxygen than erythrocytes. The dispersed fluorochemicals are adequate substitutes for perfusion of isolated preparations of mammalian brain, heart kidney, lung and liver. However, when put into the circulation of the intact animal, the dispersed fluorochemicals tends to produce lesions of the lungs, dilation of the right heart, and ultimately fatal hypoxia. It is suggested that the course of events following intravenous injection of dispersed fluorochemical is initiated by an interaction of the perfluoro particles with blood platelets or blood clotting factors. The ensuing intravascular clotting could then cause the changes in the lungs which lead to a marked increase in pulmonary artery pressure and dilation of the right heart. These events would terminate in fatal hypoxia due to pulmonary pathology and heart failure.


Case report: mechanical failure of a spinal transabdominal teflon stent in tuboplasty. Intraperitoneal breakage of a transabdominal tuboplastic prosthesis was reported. Incomplete small bowel obstruction may be predisposed by the open loop formed by transperitoneal placement. Avoidance of re-exploration, an intent of the technique, is obviated by stent breakage.


Reconstruction of fallopian tubes in previously sterilized patients. Restoration of tubal patency after surgical sterilization in which the luminal continutiy is interrupted requires either uterine implantation of the patent distal segment or resection and end-to-end anastomosis of patent adjacent segments. Although it is logical to assume that after most tubal ligations the intramural segment remains normal and end-to-end anastomosis is possible, both segments should be evaluated and tested prior to the plastic reconstruction. The results of 178 operations collected from the literature and the author's 23 attempts at surgical reversal of previous tubal operations for surgical sterilization were described. The over-all pregnancy rate after resection and end-to-end anastomosis was 39.0%; after uterotubal implantation, it was 19.4%. The latter procedure was performed in 60% of the patients. The small series of reports makes it difficult to evaluate conception rates or to judge the merits of specific reconstructive operative techniques.


Genetic control of phosphate-metabolizing enzymes in Neurospora crassa: relationships among regulatory mutations. In Neurospora crassa, the phosphate-metabolizing enzymes are made during phosphate starvation, but not under phosphate sufficiency. The synthesis of these enzymes is controlled by three regulatory genes: pcon-nuc-2, preg and nuc-1, pcon-nuc-2 and preg are closely linked. A model of the hierarchical relationships among these regulatory genes is presented. Studies of double mutants and revertants confirm several predictions of the model. It has been found that nuc-2 (null) and pcon-c (constitutive) mutations reside in the same cistron. preg-c (constitutive) mutations are epistatic to nuc-2 mutations. nuc-1 (null) mutations are epistatic to all others.


The value of echocardiography in older patients. Many of the diseases for which echocardiography has proved to be of value are not common in geriatric patients. In addition, adequate echocardiographic examination is technically difficult in this group of patients. However, despite these facts, echocardiography is most helpful in several specific situations in older patients. These patients often are brittle, have multiple problems, and obviously should not be subjected to an extensive or a hazardous examination. In this regard, echocardiography is ideal, as it causes no discomfort or hazard to the patient. As the indications for this technique increase, it may be used more extensively as a diagnostic tool in elderly patients.


[Comparative studies on the drug therapy of gonarthroses]. The clinical effectiveness of an anti-arthrotic drug was investigated in a comparative trial. 106 patients suffering from moderate arthrosis of the knee were placed into two groups in accordance with a random table and treated by intramuscular injection of Arteparon forte or an analogous control product. The criteria of assessment were pain on movement, pain at rest, limitation of active and of passive movement. The number of patients who at the end of treatment and at the time of follow up, three months later, had no or insignificant residual symptoms and signs, were greater in the Arteparon forte group than in the control group. This difference was statistically significant two months after the beginning of treatment for the criterion pain at rest, and five months after beginning of treatment for all criteria. A global assessment of the results was carried out according to the number of pronouncedly improved criteria in the individual patients. According to this assessment, the results of treatment at the termination of treatment amounted to 73.6% in the Arteparon forte group and 56.6% in the control group. At the time of follow up the corresponding value of the Arteparon forte group was 68.4%, thus significantly greater than that of the control group (29.4%).


Patient rehabilitation through hospital work under Fair Labor Standards. Payment of patients for hospital work assignments has become a matter of great concern for mental health institutions since the 1973 federal court ruling requiring the Department of Labor to enforce the 1966 amendments to the Fair Labor Standards Act. A work program in compliance with Fair Labor Standards has been operating at Binghamton (N.Y.) Psychiatric Center since 1971. Certificates from the labor department permit the hospital to pay less than the minimum wage for patients in occupational training or in sheltered employment in a regular job. The authors believe that work therapy has important clinical and rehabilitative functions, and that patients should not be denied the opportunity for such work because of the court ruling.


Histochemical profiles of rat soleus intrafusal fibres after chronic exercise. Intrafusal fibres from the rat soleus were investigated for representative histochemical profiles in sedentary animals and animals chronically exercised for 17 weeks on a treadmill. The pattern of myosin adenosine triphosphatase (ATPase) activity in the polar region revealed three intrafusal fibre types: (1) myosin ATPase-dark (MD) fibres, alkali- and acid-stabile; (2) myosin ATPase-light (ML) fibres, alkali- and acid-labile; and (3) myosin ATPase-reversible (MR) fibres, alkali-stabile and acid-labile. The three fibre types were correlated with the level of reduced NADH diaphorase activity, with MR, ML and MD fibres staining dark, moderate and light, respectively. In the equatorial region the morphological features of representative ML and MD fibres revealed that they were nuclear bag fibres, while representative MR fibres were identified as nuclear chain fibres. The MR fibres in the exercised animals had higher levels of myosin ATPase alkaline stability and acid lability than MR fibres in the sedentary animals, suggesting the MR fibre profiles are selectively influenced by chronic exercise. The mean cross-sectional area of MR fibres from the exercised animals was significantly less than the MR fibres from the sedentary animals. In contrast to the effect of endurance training on NADH diaphorase activity in extrafusal muscle fibres, there was evidence of less activity in the MD fibres of the exercised animals.


Electron microscopic visualization of proteoglycans with Alcian Blue. The intercellular matrices of bovine nasal cartilage, chick embryo perichordal cartilage, and chick embryo mesenchymal cells cultured in vitro have been examined by electron microscopy after staining them with Alcian Blue in salt solutions according to Scott & Dorling (1965). Matrix granules, which are typical components of cartilage at the ultrastructural level, are not visible after Alcian Blue staining and are replaced by alcianophilic rod-like particles, varying in length and width. With tissue cultures, Alcian Blue stains 40-120 A thick filaments which display an orthogonal and longitudinal relationship to collagen fibrils. We assume that cartilage matrix granules represent linear proteoglycans that are coiled as a consequence of the usual glutaraldehyde-osmium fixation. It is thought that Alcian Blue, on the other hand, contributes to the stabilization of the proteoglycans in their original structural arrangement. This stabilizing property presumably also results in the sharp visualization of fine filaments in the tissue culture matrix.


Muscle fibre type populations of human leg muscles. Four selected leg muscles (gastrocnemius, soleus, vastus lateralis and intermedius) from thirty-two humans were autopsied within 25 hr of death and examined histochemically. The results of histochemical myofibrillar adenosine triphosphatase activity demonstrated that the soleus and vastus intermedius muscles have a higher proportion of slow twitch fibres (70%, 47%) than their synergists, gastrocnemius and vastus lateralis, respectively. The gastrocnemius contains about 50% slow twitch fibres and the vastus lateralis about 32%. Similar proportions of slow and fast twitch fibres have been reported for these hindlimb muscles in other mammals. Human muscles, however, differ from other mammalian muscles in that the proportion of slow and fast twitch fibres were similar in the superficial and deep regions of the muscles examined. Fast twitch oxidative glycolytic fibres in sedentary humans were observed less frequently, and they are less prominent in terms ofoxidative enzymatic activity when compared to similar fibres of several laboratory mammals studied previously.


Absorption, distribution and excretion of neocarzinostatin (NCS) in mice after oral administration. Distribution, excretion and toxicity of an antitumor protein, neocarzinostatin (NCS) were examined in mice after oral administration. The oral LD50 1 g/kg after intravenous injection. After oral administration of 200 mg/kg of NCS, the tissue level was low but detectable in lung, skin and pancreas in addition to the tissues of the gastrointestinal tract. The NCS level in lung and skin remained constant through 6 hours. In gastrointestinal tissues after oral administration the level was higher in the stomach than the large intestine or small intestine. The total recovery of orally administered NCS in feces of mice was 26.5% of the given dose during the first 12 hours. Inactivation of NCS by homogenates of small and large intestines (about 50%) was found in in vitro experiments.


Mechanism of DNA degradation induced by neocarzinostatin in Bacillus subtilis. When logarithmically growing Bacillus subtilis cells were exposed to the antitumor protein, neocarzinostatin (NCS), at a concentration of 50 mug/ml, cellular DNA was gradually degraded into an acid-soluble form (up to 60 percent of total DNA). The degradation appeared to initiate at the growing regions of DNA and to proceed sequentially from the nascent regions to preexistent DNA. Concomitantly with, or perhaps as a consequence of, the degradation of growing regions, DNA detached from the cell membrane and started to show single-strand nicks within 30 minutes after exposure of the cells to NCS, whereas double strand scission in the DNA became detectable in about 90 minutes. Such endonucleolytic breaks in DNA eventually gave rise to the formation of double-stranded DNA fragments of a single-size class (30-S) as determined by sedimentation in either neutral or alkaline sucrose gradients. In contrast to previous results with Sarcina lutea, the NCS-induced DNA degradation was stimulated by chloramphenicol in B. subtilis and the DNA fragment were not the final breakdown products, but were further degraded into acid-soluble materials.


Methyltrophic enzyme distribution in Methylosinus trichosporium. Key enzymes involved in the oxidation and fixation of methane by Methylosinus trichosporium were examined for localization within the bacterial cells. A differential centrifugation scheme following cell disruption was used to provide membrane and soluble fractions for the enzyme assays. All the methylotrophic enzymes examined were found to be soluble with this fractionation scheme. Electron transport involving a cytochrome c2 with absorption peaks at 416, 522, and 550 nm and oxidative phosphorylation were found in the membrane fractions. Mixed soluble and membrane fractions coupled the oxidation of emthanol and formate with cytochrome reduction.


Biphasic ATP splitting of myosin at low temperature. Studies were carried out to elucidate the nature of biphasic ATP hydrolysis by myosin at low temperature.  1. The rate of ATP splitting decreased sharply at 3--5 min after initiation of the reaction below a critical temperature (25 degrees and 30 degrees in the presence of Ca-2+ and EDTA, respectively).  On the other hand, Mg-2+-ATPase [ED 3.6.1.3] did not exhibit such biphasic kinetics.  2. The Arrhenius plot of the second phase of the reaction after the rate transition gave a straight line whether the temperature of assay was above or below the critical one, giving 5.7 kcal/mole as the activation energy of Da-2+-ATPase showed features similar to those of Ca-2+-ATPase.  3. Michaelis constants for the two phases at 8 degrees were also different.  In addition, the first phase of EDTA-ATPase was shown to have two different constants, depending on ATP concentration.  4. The profiles of the dependence of ATPase activity on KCl concentration were essentially the same for both phases, while bending of the time curve was scarecly observed obove pH 8 for Ca-2+-ATPase or at pH 6 for EDTA-ATPase.  5. 2, 4-Dinitrophenol abolished the phase transition for Ca-2+-ATPase and EDTA-ATPase, and heat treatment also minimized the transition for the former.


Effect of phospholipase A on active transport of amino acids with membrane vesicles of Mycobacterium phlei. Active transport of proline remained unaffected in phospholipase A-treated electron transport particles from Mycobacterium phlei. However, the steady state level of proline was reduced 50 to 60% in phospholipase A-treated depleted electron transport particles that were devoid of membrane-bound coupling factor-latent ATPase activity. The decrease in the uptake of proline in the phospholipase A-treated depleted electron transport particles was not due to a change in the apparent K-m for proline, but it was related to the amount of phospholipid cleaved from the membranes. Restoration in the level of proline transport in phospholipase A-treated depleted electron transport particles was achieved by reconstituting these vesicles with diphosphatidylglycerol and phosphatidylethanolamine liposomes. Diphosphatidylglycerol was found to be most effective in the restoration of proline uptake. In contrast to the effect of phospholipase A treatment on proline transport, similar treatement of the electron transport particles or depleted electron transport particles failed to inhibit the active transport of either glutamine or glutamic acid. Studies with phospholipase A-treated membrane vesicles confirmed earlier findings that a proton gradient is not required for active transport of amino acids.


Reconstitution of active ion transport by the sodium and potassium ion-stimulated adenosine triphosphatase from canine brain. Sodium and potassium ion-stimulated adenosine triphosphatase ((Na+ + K+)-ATPase) was partially purified from canine brain gray matter and reconstituted into vesicles of phosphatidylcholine. A proportion of the enzyme molecules was reconstituted into sealed vesicles with the ATP-hydrolyzing site facing the outside of the vesicles. ATP was added to the outside of the vesicles after they had equilibrated with radioactive tracer, and the resulting active transport of Na+ and K+ was followed. Unlike the purified kidney renal medulla enzyme used in an earlier study, the brain enzyme transports both Na+ and K+(Rb+). Vesicles were made in solutions with different proportions of NaCl and KCl, and over the range studied, an average of 1.8 Rb+ ions were transported for every 3 Na+ ions. When ATP is depleted, the transported ions diffuse back to their equilibrium level in the vesicles.


Physical and chemical properties of the NH2-terminal glutamic acid and lysine forms of human plasminogen and their derived plasmins with an NH2-terminal lysine heavy (A) chain. Comparative physical and chemical data are described for the human NH2-terminal Glu-plasminogen and Lys-plasminogen forms in order to determine the exact relationship between these two types of the zymogen. The molecular weights of Glu-plasminogen and Lys-plasminogen were similar and were determined to be 83, 800 plus or minus 4, 500 and 82, 400 plus or minus 3, 300, respectively, by sedimentation equilibrium methods. The molecular weights were identical in dodecyl sulfate solutions, approximately 83, 000, by sedimentation equilibrium methods. The sedimentation coefficients, s-020, w of Glu-plasminogen and Lys-plasminogen were determined to be 5.0 S, and 4.4 S, respectively. These two plasminogen forms had different partial specific volumes, and calculations of the frictional coefficients from sedimentation coefficients and molecular weights indicated conformation differences. Glu-plasminogen appeared to be larger in size than Lys-plasminogen in acrylamide gel-dodecyl sulfate electrophoresis. The amino acid compositions of Glu-plasminogen and Lys-plasminogen, and their major isolated isoelectric forms, were found to be similar, but several amino acid residues (glutamic acid, alanine, isoleucine, phenylalanine, and lysine) were found to be significantly higher in the Glu-plasminogen forms. The derived plasmins from both the Glu- and Lys-plasminogens with an nh2-terminal Lys- heavy (A) chain were found to have identical molecular weights of 76, 500 plus or minus 2, 500, and sedimentation coefficients, s-020, w of 4.3 S.