A novel approach to the identification of surface receptors. The use of photosensitive hetero-bifunctional cross-linking reagent. Methyl 4-azidobenzoimidate, a photosensitive hetero-bifunctional cross-linking reagent, was synthesized and characterized. This reagent has an imidoester at one end, which reacts spontaneously with primary amines, and an arylazide at the other end, which reacts with a variety of chemical groups upon photolysis by ultraviolet radiation. The reagent molecules were attached to concanavalin A by reactions between imidoester groups of the reagents and free amino groups of the lectin. These activated lectins were purified on a Sephadex G-25 column and showed the binding affinity to an affinity column, glucosylated Sepharose, and to the human erythrocyte ghost membrane. The activated lectins were incubated with the membranes and then unbound lectins were removed by washing. The lectins bound to receptors in the membranes were irradiated with a shortwave ultraviolet lamp to photolyze arylazides attached to the lectins, thus cross-linking the lectins and receptors together. Then the membranes were solubilized and electrophoresed. On gels, the intensity of the lectin receptor band diminished slightly and concomitantly a new band of a higher molecular weight appeared. When 125I-labeled concanavalin A was used, the new band contained the radioactivity. The extent of the appearance of the new band and the decrease of the receptor band were reduced significantly when the ultraviolet irradiation was omitted or the activated lectins were incubated with the membranes in the presence of the lectin inhibitor, alpha-methylmannoside. The irradiation of nonactivated, receptor-bound concanavalin A did not cause those changes. When the activated lectins alone were irradiated with ultraviolet, the band of the lectin dimer appeared whereas nonirradiated lectins appeared mostly as monomers. It is concluded that a small fraction of the activated lectins were cross-linked to receptors in the membrane upon photolysis. In this study, only 8 reagent molecules were attached to a tetramer of the lectin, compared with the presence of approximately 40 available free amino groups. The efficiency of such cross-links of ligands to receptors may be increased by employing longer versions of the hetero-bifunctional cross-linking reagents and also by attaching more of the reagent molecule to ligands.


Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions. The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions  stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.


Purification and properties of the urea amidolyase from Candida utilis. Urea amidolyase was purified to homogeneity from extracts of Candida utilis. The purification involves protamine sulfate precipitation, ammonium sulfate precipitation, polyethylene glycol precipitation, Sepharose 6B gel filtration, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography. The final preparation is pure as judged by disc-gel electrophoresis. The molecular weight of urea amidolyase, as determined by gel filtration and disc-gel electrophoresis, is between 500,000 and 520,000. Treatment with sodium dodecyl sulfate results in two peptides with molecular weights of 70,000 and 170,000. The urea carboxylase and allophanate hydrolase activities of urea amidolyase may be distinguished from one another on the basis of (a) the effect of the stabilizers, urea and glycerol, (b) the effect of storage pH on activity, and (c) selective inhibition by sulfhydryl reagents.


Mechanisms of acylation of chymotrypsin by phenyl esters of benzoic acid and acetic acid. The kinetics of the acylation of alpha-chymotrypsin by a series of substituted phenyl p-nitrobenzoates have been studied by stopped flow and conventional spectrophotometry. Electron withdrawal in the leaving group accelerates the rate of acylation, and the p value obtained for eight esters is +1.96. The pH- and pD-independent acylation rate constants are, respectively, 1.40 X 10(4) M-1S-1 and 1.23 X 10(4) M-1S-1 for p-nitrophenyl p-nitrobenzoate, and, respectively, 2.19 X 10(3) M-1S-1 and 1968 X 10(3) M-1S-1 for p-nitrophenyl benzoate at 25 degrees. An analysis of structure-reactivity results and kinetic solvent isotope effects indicates a mechanism for acylation by phenylbenzoates in which initial reaction is a nucleophilic attack by an imidazole of the enzyme (His 57). Subsequently, there is rapid transfer of the acylating group to the serine 195 from the acylimidazole species. The kinetic solvent isotope effects for acylation by p-nitrophenyl phenyl acetate and p-nitrophenyl phenyl acetate and p-nitrophenyl hydrocinnamate, in 5%, v/v, acetonitrile, are 1.3 and 2.0, respectively. The latter ester is inhibited more than is p-nitrophenyl benzoate when 5%, v/v, dioxane is substituted for 5%, v/v, acetonitrile as co-solvent. In the presence of 5%, v/v, dioxane a change in the kinetic solvent isotope effect to 1.7 is found for p-nitrophenyl benzoate and p-nitrophenyl phenylacetate while that for the analogous hysdrocinnamate ester is unaffected. The results for the latter substrate are in accord with a general base-catalysed mechanism. Electron-withdrawal groups in the phenyl ring of phenyl acetates accelerate the enzyme acylation yielding a leaving group p of 2.05. The kinetic solvent isotope effects for acylation by p-nitrophenyl thiolacetate and by p-nitrophenyl acetate are close to 2.0. The mechanism of acylation of chymotrypsin by phenyl acetates is not unambiguously defined using these data.


Endonuclease V of Escherichia coli.

Endonuclease V of Escherichia coli. A small endodeoxyribonuclease )2.3 S) that is active on single-stranded DNA has been extensively purified from Escherichia coli so as to be free of other known DNases. It has an alkaline pH optimum (9.5), requires Mg2+, and makes 3'-hydroxy and 5'-phosphate termini. The nuclease nicks duplex DNA, particularly if treated with OsO4, irradiated with ultraviolet light, or exposed to pH 5. The uracil-containing duplex DNA from the Bacillus subtilis phage PBS-2 is an especially good substrate; it is made acid-soluble by levels of the enzyme which fail to produce any acid-soluble material in other single-stranded or duplex DNAs. Neither RNA nor RNA-DNA hybrid are degraded by the enzyme. The enzyme specificity suggests that it might act at abnormal regions in DNA, so that its in vivo function could be to initiate an excision repair sequence. Its high activity on uracil-containing DNA could imply that the enzyme provides an alternative mechanism for excising uracil residues from DNA to the pathway utilizing uracil-DNA N-glycosidase. We suggest that this enzyme be designated as endonuclease V of E. coli.


Human angiotensinogen. Purification partial characterization, and a comparison with animal prohormones. The renin-angiotensin system appears to play a major role in the regulation of sodium excretion and fluid intake in a wide variety of animal species from mammals to teleosts. In mammals the system has evolved further importance in terms of blood pressure homeostasis. This hormonal system in all species appears to involve a serum protein prohormone, angiotensinogen, a proteolytic enzyme, renin, and angiotensin I, the decapeptide product of the reaction between renin and angiotensinogen. The importance of this system to the organism appears to correlate directly with the necessity to conserve sodium while an abnormality of this process may underlie the development of hypertension in man. As the starting point of the system, angiotensinogen assumes special importance as a possible index of evolutionary development. In addition, it has been known for many years that human (viz. primate) angiotensinogen differs from that found in other mammals in its inability to be a substrate for animal renins while animal angiotensinogens readily react with human renin. Thus, the enzymatic specificity appears to reside with the prohormone. The biochemical basis for this difference is unresolved due primarily to the lack of purified human angiotensinogen. In this paper we describe methods for the purification of human angiotensinogen which have direct applicability to animal angiotensinogens. Our approach utilizes ammonium sulfate precipitation, Sephadex G-150 chromatography, multiple isoelectric focusing, and concanavalin A-Sepharose affinity chromatography. With the availability of highly purified human angiotensinogen we compared the molecular weights, heterogeneity, isoelectric points, and thermal lability of hog, rabbit, and human angiotensinogen in order to define the biochemical basis of the species variation in renin reactivity...


Studies of individual carbon sites of hen egg white lysozyme by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Assignment of the nonprotonated aromatic carbon resonances to specific residues in the sequence. The resonances of nonprotonated aromatic carbons in natural abundance 13C NMR spectra of hen egg white lysozyme are assigned to specific residues of the amino acid sequence. Chemical shift considerations, the effect of pH, and partially relaxed Fourier transform NMR spectra are used to assign each resonance to one of the seven types of nonprotonated aromatic carbons of amino acid residues. Spectra of chemically modified lysozyme samples yield various assignments to specific residues in the sequence. Line-broadening effects caused by binding of the relaxation probes Gd3+ and 4-N-acetamido-2,2,6,6-tetramethylipiperidine-1-oxyl yield specific assignments which are fully consistent with those based on chemical modifications. The effects of paramagnetic shift reagents and amino sugar inhibitors do not yield any obvious specific assignments. The effect of pH on the chemical shift of Cgamma of His-15 yields a pKalpha in agreement with published values, and indicates that the imidazole form of His-15 exists mainly (or entirely) as the Nepsilon3-H tautomer. The effect of pH on the chemical shifts (measured up to pH 8.8, at 38 degrees) of Czeta and Cgamma of the 3 tyrosine residues yields crude pKalpha values of 9.5 and 10 for Tyr-23 and one of the other tyrosines, respectively. The 3rd tyrosine residue does not exhibit titration behavior.


Participation of tryptophan 62 in the self-association of hen egg white lysozyme. Application of natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Self-association of hen egg white lysozyme in solution of 38 degrees) is examined by means of natural abundance 13C nuclear magnetic resonance spectroscopy. The effect of pH on the resonances of the nonprotonated aromatic carbons of 9 mM lysozyme, and the effect of protein concentration (at pH 7) on these resonances, both indicate that self-association significantly affects the chemical shift of Cgamma of Trp-62, but not the chemical shifts of the other nonprotonated aromatic carbons. This result is consistent with the reported participation of Trp-62 in the intermolecular contact (Banerjee, S.K., Pogolotti, A., and Rupley, J.A. (1975) J. Biol. Chem. 250, 8260-8266). Our results indicate that the resonance of Cgamma or Trp-62 is a convenient monitor of lysozyme self-association. The chemical shift of this resonance reflects the extent of aggregation, while the line width yields information about the lifetime of the intermolecular contact. This lifetime is 1 to 2 ms at 38 degrees (9 mM protein, 0.1 M NaCl, pH 7). Our results also indicate that self-association of lysozyme is not accompanied by any general conformational change, and that binding of a lanthanide ion (at the metal ion binding site near the carboxylate groups of ASP-52 AND Glu-35) strongly suppresses self-association.


Galactosyl transferase of a Golgi fraction from cultured neoplastic mast cells. Suspension cultures of neoplastic mouse mast cells were used to obtain large quantities of a homogeneous cell population as starting material for cell fractionation. A Golgi fraction was prepared by slight modification of established techniques and identified by electron microscopy. Assay of galactosyl transferase activity using ovalbumin, desialylated degalactosylated orosomucoid, and N-acetylglucosamine as galactose acceptors showed that the Golgi fraction was enriched in specific activity over the homogenate. The Golgi galactosyl transferase was examined in detail. Acceptor concentrations for optimal galactose incorporation were determined, and substrate inhibition effects were shown with higher concentrations of all three acceptors. Manganese was shown to be necessary for galactose incorporation. A higher concentration of manganese afforded some protection from substrate inhibition by acceptors, but at the same time was itself inhibitory. All three acceptors competed with one another for galactose incorporation, indicating that a single enzyme catalyzed the transfer of galactose for all acceptors.


The pH and temperature dependence of the interaction of steroid hormones with the transport system of glucose in human erythrocytes. The thermodynamic parameters deltaH degrees, deltaF degrees, deltaS degrees and the pH dependence of the interaction between steroids and the glucose carrier in human erythrocytes have been studied. The results indicate that, according to the structure of the steroids, hydrophilic as well as hydrophobic bonds can be involved in the association. For the binding of the C-21-steroids to the carrier the polarity rule has been observed to be valid. On the basis of the thermodynamic parameters it has been found that in the association process of the steroids with the carrier, simultaneous randomization of ordered water molecules is important.


The in vivo effect of dimethyl sulphoxide (DMSO) on protein synthesis and the polyribosome profile in Paramecium. When Paramecium tetraurelia in log phase growth is treated with 4% dimethyl sulphoxide (DMSO) for five minutes the amount of polyribosomes is reduced 3- to 4-fold while there is a corresponding increase in 80s ribosomal material. Reducing the concentration of DMSO to 1% allows immediate reversal of the condition. Paramecium polyribosomes subjected to 4% DMSO either in whole cell homogenates or during purification through sucrose density gradients appear unaffected while cycloheximide at concentrations up to 100 mug/ml did not prevent DMSO from exerting its effect in vivo. Analyses of 14C amino acid incorporation experiments indicated a strict correspondence between the effect of DMSO on polyribosomes and overall protein synthesis. The reduction of acid precipitable radioactivity in the polyribosomal region after DMSO treatment was associated with a corresponding increase in radioactivity in the 80s region. There was no comparable increase in the acid precipitable radioactivity in the soluble fraction. The overall results of the study suggest that DMSO acts on polyribosommes indirectly through some unknown primary reaction with cell constitutents, and that the mode of action is such as to cause the release of ribosomes from messenger RNA (mRNA) rather than to prevent initiation of the ribosome-mRNA complex. Our data suggest that the effect may be selective. Finally, it is of interest that high concentrations of DMSO (above 8%) appear to have the opposite effect of lower concentrations of DMSO, i.e., they appear to "freeze" the ribosomes to mRNA.


Enzyme inactivation by a cellular neutral protease: enzyme specificity, effects of ligands on inactivation, and implications for the regulation of enzyme degradation. A protease from Tetrahymena pyriformis inactivated eight of nine commercially available enzymes tested, including lactate deyhdrogenase, isocitrate dehydrogenase (TPN-specific), glucose-6 phosphate dehydrogenase, D-amino acid oxidase, fumarase, pyruvate kinase, hexokinase, and citrate synthase. Urate oxidase was not inactivated. Inactivation occurred at neutral pH, was prevented by inhibitors of the protease, and followed first order kinetics. In those cases tested, inactivation was enhanced by mercaptoethanol. Most of the enzyme-inactivating activity was due to a protease of molecular weight 25,000 that eluted from DEAE-Sephadex at 0.3 M KCl. A second protease of this molecular weight, which was not retained by the gel, inactivated only isocitrate dehydrogenase and D-amino acid oxidase. These two proteases could also be distinguished by temperature and inhibitor sensitivity. Two other protease peaks obtained by DEAE-Sephadex chromatography had little or no no enzyme inactivating activity, while another attacked only D-amino acid oxidase. At least six of the enzymes could be protected from proteolytic inactivation by various ligands. Isocitrates dehydrogenase was protected by isocitrate, TPN, or TPNH, glucose-6-dehydrogenase by glucose-6-P or TPN, pyruvate kinase by phosphoenolypyruvate or ADP, hexokinase by glucose, and fumarase by a mixture of fumarate and malate. Lactate dehdrogenase was not protected by either of its substrates of coenzymes. Citrate synthase was probably protected by oxalacetate. Our data suggest that the protease or proteases discussed here may participate in the inactivation or degradation of a least some enzymes in Tetrahymena. Since the inactivation occurs at neutral pH, this process could be regulated by variations in the cellular levels of substrates, coenzymes, or allosteric regulators resulting form changes in growth conditions or growth state. Such a mechanism would permit the selective retention of enzymes of metabolically active pathways.


Quantitative gas chromatographic analysis of flunitrazepam in human serum with electron-capture detection. A rapid method for the determination of flunitrazepam and desmethylfflunitrazepam in human serum in the range 10-300 ng/ml is described. Both drugs are isolated from biological material by means of a single extraction, part of the organic phase is evaporated to dryness and the residue is dissolved in a small volume of benzene. Without further purification, the substance is determined gas chromatographically with an electron-capture detector configuration of 63Ni-type. The method permits the quantitative determination of at least 25-300 ng/ml with an overall recovery of flunitrazepam of 99.7 +/- 4.9% and of desmethylflunitrazepam of 98.6 +/- 7.8% from serum. All calculations were carried out by a data system that was programmed for this purpose. The limit of detection for flunitrazepam is of the order of 1 ng/ml in serum. The method is sufficiently sensitive and specific for therapy control purposes. The time needed for an analysis is less than 1 h.


Mass fragmentographic quantitation of ethotoin and some of its metabolites in human urine. A method for the determination of ethotoin and its p-hydroxylated and dealkylated metabolites in urine has been developed. Ethotoin and the metabolites were extracted from acidified urine with ethyl acetate and silylated before injection into a combined gas chromatograph--mass spectrometer. Four partly identified metabolites were recorded, but their exact quantitation was not possible as pure reference substances were not available. The limit of sensitivity was far below the amounts of ethotoin and of its metabolites found in urine from patients treated with therapeutic doses of ethotoin.


Evidence for the existence of a large molecular weight protein in human pituitary tissue having follicle stimulating hormone activity. A large molecular weight protein fraction was isolated during purification of follicle stimulating hormone (hFSH) from acetone preserved human pituitary glands. This large molecular weight fraction, F-1, was not retarded by Sephadex G-100 as was hFSH, nor did it penetrate 7.5% acrylamide gel upon electrophoresis in sodium dodecyl sulfate as did hFSH. There was no evidence of dissociation of F-1 into smaller molecular weight fractions upon its refiltration through Sephadex G-100 or upon electrophoresis, suggesting F-1 was not an aggregate of smaller size proteins. Fraction F-1 stimulated ovarian growth in rats, inhibited binding of 125I-hFSH to rat testes membranes and cross-reacted with antiserum to hFSH, hFSH-alpha subunit and hFSH-beta subunit as determined by radioimmunoassay. Based on gel filtration experiments, it seemed unlikely that immunologic activity against subunit antisera could be attributed to contamination of F-1 by free FSH subunits. The membrane binding activity of F-1 decreased after incubation with 8M urea, but at a rate less than 1/2 that seen with intact hFSH similarly treated, indicating the two activities were not identical. These results suggest that human pituitary tissue contains a large molecular weight protein with FSH-like biologic and immunologic activity, but which is distinct from native hFSH.


Mechanism for calcium urolithiasis among patients with hyperuricosuria: supersaturation of urine with respect to monosodium urate. Since monosodium urate (NaU) may play an important etiologic role in the formation of renal stones containing Ca in patients with hyperuricosuria, the current studies were undertaken to define some of the physiocochemical factors which determine the formation of NaU. In solutions containing Na, uric acid was rapidly transformed to NaU at pH greater than 6. The results indicated that NaU, and not uric acid, was the stable phase above this pH. A reliable and simple method for the calculation of the state of saturation of urine with respect to NaU was developed from the ratio of concentration products of Na and total dissolved urate (Upi) in the ambient fluid before and after incubation of urine with synthetic NaU. The concentration product ratio closely approximated the ratio of activity products of Na+ and acid urate ion. In contrast, the relative saturation ratio, or the ratio of activity product of original sample and the thermodynamic solubility product of NaU, often differed from the activity product ratio in the individual urine samples. With the concentration product rate, it was found in 45 urine samples that a critical determinant for the supersaturated state with respect to NaU was the high concentration of UT. At UT greater than 300 mg/liter, urine samples were invariably supersaturated with respect to NaU. These results suggest that the nidus of NaU could potentially form in the urine of patients with hyperuricosuria and Ca stones.


Fluorescence polarization studies of rat intestinal microvillus membranes. Rat intestinal microvillus membranes and lipid extracts prepared from them have been studied by fluorescence polarization with three lipid-soluble fluorophores: diphenylhexatriene, retinol, and anthroyl-stearate. The degree of fluorescence polarization of diphenylhexatriene, which provides an index of the "microviscosity" of the lipid regions of the membrane, is exceptionally high in microvillus membranes, the highest yet reported in normal biological membranes. Both the membrane proteins and lipids were found to contribute to the high values. With each of the three probes the polarization values are higher in ileal microvillus membranes as compared to membranes from proximal intestinal segments. Temperature-dependence studies of the fluorescence polarization of diphenylhexatriene and anthroylstearate demonstrate a phase transition in microvillus membranes and in liposomes prepared from their lipid extracts at approximately 26+/-2 degrees C. Ambient pH influences markedly the diphenylhexatriene fluorescence polarization in microvillus membranes but has little effect on that of human erythrocyte ghost membranes. The "microviscosity" of jejunal microvillus membranes is maximal at pH 6.5-7.0 and decreases as much as 50% at pH 3.0, an effect which depends largely upon the membrane proteins. Addition of calcium ions to suspensions of microvillus membranes increases the fluorescence polarization of retinol and anthroyl-stearate, but not that of diphenyl-hexatriene. This confirms the localization of the last compound to the hydrophobic interior of the membrane, relatively distant from the hydrophilic head groups of the polar lipids. Microvillus membrane proteins solubilized with Triton X-100 give relatively high fluorescence polarization and intensity values with retinol, suggesting the presence of binding proteins which could play a role in the normal absorptive mechanism for the vitamin.


Seminal quality, spermatozoal outpost, and testicular changes in growing Holstein bulls. Nine Holstein bulls, initially between the ages of 17 and 22 mo, awaiting proofs in artificial breeding, were ejaculated each summer for 3 yr, and 8 and 7 survivors for 4 and 5 yr, respectively, to study changes in the same group of bulls and determine the predictive value of early measurements. Semen was collected twice a day, 2 days per wk, for 4 wk. Bulls differed in testicular size, consistency, ejaculate volume, total spermatozoal output, percentage of unstained spermatozoa and abnormal spermatozoa, and in several storage and freezing tests. The largest yearly effects were on testis size and consistency, ejaculate volume, and total output of spermatozoa. The latter increased per bull from 28.3 X 10(9) per wk at 17 to 22 mo of age to 40.9 X 10(9) per wk 4 yr later, representing a high output of spermatozoa with a total of only 20 min of intensive sexual preparation per wk. The correlation between testis size and spermatozoal output was .72. Testicular consistency was indicative of semen quality measured simultaneously as judged by correlations with spermatozoal characteristics ranging from .61 to .95. These characteristics are believed to be representative of those Holstein bulls in artificial breeding which are ejaculated frequently.


Extracellular dextran hydrolase from Streptococcus mutans strain 6715. Streptococcus mutans strain 6715 was shown to produce an extracellular dextramase with endohydrolytic alpha-(1 leads to 6)-glucan-6-glucanohydrolase activity. The enzyme degraded soluble polymers produced by some oral streptococci but did not disperse the streptococcal plaques tested. Enzyme levels in the culture supernatant were elevated in sucrose and sucrose plus glucose cultures but remained at basal levels in glucose and dextran plus glucose cultures.


Compratative distortion in three-unit fixed prostheses joined by laser welding, conventioanl soldering, or casting in one piece. This study evaluated three different joint-forming procedures: laser welding, soldering, and one-piece casting. Fifteen three-unit bridges were fabricated. The findings showed post-jointing distrotion to be significantly less for laser-welded and one-piece  castings than for soldering; and significantly greater reliability (least error variance) in joining with the laser than either one-piece casting or  soldering.


Reduction of adenylate cyclase activity in lysates of human platelets by the alpha-adrenergic component of epinephrine. The effects of the alpha- and beta-adrenergic components of epinephrine on adenylate cyclase activity were studied in lyastes of human platelets. The beta-adrenergic component increased enzyme activity between 20 and 50%, whereas the alpha-adrenergic component caused a concentration-dependent reduction of adenylate cyclase activity. The maximal inhibition of adenylate cyclase was 50 to 60% with about 100 muM epinephrine; about 1 muM epinephrine caused half-maximal inhibition. The alpha-adrenergic reduction of adenylate cyclase activity appeared to be immediate and was specific for platelets from species in which alpha-adrenergic stimulation caused aggregation. Adenylate cyclase activity measured in the presence of prostaglandins of fluoride was also reduced by the alpha-adrenergic component of epinephrine, but the inhibitory effect was less than on basal activity. In contrast, the 5'-guanylylimidodiphosphate-stimulated enzyme was not affected by stimulation of alpha-adrenergic receptors.


The distribution of acid and alkaline ribonuclease activities in preneoplastic and neoplastic rat livers. Ribonuclease (RNase) activities revealed by the substrate film method were compared with reactions for acid and alkaline RNase obtained by lead precipitation technique in serial sections of preneoplastic livers and hepatomas. The preneoplastic parenchymal tissue giving positive reactions with ribonucleic acid films showed both acid and alkaline RNase activities by lead precipitation technique, and the area of hyperplastic nodules nonreactive against substrate films were deficient in acid and alkaline RNase activities. Preneoplastic hyperbasophilic foci and hepatoma gave weak or negative reactions by either method, but necrotic areas and stromal tissue showed appreciable RNase activities. Thus a good correlation was observed in these tissues between the RNase activities revealed by the film method and those demonstrated by lead precipitation.


Surface localization of 5'-nucleotidase on the mouse lymphocyte. The optimal conditions for the cytochemical localization of 5'-nucleotidase (AMPase) in the mouse lymphocyte have been established. Quantitative monitoring of the effects of fixation and the components of the cytochemical medium showed that the cytochemistry can be performed under conditions that do not lead to loss of AMPase activity, and also under conditions where penetration of the substrate into the cell has occurred. The cytochemical reaction product was seen only on the surface of a proportion of splenic lymphocytes, regardless of the fixative used. Biochemical data confirmed that AMPase is an ectoenzyme and is the only protein in splenic lymphocytes capable of catalysing the hydrolysis of AMP. The activity of 5'-nucleotidase was studied also by harvesting cells either from thymus or spleen of A/ST or Cd-1 mouse strains. The enzymatic activity in splenic lymphocytes was more than six time higher than the activity of intact thymus cells. Cytochemically it was evident that within splenic lymphocytes there was a distinct population of lymphocytes with readily demonstrable AMPase activity, and another with no cytochemically demonstrable AMPase activity. It was concluded that murine lymphocytes vary in their activity of AMPase, and that the enzyme is exclusively confined to the cell surface.


Activation of the alternative pathway by pneumococcal cell walls. The present studies were performed in order to identify the pneumococcal subcellular component responsible for activating the alternative pathway. Purified pneumococcal cell walls were able to activate the alternative pathway at a concentration as low as 5 mug/ml and were more active than crude cell walls, which in turn were more active than the whole organism. Purified pneumococcal cell membranes also were able to activiate the alternative pathway but had less than 10% of the activity of the purified walls. Thus, the cell wall appears to play a major role in pneumococcal activation of the alternative pathway. Pneumococcal cell walls containing ethanolamine were as effective as cell walls containing choline in activating the alternative pathway. Since C-reactive protein binds specifically to the phosphorylcholine residue of pneumococcal C-polysaccharide, it is unlikely that pneumococcal cell walls must combine with C-reactive protein in order to activate the alternative pathway.


Differential effects of alloimmunization on T cells mediating graft-vs-host splenomegaly or the allogeneic effect in F1 mice. Spleen cells from normal BALB/c mice or mice immunized 10 or 30 days previously with C57BL/6 spleen cells were tested for a) their capacity to produce graft-vs-host (GVH) reactions in newborn F1 mice and b) their capacity to produce an allogeneic effect in adult F1 mice immunized with Type III pneumococcal polysaccharide. GVH reactivity of alloimmune spleen cells obtained 10 or 30 days after immunization was significantly increased as compared to the reactivity of normal spleen cells in that a) at comparable cell doses, higher spleen indices were obtained with alloimmune cells than normal cells, and b) alloimmune cells produced severe runting at lower cell doses than normal cells. By comparison, the capacity of alloimmune spleen cells to produce an allogeneic effect was reduced 50% on a per cell basis as compared to normal spleen cells at both 10 and 30 days after immunization. These results give further evidence that T cells producing the allogeneic effect are distinct from the T cell populations which interact to produce GVH splenomegaly.


Suppressor T cells arising in mice undergoing a graft-vs-host response. We investigated the ability of mice to generate antibody-forming cells when undergoing a graft-vs-host reaction. (C57BL/6 X DBA/2)F1 mice (BDF1) injected with C57BL/6 spleen cells generated suppressor T cells which inhibit antibody synthesis by BDF1 spleen cells in vitro. These T cells arose from the donor inoculum. They differ from helper T cells in size and they act directly on antigen reactive B cells. The suppressor T cells were specifically directed against components of the H-2 region of the reciprocal parental strain (DBA/2 = H-2d) in the hybrid F1 mouse.


Immune competence of splenic lymphocytes following graft-vs-host disease in mouse allogeneic radiation chimeras. The abnormal immune response of long-term mouse allogeneic chimeras is reflected by qualitative deficiencies in either T or B lymphocytes. The present study was undertaken to determine if a relationship existed between the severity of graft-vs-host disease (GVHD) that these animals had experienced and a functional defect in either the T or B cell population. The in vitro PFC response of chimera spleen cells to sheep red blood cells (SRBC) was evaluated in the presence of normal T or B lymphocytes 4 to 8 months after marrow transplantation and well beyond the GVHD period. In an analysis of several different allogeneic radiation chimeras, our results showed no relationship between the severity of GVHD experienced and the immunologic capacity of either T or B cells. Thus, different chimera combinations showing similar degrees of GVHD were functionally deficient in one or the other of these two cells types or both with no apparent predilection for abnormality in either population. In examining the quantitative in vitro PFC response to sheep RBC by spleen cells from individual chimeras, we found that the number of PFC formed was related to the severity of GVHD experienced by that animal. A general relationship between severity of GVHD and PFC capacity may also exist between chimeras of different genetic combinations. However, this relationship is not precise since gross exceptions occur. Our results, although documenting further the qualitative abnormalities in T and/or B lymphocytes of radiation chimeras, do not reveal the factor or mechanisms by which these cells are made unresponsive. It is suggested that the tolerance-inducing mechanism of these animals, whether it be humoral blocking factors or suppressor cells, is in some way interfering with the collaboration of T and B cells for antibody production.


In vitro inhibition of collagen cross links by catechol analogs. Catechol analogs inhibit the formation of hydroxylysine-derived intermolecular collagen cross links in tissue cultures of chick embryo calvaria. Formation of intermolecular collagen cross links was measured following incorporation of [14C]lysine, reduction with sodium borohydride, and elution from an ion exchange column with a pyridine-formate gradient. Cultures grown in the presence of 10(-3) M catechol, 10(-3) M dopamine, 10(-3) M L-dopa, or 10(-3) M D,L-serine-(2,3,4-trihydroxybenzyl)-hydrazide demonstrated between 43 and 84% inhibition of hydroxylysine formation. Collagen biosynthesis was not diminished in these cultures as compared to controls without additions or with beta-aminopropionitrile when measured by collagenase digestion. The formation of hydroxylysine-derived intermolecular cross links was inhibited 34 to 93% for 5,5'-dihydroxylysinonorleucine and 7 to 71% for 5-hydroxylysinonorleucine. The catechol analogs also inhibit the activity of lysyl hydroxylase as measured by specific tritium release as triated water from an L-[4,5-3H]lysine-labeled unhydroxylated collagen substrate prepared from chick calvaria. Since catechol analogs inhibit the formation of hydroxylysine in a cell-free assay, these compounds must pass into the cells of calvaria in this culture system to inhibit intracellular hydroxylysine formation and subsequently to diminish the reducible intermolecular cross links of the newly synthesized collagen.


Photoreactions of 3,3',4',5-tetrachlorosalicylanilide with proteins. The photoreactions of 3,3',4',5-tetrachlorosalicylanilide anion (TCSA-) with two serum proteins were studied. TCSA- and anions of two similar compounds, N-ethyl-3,5-dichlorosalicylamide and salicylanilide, bind noncovalently to human serum albumin (HSA) without irradiation in aqueous pH 7.4 buffered solutions. TCSA- noncovalently bound to HSA yields three types of photoproducts when irradiated with ultraviolet light (lambda greater than 360 nm). A covalently bonded photoadduct between TCSA- and HSA is formed and histidines in HSA are chemically modified. In addition to these two types of photoproducts which involve HSA, two of the four TCSA- photoproducts which form when HSA is absent are also formed when the TCSA-/HSA complex is irradiated. The results presented indicate that not all proteins in the skin are capable of being the carrier protein in photoallergy of TCSA- and that cross reactivity to other halogenated salicylanilides can be explained by further photochemical reactions of TCSA- photoproducts.


Prolonged maintenance of 2,3-DPG in liquid blood storage: use of an internal CO2 trap to stabilize pH. A close relationship exists between the decrease in concentration of 2,3-diphosphoglycerate (2,3-DPG) and a fall in the pH of stored blood. Buffering the stored red cells with bicarbonate is one solution to the problem of maintaining pH during storage. The effectiveness of this buffer depends upon loss from the stored blood of carbonic acid in the form of CO2. We describe a system in which the CO2 is trapped in a small internal package which contains calcium hydroxide, or calcium hydroxide embedded in Silastic. A medium containing bicarbonate, adenine, glucose, phosphate and mannitol (BAGPM) is added after initial packing of the erythrocytes. With this approach, it has been possible to maintain 2,3-DPG at 92 percent of original, and ATP was approximately 62 percent of initial levels at the end of 42 days of storage if an internal Silastic bag containing calcium was used in bags agitated once weekly. More frequent agitation (five times weekly) produced acceptable maintenance of both 2,3-DPG (78 percent of original) and ATP (44 percent of original) after 42 days of storage when a Silastic block impregnated with calcium hydroxide was utilized to absorb CO2.


Delayed initiation of SS1 pulses in the sea anemone Calliactis parasitica: evidence for a fourth conducting system. 1. Single electrical shocks to the column sometimes elicit a series of 1-6 pulses in the SS1 (ectodermal slow system) but the first pulse does not appear until 5-28 s after stimulation. These pulses occur in addition to the early SS1 pulse which follows every shock and which has a conduction delay of less than 1 s. 2. The threshold of the delayed SS1 response is different from the thresholds of the three known conducting systems (through-conducting nerve net, SS1, and SS2). 3. In the case of stimulation of the column, the delayed SS1 pulses do not arise at the point of stimulation but probably originate in the tentacles or upper column. The pulse origin can shift during a single burst. 4. The pathway from the point of stimulation to the site of origin of delayed SS1 pulses is endodermal. We propose that this pathway represents a fourth conducting system (Delayed Initiation System--DIS). The DIS must connect, across the mesogloea, with the ectodermal SS1. The long pulse delay and repetitive firing may derive from pacemaker activity in the DIS. The DIS pacemakers closely resemble the pacemakers connected to the through-conducting nerve net. The DIS may be neuronal. 5. Delayed SS1 pulse bursts from unattached anemones showed an earlier onset, and more pulses/burst, than those from attached anemones. 6. Delayed SS1 pulses can also be evoked by electrical, and in some cases mechanical, stimulation of the pedal disc, tentacles, and pharynx, but there are regional differences in the number of pulses evoked, in their delay, and in their site of origin.


Oxygen-binding properties of hemoglobins from estivating and active African lungfish. The oxygen-binding characteristics and the multiplicity of the stripped hemoglobiin from active lungfish Protopterus amphibius, are the same as in specimens that have been estivating for about 30 months, showing that alteration in the hemoglobin molecules is not involved in the earlier reported increase in oxygen affinity of whole blood during estivation (Johansen et al., '76). At pH 7.0 and 26 degrees C the hemolysates show a high oxygen affinity (P50 = 3.1 Torr), a Bohr factor (delta log P50/delta pH) of - 0.33, and a cooperativity coefficient (n) of 1.7. Between 15 and 26 degrees C, the apparent heat of oxygenation (delta H) is - 8.6 Kcal-mole-1 at pH 7.0, corresponding with data for other fish. A low sensitivity of oxygen affinity to urea appears to be adaptive to the high urea concentrations in estivating lungfish. The salt sensitivity is, however, similar to human hemoglobin. The hemoglobin consists of two major (electrophoretically anodal) components, which differ slightly in oxygen affinity but are both sensitive to pH and nucleoside triphosphates (NTP). Guanosine triphosphate (GTP), the major erythrocytic organic phosphate, however, depresses the oxygen affinity of the composite and separated hemoglobins more effectively than ATP suggesting that GTP is the primary modulator of oxygen affinity. Comparative measurements reveal only one major hemoglobin component in P. annectens which has a markedly lower oxygen affinity and phosphate sensitivity than P. amphibius hemoglobins and thus seems less pliable to phosphate-mediated variation in oxygen affinity. The data are discussed in relation to the hemoglobin systems of other fish.


Correlation of structural transitions in coliphage R17 with its loss of infectivity. A circular dichroism comparative study of isolated and in situ phage R17 RNA reveals in both cases the same degree of base pairing. However, thermal circular dichroism melting profiles exhibit the presence of free energy of interaction between RNA and capsid protein. It is apparent that the capsid stabilizes the RNA structure with and without the addition of Mg2+. A close RNA capsid association is also derived from pH titration circular dichroism studies. The pH melting of the RNA in situ starts to occur about 0-5 pH unit higher with and without the addition of Mg2+ than the acid denaturation of isolated RNA. A direct correlation between bathochromic CD peak shift to the main position band and loss of survivors is noted for the thermal melting as well as pH titration experiments. It is suggested that the heat and pH induced conformational alterations of R17 RNA in situ coinciding with loss of infectivity occur after an in situ alteration of nucleic acid-capsid protein interaction.


The use of tris-HCl buffer for inoculation of tomato protoplasts with tobacco mosaic virus. When tris-HCL was used as the buffer for inoculation of tomato protoplasts with tobacco mosaic virus, a greater proportion became infected than when phosphate was used. Using 0-05 M-tris-HCl buffer, pH 8-0, in the presence of poly-L-ornithine or poly-D-lysine, 50 to 80 % infection was obtained.


Adsorption of phage P22 to Salmonella typhimurium. Adsorption of phage P22 to its receptor in the lipopolysaccharide (LPS) of the envelope of Salmonella typhimurium is accompanied by a hydrolytic cleavage of the O polysaccharide chain. The enzyme, and endorhamnosidase, is found in the phage tail. Propagation of a mutant of phage P22, containing two amber mutations, under restrictive conditions permitted isolation of phage tail parts with endorhamnosidase activity. The tail parts, purified by ion exchange chromatography, were shown to be homogenous by polyacrylamide gradient gel electrophoresis, isoelectric focusing in polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. The mol. wt. was estimated to 240000. The optimal pH range for glycosidase activity was 5 to 7 and optimal temperature 37 degrees C. Hydrolysis of the O polysaccharide chain, when estimated with whole bacteria as the substrate, did not seem to be influenced by the cation concentration. Eclipse of P22 phage particles to whole bacteria was likewise uninfluenced by the cation concentration in the reaction mixture, but eclipse by isolated receptor containing LPS required cations. The optimal concentration for divalent cations was 2 X 10(-3) M, for trivalent cations 1 X 10(-3) M.


Traditional versus individualized nursing instruction: comparison of State Board Examination scores as a result of these two methods of nursing instruction. There is no evidence as a result of this study to preclude that the LEGS Program is not worthwhile. Achievement of State Board Examination Scores have not suffered as a result of this method of instruction, at least in the three schools studied. Statistically, there is no significant difference in mean achievement of State Board Examination Scores as a result of the two methods of nursing instruction. Efforts should be made to improve upon the further investigate this new method of nursing instruction.


Teaching patient assessment: the pros & cons of clinical rounds. The first step in the nursing process is assessment. Facility with patient assessment skills precludes planning, implementing and evaluating patient care. Teaching patient assessment is time consuming and potentially anxiety-provoking. Classroom lectures and practice labs are not enough if students are to learn how to assess patients with pathophysiological responses. We found that clinical rounds on select patientw was a most important adjunct. Coordinating and conducting clinical rounds presents many problems but faculty and students are in complete agreement that the advantages far outweigh the disadvantages. We are hoping that others can "go to school" on some of our experiences as we will continue to do so.


Instructor and course evaluation based on student-identified criteria. Students have come to school for an education and it is their right to evaluate the quality of the education they are receiving. They should not have to demand or even ask for the privilege of saying what they think. Instructors should be providing the opportunity for evaluation by requesting that information from the students. No value judgment can be totally objective, but an instrument composed of mutually agreed upon statements should encourage the greatest possible degree of objectivity. Using one accepted form throughout the school, all students would be considering the same characteristics and traits for every instructor and course evaluated. Each instructor would receive similar information about personal performance and about the course presented. Students would be free to talk to the faculty or to add comments if they so desired; but, a questionnaire used in every course would allow and even encourage responses from every student enrolled. Faculty responsibility would not end with the preparation and implementation of an evaluation instrument. Instructors would have to let the students know their opinions are important and will be considered in curricular and instructional decisions. Faculty and students would be communicating and hopefully fulfilling the needs of and responsibilities to each other.


An experience with a systems approach to curriculum design. Learning system design, based on general systems theory, is a powerful facilitator for planning and implementing programs of curriculum and instruction. Curriculum planners are directed to a practical consideration of all systems components, and an awareness of their interrelatedness. Specifically they must deal with: Problem areas or barriers to success; the need to define and choose explicit outcomes; ways to make the curriculum operational; and a specific plan for program assessment. The systems process is value free and content free. Values are generated as content is selected and fitted into the system components. Therefore the system input is a crucial factor in the quality of the system. I have attempted to share some aspects of systems methodology which were very useful in the experience of designing a program for the education of gerontological nurse practitioners.


Evaluating the nonverbal communication skills of nursing students. This study examined the accuracy rate of judging nonverbal skills used by nursing students in communicating with patients. Self-ratings of the students were compared with composite instructor ratings to find areas of disagreement regarding the quality of the nonverbal skills. Students who rated their nonverbal acuity higher than the instructors and students who rated their nonverbal acuity lower than the instructors were parceled out of the total sample of 92 senior nursing students enrolled in a baccalaureate program. Nonverbal acuity was measured by asking subjects to identify nonverbal information about emotional states from pictures. A November Picture Test consisting of 120 pictorial items was utilized as the dependent variable. Results showed that almost one half of the sample disagreed with the instructor rating applied to their nonverbal skills with patients. The differences between the groups was significant at the 0.05 level and the students were accurate in designating the direction of difference. Results are discussed in relation to clinical evaluation by both faculty and students in appraising communication skills. Implications such as grade inflation are discussed and recommendations for further study are covered.


The effects of feeding on arterial blood gases and lung mechanics in newborn infants recovering from respiratory disease. Fifteen infants recovering from neonatal respiratory disease had arterial blood gases and lung mechanics measured 5 minutes before bolus feeds and at 5, 10, 20, and 30 minutes after feeding to determine physiologic effects of feeding. PaO2 fell significantly from prefeeding values at 5, 10, and 20 minutes after feeds. Mean prefeeding pH and base excess values were significantly different from mean postfeeding values at 5, 10, 20, and 30 minutes, respectively. PaCO2 remained unchanged before and after feeding. Heart rate and systolic and diastolic blood pressure did not change throughout the study. Dynamic lung compliance, respiratory, rate, and tidal volume did not change significantly but there was a trend toward increase in tidal volume. Mean minute volume rose with time as a consequence of the increased tidal volume. Work of breathing remained unchanged at 10 and 20 minutes postfeed and increased slightly at 30 minutes; this was due to a small increase in both elastic and viscous work components at this time.


Efficacy of caffeine in treatment of apnea in the low-birth-weight infant. The efficacy of caffeine citrate in the management of apnea in the newborn infant was evaluated. Caffeine citrate was given to 18 preterm neonates with recurrent apneic spells. Mean (+/- SE) birth weight and gestational age were 1,065.0 +/- 71.9 gm and 27.5 +/- 0.6 weeks, respectively. Mean age at onset of apnea and at initiation of caffeine treatment was 6.5 +/- 3.7 days and 18.2 +/- 4.9 days, respectively. Caffeine citrate was administered with a loading dose of 20 mg/kg intravenously followed within two to three days by 5 to 10 mg/kg once or twice daily. All infants except one showed a significant decrease in the frequency of apneic episodes associated with caffeine therapy. Mean frequencies of apneic spells were 13.6 +/- 2.5 and 2.1 +/- 0.6 apnea per day before and after initiation of caffeine treatment, respectively. Respiratory rate was increased, and blood [h]+ion concentration and Pco2 were decreased. The data suggest that caffeine is an effective pharmacologic respirogenic agent in the preterm infant with apnea.


Nucleophilic addition of bisulfite ion to prostaglandins E2 and A2: implication in aqueous stability. Evidence is presented to indicate that the bisulfite ion (HSO3-) adds across the C-9 carbonyl group of dinoprostone (prostaglandin E2) and across the delta 10,11- bond of prostaglandin A2. At room temperature, the apparent equilibrium constant, determined by phase solubility analysis, circular dichroism, UV spectroscopy, and partitioning, for the formation of the bisulfite adduct of dinoprostone is about 7.5 M-1 at neutral pH. From this result and a free energy relationship reported in the literature for the thermodynamics of nucleophilic addition to carbonyl groups, it is concluded that the chemical reactivity of the C-9 carbonyl group of dinoprostone is not high enough to improve aqueous stability through reversible one-step nucleophilic reactions. However, from a series of kinetic experiments, it is concluded that the equilibrium is extremely favorable for the formation of the bisulfite adduct of prostaglandin A2 over pH 4-8 at room temperature. The second-order rate constant for the attack of sulfite ion (SO3 2-) to prostaglandin A2 is 1.75 sec-1 M-1.


Use of microcapsules as timed-release parenteral dosage form: application as radiopharmaceutical imaging agent. The development of a new type of parenteral dosage form is described. A system of microencapsulation was formulated which produced microcapsules containing a water-soluble core material. The basic microencapsulation system could be altered to produce microcapsules with varied timed-release characteristics. Tracer methodology was employed as a sensitive and versatile analytical tool for the development and evaluation of the microencapsulation system. The core material was labeled by neutron activation after microcapsule formulation, which eliminated the radiation hazard and contamination problems that could occur during formulation with a labeled core material. Both in vitro and in vivo testing showed that the release patterns of labeled core material could be altered and detected. The microcapsules developed have potential as a timed-release parenteral dosage form and as an organ-imaging radiopharmaceutical.


Oxidation photosensitized by tetracyclines. Irradiation with 365-nm UV light of aerated aqueous solutions of tetracycline gives rise to oxygen uptake when the pH of the solution is above 7.5. The kinetics of the reaction were followed using a polarographic oxygen electrode at a range of pH values for seven currently prescribed tetracyclines. Variation of tetracycline concentration, UV light intensity, and temperature showed the characteristics normally associated with a sensitized photo-oxygenation mechanism rather than a free-radical process. Copper(II) ions inhibited the photo-oxidation of tetracycline, apparently by complex formation. The tetracyclines were tested for photosensitizing capability with oxidizable acceptors. In aqueous solution, no photosensitizing effect could be seen, but methanol solutions of 2,5-dimethylfuran and dl-limonene were oxidized at considerably increased rates when small amounts of tetracyclines were present. This observations has implications for the mechanism of in vivo photosensitivity reactions that occur when tetracyclines are taken internally.


Chemical ionization mass spectra of phenothiazine derivatives and their oxygenated analogs. Twenty-nine derivatives of phenothiazine formed relatively stable ions by proton capture under conditions of chemical ionization, using methane or isobutane as reagent gas. Fragments of generally low abundance formed by simple bond cleavage in the trimethylene portion of the side chain and by loss of water or hydrogen halide. Mass spectra obtained from pyrolyzates of amine salts and from the corresponding free bases were essentially identical.


Effect of chloroquine adsorption on acid reactivity of magnesium trisilicate. Due to the adsorption of chloroquine by magnesium trisilicate, both the BP acid absorption test and the rate of hydrochloric acid uptake, as monitored by pH measurements, were significantly reduced. This reduction was dependent on the amount of chloroquine adsorbed, since multilayer adsorption produced relatively more suppressive effects than did monolayer adsorption. The presence of adsorbed chloroquine also decreased the amounts of magnesium released in an acid medium. The inhibition of the antacid property due to chloroquine adsorption may be attributed to the occupation of the reactive sites of the antacid surface by chloroquine and to a reduction of the surface of the antacid due to flocculation of the particles.


Prenatal administration of levorphanol or dextrorphan to the rat: analgesic effect of morphine in the offspring. We have recently demonstrated that prenatal administration of morphine to the rat results in tolerance to the analgesic effects of morphine in the offspring at 3 to 11 weeks of age. To extend these findings, levorphanol or dextrorphan was administered to female CFE rats during days 5 to 12 of gestation. Control animals were injected with 0.9% saline on the same schedule. At 5 weeks of age all offspring were tested with graded doses of morphine in the hot-plate test for analgesia. Morphine produced a dose-related increase in analgesia in all offspring, but the effect of morphine in the offspring of levorphanol-treated females was significantly reduced compared to the offspring of saline-treated females; the analgesic effect of morphine did not differ between the offspring of the dextrorphan- and saline-treated females. The analgesic effect of morphine remained reduced in 9-week old offspring of levorphanol-treated females compared to the corresponding offspring of females that had received saline. Diminished analgesic activity of morphine in the offspring of levorphanol-treated females compared to the offspring of females that had received dextrorphan or saline was still observed even when the offspring were rendered tolerant to morphine by daily drug injections over a period of 5 days. Thus, the protracted tolerance to the analgesic effects of morphine can also be produced by a morphine congener (levorphanol), but not by its analgesically inactive (+) isomer (dextrorphan).


Effects of maternal ethanol ingestion on amine uptake into synaptosomes of fetal and neonatal rat brain. Pregnant rats were kept on an ethanol-containing (6.8% v/v) liquid diet either from the 13th or 18th day of gestation. In pups exposed to ethanol from the 13th day of gestation, synaptosomal uptake of 3H-tyramine and its conversion to 3H-octopamine were increased initially and exposure to ethanol from the 18th day of gestation produced increases in synaptosomal uptake and conversion for longer periods. These changes were not observed in adult rats administered ethanol. Brain tyrosin hydroxylase activity in the developing rats was unaltered by continuous exposure to ethanol. Withdrawal from ethanol either at 0, 3 or 5 days of age enhanced the tendancy toward increased synaptosomal uptake and conversion, but the magnitude of change depended upon the time of exposure and the age at which withdrawal was initiated. These results suggest that maternal ethanol ingestion can affect synaptic function in the developing central noradrenergic system, with consequent alterations in neurotransmitter uptake and storage.


Saluretic and uricosuric effects of (6, 7-dichloro-2-methyl=1-oxo-2-phenyl-5-indanyloxy) acetic acid (MK-196) in the chimpanzee. The saluretic and uricosuric responses elicited by a novel agent, MK-196, have been studied in a great ape, the chimpanzee. This agent is orally active at very low doses and has a prolonged duration of action. Probenecid does not appear to influence the saluretic and uricosuric properties of MK-196. Net tubular secretion of urate was reduced by MK-196. Urinary pH changes did not compromise the efficacy of this new agent. On a dose basis, MK-196 was more saluretic (and uricosuric) than ethacrynic acid or furosemide and possessed a longer duration of action. Because of the marked natriuresis caused by MK-196, some increase in potassium excretion occurred.


Renal excretion of a slauretic-uricosuric agent (MK-196) and interaction with a urate-retaining drug, pyrazinoate, in the chimpanzee. The excretory pattern for MK-196 is ocmpatible with that of other weak organic acids such as salicylate and probenecid. Tubular secretion of MK-196 is strongly inhibitied by probenecid and high loads of p-aminohippurate. Urinary excretion of MK-196 is increased 10-fold when the urine is alkaline. Clearances of MK-196 were not corrected for plasma protein binding of the drug which is very high (greater than 99%). Bidirectional transport processes are operative in that MK-196 is secreted by the renal tubule and passively back diffuses across the tubular epithelium by a pH-dependent process. MK-196 is able to overcome pyrazinoate-induced urate retention, whereas probenecid is not when studied by conventional clearance techniques. The uricosuric activity of MK-196 appears to be somewhat less with pyrazinoate than in its absence. When MK-196 is administered prior to pyrazinoate an attenuated uricosuric response was observed. This finding cannot be ascribed to a temporal decline in uricosuric action. Diuresis and saluresis produced by MK-196 are not influenced by pyrazinoate. The interaction of MK=196 and pyrazinoate on urate excretion is in direct contrast to results obtained with probenecid and pyrazinoate. A model has been proposed to explain this unique finding.


Potential difference and the distribution of ions across the human red blood cell membrane; a study of the mechanism by which the fluorescent cation, diS-C3-(5) reports membrane potential. 1. The mechanism by which the fluorescent, cationic dye diS-C3-(5) responds to the membrane potential of red blood cells has been investigated. 2. The dye in aqueous solution absorbs most strongly at 650 nm. Addition of white, haemoglobin-free membranes red shifts the absorption maximum ca. 20 nm, while addition of membrane-free cell lysate results in the appearance of a new dye absorption peak at 590 nm. Thus the dye binds both to cell membranes and to cell contents. The component of the cytoplasm which binds the dye is non-dialysable, presumably haemoglobin. 3. Dye added to a suspension of intact cells shows a strong absorption at 590 nm indicating that the dye has bound to the cell contents and that the membrane is permeable to the dye. 4. The amount of dye which partitions into (and on to) the cells can be determined, as reported by Sims, Waggoner, Wang & Hoffman (1974), from the fluorescence of the dye remaining in the supernatant after the cells are centrifuged to the bottom of the suspension. In most conditions the proportion of the cell associated dye which is either free inside the cell or bound to the outside face of the membrane is negligible compared to the proportion bound to the cell contents. 5. On the assumption that the dye is not actively transported, the ratio of the equilibrium dye activities inside and outside the cell, ai/ao, is determined by the membrane potential according to the Nernst relation. Driving the membrane potenial negative then increases the cell associated dye by increasing the activity ratio and hence ai and the amount of dye bound to cell contents. 6. At the known Donnan equilibrium potential the internal dye activity can be calculated from the external activity. An empirical relation between cell associated dye and internal activity has been determined by measuring the dye partition between cells and medium at different external activities. 7. Using this empirial relation, and providing that any changes in cell composition do not affect the dye binding, the internal activity at any potential can be calculated from the measured amount of cell associated dye. The external activity can be estimated fluorimetrically. The membrane potential is then calculated from the activity ratio. 8. The membrane potenial of cells has been altered by adding valinomycin in the presence of different K gradients. Under the conditions used, the 'constant field' permeability for K-Val is 15-20 times that of Cl. 9. Dye binding to haemoglobin is influenced by pH and thus dye partitioning into cells changes with intracellular pH. Increasing intracellular pH increases the amount of dye partitioned, while decreasing pH decreases this amount. 10. When large potentials are produced with valinomycin there is no change in intracellular pH. This result indicates that in red blood cells intracellular pH is determined by the external pH and the Cl concentration ratio and not by the membrane potentials. 11. DiS-C3-(5) can be used to estimate potentials across resealed ghost membranes...


The Bohr effect on the reaction of carbon monoxide with fully oxygenated haemoglobin. 1. The rate at which CO displaces O2 from its combination with haemoglobin in solution, has been measured spectrophotometrically, using a rapid-mixing stopped-flow technique. 2. In the presence of CO2, the reaction proceeds by a unimolecular dissociation, with a rate constant r. 3. The relationship of the reciprocal of r to the ratio PO2/PCO is nonlinear, and a different curve is obtained at each CO concentration. 4. Measurements were made of the rate of the reaction when the pH was varied, with constant or varying PCO2. In both situations the value of r was found to have a miximum, for a given PO2/PCO ratio and CO cencentration, at pH 7.2. 5. An analysis of these results suggest that the Bohr effect, of pH and PCO2, is a dynamic equilibrium between four stable tertiary states of each of the alpha and beta chains. Each intermediatory complex has different rate constants for CO and O2.


Interprofessional education of the new health practitioner. This is a report of a survey of the interprofessional education activities included in 54 physician's assistant and 60 nurse practitioner programs. In a majority of such new health practitioner programs, the importance of interprofessional education as a part of training for team health care delivery has been recognized, and some structured interprofessional activity has been provided. A variety of methods are being used to achieve the objectives of effective team delivery of primary health care, including the mixing of students in both classroom and clinical settings and the inclusion of specific courses on role and professional identity. Seventy percent of physician's assistant students and 38 percent of nurse practitioner students in the programs included in the study have at least one classroom activity shared with another health profession group, most commonly medical students. Only a surprisingly small percentage of programs were involved in any evaluation of their interprofessional activities. In spite of the primary care orientation of new health practitioner programs, less than 40 percent offer clinical experience on a health team in a primary care setting.


Role of bacterial microflora in development of intestinal lesions from graft-versus-host reaction. Acute secondary disease was induced in (C57BL X CBA)F1 mice by transplanting CBA bone marrow and spleen cells following lethal whole-body irradiation. The lesions of graft-versus-host (GvH) disease were scored quantitatively by counting of degenerated crypts in subcutaneous fetal gut implants that were free of bacteria. In conventional F1 mice the damage in F1 fetal gut was twice as great as in F1 fetal gut implants carried by decontaminated chimeras. CBA fetal gut implants developed substantial damage when present in conventional chimeras, but not when present in decontaminated chimeras. These results could be explained by assuming the presence of cross-reacting antigens on intestinal bacteria and in the gut epithelial tissue. They also explained the profound protection against delayed GvH mortality provided by removal of the intestinal microflora.


Observations upon calcium oxalate crystallization kinetics in simulated urine. Two major etiologic theories of urinary stones are excessive saturation of urine with crystallizable substances or defects in inhibitors that allow relative supersaturation to occur. To date, it has been difficult to confirm the supersaturation theory in experiments using diffusion-limited crystallization systems because direct measurements of the nucleation process of crystallization could not be performed. We used well developed, continuous crystallizer techniques and adapted them from industrial use to the study of stone disease. Data derived from the experiments allow the absolute measurement of crystal growth rate and determination of nucleation rate. These methods were applied to study the calcium oxalate dihydrate (weddellite) system in artificial urine that lacked only proteinaceous components. Based on these experiments it was not possible to grow crystals large enough within 5 to 20 minutes to obstruct the collecting ducts of the kidney. Therefore, it appears that other processes, such as aggregation or stasis within tissues, may well be related to initiation of stone disease. Under the experimental conditions of this study nucleation rate exceeded growth rate. Therefore, multiple small particles are created at the expense of allowing larger particles to grow. Inhibitors can be tested rapidly in this system by adding them in concentrations compatible with those found in urine.


[Bacteriological evaluation of clindamycin-2-phosphate (author's transl)]. Clindamycin-2-phosphate, a newly developed injectable antibiotic, was bacteriologically evaluated in comparison with the parent compound, clindamycin. Clindamycin-2-phosphate has a rather weak antibacterial activity. However, it is metabolized into clindamycin in vivo. And this parent compound shows strong activity against both gram-positive bacteria and gram-negative cocci. In vitro studies demonstrated that clindamycin-2-phosphate and clindamycin are affected by the pH of the medium used--the antibacterial activity becomes stronger as the medium is made more alkaline--while they are little affected by the size of the inoculum. In mice with experimental infections, clindamycin-2-phosphate showed about the same therapeutical effect as clindamycin in contrast with the former's inferior in vitro activity as compared with the latter.


[Use of clindamycin-2-phosphate in ophthalmology (author's transl)]. Clindamycin-2-phosphate was studied to evaluate its possible use in ophthalmology. 1. Sensitivity of organisms isolated from human clinical materials: Of 44 staphylococcal strains in vitro studied 13 were highly sensitive to 0.1 mug/ml and 28 were resistant to 100 mug/ml or more of clindamycin-2-phosphate. 2. In experiments with rabbits, clindamycin-2-phosphate showed superior penetration into the ocular tissues after intravenous injection than after intramuscular injection. 3. Clindamycin-2-phosphate was administered intramuscularly at the dose of 300 mg to 6 healthy volunteers. The blood concentrations of 6 cases averaged 3.2 mug/ml after 1 hour, and 0.8 mug/ml after 6 hours. 4. Fourteen patients with extraocular infection were treated with daily intramuscular administration of 300 mg of clindamycin-2-phosphate. All of them improved by the treatment, and no side effect was observed.


Experimental models for prevention of graft-versus-host reaction in bone marrow transfusion. I. Selective suppression and augmentation of splenomegaly and cytotoxicity. Induction and suppression of splenomegaly and cytotoxicity against C57BL/L cells were studied in (AKR X C57BL/6) F1 hybrid adult mice after the transfer of AKR lymphoid and bone marrow cells. 1) Splenomegaly and cytotoxicity were dissociated in the developmental stages of the graft-versus-host reaction. When lymphoid and bone marrow cells of normal AKR mice were injected into F1 recipients, splenomegaly was prominent on days 5 and 7, but cytotoxicity of spleen cells was not detected. Splenomegaly became less prominent but the cytotoxicity became detectable on day 14 after the injection. 2) Cytotoxic activity of spleen cells of F1 recipients was suppressed by the treatment of AKR donors with C57BL/6 lymphoid cells in Freund's complete adjuvant. Splenomegaly, however, was substantially enhanced by such a treatment of the donors. On the other hand, induction of the cytotoxic activity was facilitated by the treatment of donors with C57BL/6 skin grafts. 3) F1 hybrid mice could be protected from the graft-versus-host reaction by the injection of AKR anti-C57BL/6 serum or pretreatment of AKR donors with sonicated cellular antigens of C57BL/6.


Germination of unactivated spores of Bacillus cereus T. Effect of preincubation with L-alanine or inosine on the subsequent germination. Heat-activated spores of Bacillus cereus T germinate rapidly in the presence of L-alanine alone or inosine alone. In contrast, unactivated spores can not germinate in the presence of either germinant alone but rapidly in the presence of both germinants. The highest level of cooperative action of L-alanine and inosine on the germination was observed when they were present in a ratio 1:1. Preincubations of unactivated spores with L-alanine or inosine had opposite effects on the subsequent germination in the presence of both germinants: preincubation with L-alanine stimulated the initiation of subsequent germination, while preincubation with inosine inhibited it. These results suggest that germination of unactivated spores initiated by L-alanine and inosine includes two steps, the first initiated by L-alanine and the second prompted by inosine. The effect of preincubation of unactivated spores with L-alanine was not diminished by washings. The pH dependence of the preincubation of unactivated spores was not so marked as that of the subsequent germination in the presence of inosine.


Iron poisoning.

Iron poisoning. Based on the metabolism, pathophysiology and clinical picture of iron poisoning, a treatment protocol for the emergency department is presented. For insignificant amounts, treatment is ipecac and oral bicarbonate. For ingestions of amounts greater than 150 mg/kg, even if the patient is asymptomatic, hospital observation is necessary. Chelation therapy with Desferal is reserved for patients with free serum iron, and probably for patients who present in coma, shock, or convulsions.


[Bone marrow transplanation by aplastic anemia and acute leukemia--chances, success, indications (author's transl)]. The experimental background, methods of preventing immunological complications and clinical results of bone marrow transplantation in cases with aplastic anemia and acute leukemia in relapse are reported. The causes for failure in clinical bone marrow transplantation insert: are discussed. The possibilities of bone marrow transplantation appear superior to conventional therapeutic measures, if a histocompatible sibling donor is available.


[Long space flights and the human habitat]. The paper discusses the generation of an environment which may adequately meet human requirements that have developed as a result of evolution in the Earth biosphere as applied to long-duration manned space flights. This environment must exert functional effects on physiological systems of the human body which appear to be underloaded in space flight.


Otitis media of guinea pigs.

Otitis media of guinea pigs. Otitis media occurred in 177 of 1373 guinea pigs necropsied during a six-year period. Streptococcus pneumoniae (20%), Streptococcus zooepidemicus (15%), Bordetella bronchiseptica (12%), and Pseudomonas aeruginosa (11%) were the most common bacteria isolated from affected tympanic bullae. Radiology and otoscopy were tested as means of antemortem screening for affected guinea pigs. Radiology gave 96% accuracy in diagnosing otitis media and proved to be a more satisfactory technique than otoscopy.


[Patterns in the change in the body state of dogs during a breakdown of the atmosphere regeneration system in a pressurized space]. Experiments were carried out on dogs kept in a sealed chamber. Changes in the O2 and CO2 concentrations as well as variations of physiological functions, the so-called survival curves, were studied under the conditions of terminated O2 supply and CO2 utilization. The criteria of investigation, mathematical and physiological analysis were chosen from the point of view of predicting hazardous states during failures of the environmental control system. Tolerance limits during slow and rapid changes of the environment, phases of changes of the body state and mechanisms of a combined effect of increasing hypercapnia and hypoxia were considered.


Effects of ventricular fibrillation on coronary blood flow and myocardial metabolism. Ventricular fibrillation is frequently induced during cardiac surgery to quiet the operative field. The reported effects of fibrillation on the myocardium vary considerably. In an attempt to better define these effects, we subjected 28 dogs to one hour of total normothermic bypass. Myocardial blood flow, lactate, adenosine triphosphate (ATP), oxygen consumption, and left ventricular fibrillation was induced in 5 dogs and continuous electrical fibrillation in 7 dogs. These groups were compared to two respective control groups with beating hearts of 8 animals each. Coronary sinus flow, total coronary blood flow, left ventricular flow, myocardial oxygen consumption, and myocardial tissue lactate increased significantly in the fibrillating hearts. Left ventricular dp/dt decreased with fibrillation, but not significantly. It is concluded that the metabolic demands of ventricular fibrillation exceed the increase in coronary blood flow, when compared to demands of the beating heart, and that decreased left ventricular performance may result.


Arterialized capillary blood gases in exercise studies. The PCO2, PO2 and pH of arterial blood and arterialized capillary blood from the ear lobe were compared in 13 patients at rest and during submaximal exercise. The mean capillary PCO2 was .6 mmHg lower than arterial PCO2 at rest and 1 mmHg lower in exercise. The variation in comparisons (S.D. 1.4 mmHg) at rest and exercise, was greater than between duplicate capillary samples (S.D. .7 mmHg). The mean capillary PO2 was 3.3 mmHg higher than arterial at rest, and 1.7 mmHg higher in exercise: the variation was also greater than for PCO2 (S.D. 5.2 MMHg at rest, 4.2 mmHg in exercise) and greater than in duplicate samples (S.D. 2.6 mmHg). A comparison was made of calculated cardiac output (indirect Fick method) and dead space: tidal volume ratio during exercise, using arterial PCO2 and capillary PCO2: cardiac output was systematically overestimated by 6% (S.D. 9.3%) and VD/VT ratio underestimated by 10% (S.D. 9-3%) when capillary PCO2 was used in the calculations. Capillary blood samples are an acceptable alternative to arterial blood, for measurement of blood gases and pH, as long as blood flow is brisk enough to fill a large (125 mgl) glass capillary tube within 30 seconds. The accuracy of the estimates is greatest for pH, and least for PO2. The PCO2 and PO2 measurements are sufficiently precise for their use in the calculation of cardiac output, VD/VT ratio, alveolar-arterial PO2 difference and venous admixture effect during exercise.


[Intragastric titration with extragastric pH-metry--a clinically applicable and reliable technique of quantitative analysis of gastric secretion (author's transl)]. A technique is described by which the secretory capacity of the stomach can be determined. It appears to be superior both to intragastric titration along with intragastric pH-metry and to common analysis of gastric juice obtained by aspiration. There is no essential difference when comparing results obtained intraindividually by intragastric titration with the pH being measured either inside or outside the stomach. When titrating intragastricly with the pH being measured extragastricly, a tube is used which can be check more easily, remains usable much longer, and - as a result of its smaller diameter - is tolerated much better by the patient. In contrast to common analysis of gastric juice obtained by aspiration, by intragastric titration with the pH being measured extragastricly gastric acid can be determined at its site of production (less volume loss and rediffusion of H+ -ions into tissue), the results obtained representing more accurately the true secretory capacity of the stomach. As can be seen from its results, the technique presented here is apt particularly to both testing antacids and the substances interfering with gastric secretion; it also appears to be superior to common analysis of gastric juice obtained by aspiration.


The Asian species of strychnos. Part IV. The alkaloids. Two hundred thirty-four samples from thirty-six Asian species of Strychnos have been screened for the presence of tertiary alkaloids; the data are set out in table 1. Thin-layer and gas-liquid chromatographic as well as mass-spectrometric studies have enabled alkaloids to be identified in many of the extracts prepared; the data are listed in table 2. The literature on the alkaloids and pharmacology of Asian Strychnos species is reviewed (altogether 99 plant names are considered) and compared with the findings obtained in the present work. The alkaloid economy of the plants is also discussed.


[Spermatogonia counts in descended and undescended prebuberal testes (author's transl)]. The following conclusions which show the importance of time of medical treatment resulted from the examination of 288 testicular biopsies (including 46 controls) by statistical analysis in correlation to age. 1. There is no difference in the number of spermatogonia per tubule cross section of descended and undescended testes in children from birth to 2 years of age. 2. The number of spermtogonia in descended testes is not different in children from birth to 2 years in comparison to children aged 2-6 years. 3. A statistically significant decrease of spermatogonia takes place in the undescended testes of children from 2-6 years in comparison to children from birth to 2 years. 4. The difference in the number of spermatogonia of descended and undescended testes increases in children above the age of 2. It must be concluded from this study that the pathological alterations of the germinative epithelium increase in children beyond the age of 2. Assuming that pathological alterations of the germinative epithelium are caused by the dystopy of the testes, medical treatment should be carried out before the child is more than 2 years old.


[Chemical modification of proteins. 2. Blocking of amino groups and basic amino acids and crosslinking of polypeptide chains in casein and field bean globulin by reaction with dialdehyde starch]. The reaction of dialdehyde starch with casein and field-bean globulin leads to a blocking of the protein amino groups and to a decrease of free lysine, arginine and histidine. Maximum values are reached at high protein concentrations and great molar reagent excess. At best 80-93% of the amino groups or of the available lysine may be blocked in this way. For 1% casein and I and 5% globulin solutions, the pH optimum of the reaction lies at approximately 8; for 5% casein solutions, it is shifted towards the neutral to weakly acidic range. The value for the proportion of unblocked lysine is higher (approximately 5%) when determined by amino-acid analysis after acid total hydrolysis than when measured by means of the colorimetric method according to Carpenter (20%). The difference is designed as reversibly blocked lysine proportion. There is a linear correlation between the proportion of blocked lysine and the relative nutritional value as determined by means of the test organism Tetrahymena pyriformis. Dependently on protein concentration and reagent excess, gel chromatographically detectable cross-linking products of higher molecular weight are formed by the reaction of dialdehyde starch with casein. In 5% protein solutions, such products with molecular weights of less than or equal to 900 000 are the sole detectable components.


Signs, symptoms, and pathophysiology of Pneumocystis carinii pneumonitis. In 80 patients with Pneumocystis pneumonitis, the intial signs and symptoms of infection were usually fever and cough, followed by tachypnea and coryza. Flaring of the nasal alae and cyanosis occurred later. Blood gas composition was markedly altered in its acid-base profile in most patients at admission. There was moderate to severe respiratory alkalosis and hypoxia. Clinical manifestations were correlated with the extent of histopathologic changes in the lung. (Deprivation of protein in the diet of rats provoked P. carinii infection.) P. carinii infection was found in children with kwashiorkor; evidence of protein-calorie malnutrition is closely associated with P. carinii pneumonitis in children wiht cancer and other primary diseases. P. carinii pneumonitis proved unique in that the causative organisms remained limited to the lungs even in fatal cases. No toxins have been identified, and systemic effects of the infection were only those that could be related to hypoxia and fever.


Relaxation of heart muscle by catecholamines and by dibutyryl cyclic adenosine 3',5'-monophosphate. Similarity of beta-adrenoceptors mediating contractile and relaxant effects of catecholamines in kitten pipillary muscle. 1. In isometrically contracting kitten papillary muscles, dibutyryl cyclic AMP (DBcAMP) enhanced peak tension, increased rates of contraction and relaxation, decreased tension of a phasic but not of a small tonic component of KCl-contractures, and caused aftercontractions. These effects resemble closely those of catecholamines. 2. The effects of DBcAMP on kitten papillary muscle were not influenced by (-)-bupranolol, a beta-adrenoceptor antagonist. 3. DBcAMP decreased KCl-contractures in strips of frog ventricle. 4. Phasic KCl-contractures in kitten papillary muscles were decreased by (-)- and (+)-isoprenaline. For similar effects, 100-fold higher concentrations of (+)-isoprenaline than of (-)-isoprenaline were required. 5. Increases in maximum rates of contraction and relaxation, increases in peak tension of isometric contractions and reduction of phasic KCl-contractures by catecholamines were antagonized competitively to a similar extent by (-)-bupranolol. Mean apparent equilibrium constants for the beta-adrenoceptor-(-)-bupranolol complex of 0.46-0.70 nM were estimated. These constants were quite similar irrespective of whether (-)-isoprenaline, (+)-isoprenaline or (-)-noradrenaline were used as agonists. 6. Increases in contractile strength, maximum rates of contraction and of relaxation of isometric contractions and decreases in KCl-contractures by (-)-isoprenaline were surmountably blocked by (+)-bupranolol. Mean apparent equilibrium constants for the receptor-(+)-bupranolol complex were 40-50 nM. 7. The equilibrium constants of (-)- and (+)-bupranolol for the receptors mediating positive inotropic and relaxant effects of catecholamines were not significantly different from constants for bupranolol-receptor complexes in cell-free membrane particles of kitten heart ventricle. It is suggested that the same beta-adrenoceptor triggers positive inotropic, relaxant and adenylyl cyclase-activating effects of catecholamines in kitten papillary muscle. 8. The partial agonist (-)-dichloroisoprenaline (DCI) (1 muM) reduced by 79% the phasic KCl-contractures of the kitten papillary muscles. DCI stimulates adenylyl cyclase activity of ventricle membranes to less than 1/4 of maximum stimulation by (-)-isoprenaline. If cyclic AMP produced by DCI is involved in the decrease of phasic KCl-contracture, small increase in cyclic AMP should be sufficient to induce this effect.


Activation of myocardial beta-adrenoceptors by the nitrogen-free low affinity ligand 3',4'-dihydroxy-alpha-methylpropiophenone (U-0521). The effect of 3',4'-dihydroxy-alpha-methylpropiophenone (U-0521) on the rate of spontaneously contracting cultured rat heart cells and right atria of rats and kittens was investigated. The action of U-0521 on the cellular content of cyclic AMP and on the adenylyl cyclase of heart membrane particels was also studied. 1. U-0521 caused positive chronotropic effects on single cultured heart cells and right atria of the rat. U-0521 was about 10(5) times less potent than (--)-isoprenaline. The maximum effect of U-0521 was smaller than the maximum effect of (--)-isoprenaline. A small positive chronotropic effect of U-0521 was also observed on kitten atria. 2. The beta-adrenoceptor blocker (--)-bupranolol antagonized the positive chronotropic effects of U-0521 to the same extent as the effects of (--)-isoprenaline on single cells and atria of the rat. The effects of both U-0521 and (--)-isoprenaline appear therefore mediated through the same beta-adrenoceptors. The positive chronotropic effects of U-0521 on kitten atria were also blocked by (--)-bupranolol. 3. Up to 0.1 mM U-0521 did not block the effects of (--)-isoprenaline on rat atria, not even in the presence of corticosterone or hydrocortisone. 4. 1 min incubations with equieffective (increase in cellular beating rate) concentrations of U-0521 (0.1 mM) and (--)-isoprenaline (1 nM) caused a significant increase in the cellular content of cAMP; this effect of both drugs was antagonized by 10 nM (--)-bupranolol. 5. 0.1--3.3 mM U-0521 did not stimulate the adenylyl cyclase of cell-free membrane particles of kitten ventricles. The cyclase was depressed by 10 mM U-0521. 3.3 mM U-0521 caused a 20% decrease of the maximum cyclase-stimulating effect of (--)-isoprenaline and a 1.6-fold increase of its apparent Km. 6. The results with U-0521 suggest that beta-adrenoceptors can be activated by agonists devoid of nitrogen. However, the affinity of U-0521 for the beta-adrenoceptor is very low (KU-0521 approximately 5.5 mM). The concentration of U-0521 (0.1 mM) causing maximal increases in beating rate of cultured cells probably occupies less than 7% of the available beta-adrenoceptors.


Central sites and mechanisms of the hypotensive and bradycardic effects of the narcotic analgesic agent fentanyl. In dogs, anaesthetized with chloralose, fentanyl (5 mug/kg i.v.) augmented the bradycardia produced by electrical stimulation of the carotid sinus nerves. In contrast, the same dose of the drug did not change the bradycardic response to stimulation of the nucleus of the solitary tract (NTS) indicating that a central facilitation of baroreceptor impulses occurs within the NTS, probably at the first synapse of baroreceptor reflex fibres. Bilateral destruction of the NTS caused a fulminating hypertension and tachycardia similar to that after cutting the baroreceptor afferent fibres. After both procedures, fentanyl (20 mug/kg i.v.) produced marked hypotension and bradycardia. The bradycardic effect was abolished by cutting both vagal nerves when the dogs were pretreated with a beta-adrenoceptor blocking agent (S 2395, 50 mug/kg i.v.). The results provide evidence that the NTS is not the main site of action either for the hypotensive effect or for the vagally mediated bradycardia of fentanyl. Since the dorsal nucleus of the vagal nerve was destroyed together with the NTS, this nucleus does also not appear to be a major site of the action of fentanyl. Blockade of dopamine receptors by haloperidol or pimozide or of serotonin receptors by methysergide did not change the hypotensive, bradycardic and sympathoinhibitory effects of fentanyl.


The in vitro secretion of epidermal growth factor by mouse submandibular salivary gland. A technique for the study of epidermal growth factor (EGF) secretion from salivary gland pieces in vitro is described and the responses to a variety of drugs are compared to the results of previously reported in vivo studies. Adrenaline stimulates secretion of the growth factor as does the alpha-adrenergic agonist phenylephrine, while isoprenaline is less active in stimulating EGF secretion. Phentolamine, but not propranolol, blocks the secretory activity of adrenaline and isoprenaline in this system. Pretreatment of the salivary gland pieces with colchicine reduces the secretory response to adrenaline, suggesting an involvement of microtubular proteins in the process. It is anticipated that the in vitro technique described will provide a means by which detailed investigation of the secretion of epidermal growth factor can be made.


Effect of passages of Morris hepatoma 5123D in F1 (Buffalo X Wistar) rats on permanent decrease of gamma-glutamyltranspeptidase activity. The Morris hepatoma 5123D after at least two passages in F1 (Buffalo X Wistar) rats shows quicker growth than the original tumor and bearers of it have much lower gamma-glutamyltranspeptidase activity in serum, urine, tumor and in some other organs. This new variant of the hepatoma was labeled as hepatoma 5123D/AS. Simultaneous implantation of hepatomas 5123D/AS and 5123D in the same rats prevents the increase of serum gamma-glutamyltranspeptidase activity. After surgical removal of the former tumor, the enzyme activity in serum quickly increases. No significant differences in some other peptidase activities were observed between the variant and hepatoma 5123D.


[Dynamics of synaptic changes in experimental audiogenic epilepsy]. The dynamics of ultrastructural changes in axonal endings were studied after experimental epileptic seizures. Mice and rats from strains with genetically--determined audiogenic epilepsy were used as a model of epilepsy. The animals were divided into 3 groups: in group 1 only one seizure was evoked, in group 2 eight seizures within 4 hours, group 3 served as control. The animals were killed immediately after the last seizure, 30 min. after it or 1 hour after the seizure. Hippocampal gyrus cortex was impregnated with zinc-iodide-osmium tetroxide and synapses were examined under electron microscope. The number of synaptic vesicles showing positive reaction with zinc iodide was calculated in 20 synaptic boutons in each group. A significant correlation was demonstrated between the frequency of seizures and the survival time after the seizure on the one hand, and synaptic changes, on the other. In the control group 97% of synaptic vesicles were filled with neurotransmitter substance giving positive reaction with zinc iodide. Immediately after the single seizure 46% of synaptic vesicles were found emptied, 30 min. later the neurotransmitter substance was demonstrated in 79% of vesicles, 1 hour later 82% of vesicles had normal appearance. Immediately after serial seizures 91% of vesicles were found empty, 30 min. later the neurotransmitter was present in 50% of vesicles, 1 hour later in 78%. In another group of animals seizures were evoked once daily for 40 days (chronic epilepsy model). Synaptic changes were different: the synaptic boutons were swollen, the number of vesicles was reduced, greatly enlarged vesicles and clear membrane-bound vacuoles appeared. They evidenced degenerative character of changes. It is suggested that degenerative synaptic changes may be a substrate of epileptic dementia.


[New preparation with antalgic sedative and anxiolytic activity in manifestations of interest to internal medicine. Double-blind controlled clinical study]. A controlled double-blind clinical trial using randomised blocks was carried out on 3 groups of 10 patients with internal diseases and a pain syndrome accompanied by anxiety. One group received the product under examination (FC 35aA) and the other two its components (dipotassic chlorazepate, paracetamol and codeine). FC 35aA gave the best results, pointing to synergism between its components. Increase of the pain threshold and of the actual involvement of the patient was achieved to an extent that the single components could not match when administered individually at the same doses.