Occurrence of a binding protein for 11-cis-retinal in retina.

A binding protein for retinal has been found in the soluble protein fraction of bovine retina. It was separable from the intracellular retinol- and retinoic acid-binding proteins by gel filtration on Sephadex G-100 and appeared to have a molecular weight of about 50,000. The new binding protein did not bind retinol or retinoic acid. The binding protein neither oxidized retinal in the presence of reduced pyridine nucleotides and is presumed, therefore, not to be a dehydrogenase. Bound retinal was reduced to retinol in the presence of liver alcohol dehydrogenase and NADH, indicating that the functional group remained accessible when in the protein complex. The binding protein bound cis isomers of retinal preferentially. Bound ligand was displaced most effectively by 11-cis-retinal. When individual cis-trans isomers of retinal were presented to the binding protein, binding was maximal with the 11-cis isomer. It is proposed that the protein be referred to as 11-cis-retinal-binding protein.


Structure and action of heteronemertine polypeptide toxins: disulfide bonds of Cerebratulus lacteus toxin B-IV.

The positions of the disulfide bonds in Cerebratulus lacteus toxin B-IV were investigated by hydrolysis of the unreduced protein with a variety of proteases. The resulting peptides were purified by gel filtration, ion exchange chromatography, and preparative paper electrophoresis and chromatography. Determination of the amino acid compositions of the cystine-containing peptides purified demonstrated the existence of disulfide bonds linking half-cystine residues 12 and 24, 21 and 51, and 35 and 38. The fourth bond, involving half-cystines 10 and 47, was assigned by difference.


Kinetic properties of tartrate-resistant acid phosphatase isolated from human spleen with leukemic reticuloendotheliosis.

The tartrate-resistant acid phosphatase was isolated from the spleen of a patient with leukemic reticuloendotheliosis. The unique characteristic of the enzyme is its similar reactivity toward p-nitrophenylphosphate, inorganic pyrophosphate ADP, and ATP. When ATP was incubated with the enzyme, the initial products were ADP and inorganic phosphate. The kinetic data confirmed that these substrates were hydrolyzed by the same enzyme. This type of substrate specificity is clearly different from acid phosphatase and pyrophosphatases previously described in the literature.


Measurements of half-life of rat cardiac myosin heavy chain with leucyl-tRNA used as precursor pool.

The kinetics of labeling of myosin heavy chain, following a single intravenous injection of L-[4,5-3H]leucine, were analyzed with the help of a computer, in conjunction with the labeling kinetics of the specific radioactivities of the precursor amino acid pool. As precursor we used leucyl-tRNA which, as we show here, differs significantly from the intracellular free leucine pool. The half-life of myosin heavy chain was determined from the initial period (0 to 60 min) of incorporation of label into protein after a single injection of tritiated leucine, and also from the period (7 to 14 days) when there is exponential decay of the labeled protein. Myosin heavy chain was separated from other myofibrillar proteins by polyacrylamide gel electrophoresis before measurement of leucine specific radioactivity. The specific radioactivity was measured in both protein and precursor pools by a sensitive isotope dilution procedure (range, 100 to 1500 pmol). The values for the half-life of myosin heavy chain determined at both intervals were similar (5.4 and 5.9 days). Substitution of the specific radioactivity of the intracellular free leucine pool decreased the half-life to 2.7 dyas. Similar values were obtained when the half-life was calculated by simple graphical integration of the experimental curves.


Overall mechanism and rate equation for O-acetylserine sulfhydrylase.

Saturation curves for O-acetylserine sulfhydrylase using the substrate analogues O-propionyl-L-serine, O-butyryl-L-serine, and beta-chloro-L-alanine all exhibit substrate inhibition and yield Km values comparable to O-acetyl-L-serine, except the O-butyryl derivative which has a Km 5-fold higher. Since all analogues are used as substrates and yield similar kinetic parameters in most cases, it is possible that they share a common intermediate. This evidence also suggests that specificity of O-acetylserine sulfhydrylase resides in the fact that the beta-substituted moiety on L-serine is a good leaving group. The overall rate equation for O-acetylserine sulfhydrylase was derived. A comparison of the numerical integration of the rate equation and an experimental time course is given.


Initial reactions in biosynthesis of teichuronic acid of Micrococcus lysodeikticus cell walls.

The in vitro biosynthesis of teichuronic acid, the cell wall polysaccharide of Micrococcus lysodeikticus, occurs in two stages. In the initial stage, the particulate enzyme fraction utilizes uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate N-acetylmannosaminuronic acid (UDP-ManNAcUA) to form three intermediates. N-Acetylglucosamine is transferred from UDP-GlcNAc to a carrier lipid present in the particulate enzyme fraction to form the first intermediate, GlcNAc carrier lipid. N-Acetylmannosaminuronic acid is then transferred from UDP-ManNAcUA to the GlcNAc carrier lipid to form ManNAcUA-GlcNAc carrier lipid, the second intermediate. Finally, the transfer of an additional N-acetylmannosaminuronic acid residue from UDP-ManNAcUA yields the third intermediate, (ManNAcUA)2-GlcNAc carrier lipid. Reactions involving UDP-ManNAcUA release uridine diphosphate. Concomitant synthesis of peptidoglycan or membrane mannan inhibits the synthesis of the teichuronic acid intermediates, thereby providing indirect evidence that the carrier lipid is undecaprenol monophosphate.


Reduced synthesis of [14C]mannosyl oligosaccharide-lipid by membranes prepared from concanavalin A-resistant Chinese hamster ovary cells.

We compared the synthesis of mannosyl oligosaccharide-lipid by membranes of wild type and concanavalin A-resistant Chinese hamster ovary cells during incubations with GDP-[14C]mannose and UDP-N-acetylglucosamine. The membranes prepared from exponentially growing mutant cells incorporated 10- to 70-fold less [14C]mannose into oligosaccharide-lipid than did membranes of wild type cells. The lectin-resistant cells are temperature-sensitive for growth. Using temperature-resistant revertants, we showed that the reduction in labeled oligosaccharide-lipid correlated with resistance to concanavalin A rather than with the temperature-sensitive phenotype. Tunicamycin, a specific inhibitor of oligosaccharide-lipid synthesis, reduced the synthesis of the oligosaccharide-lipid by membranes of wild type cells but did not affect the residual synthesis measured in membranes of the mutant cells. As the mutant cells were grown to high density, there was a marked increase in the synthesis of oligosaccharide-lipid by the membranes.


On the fidelity of DNA replication. Effect of metal activators during synthesis with avian myeloblastosis virus DNA polymerase.

The effect of metal activators on the fidelity of DNA synthesis has been examined. Using the DNA polymerase from avian myeloblastosis virus, the accuracy of Co2+-, M2+-, and Ni2+-activated DNA synthesis was determined with different polynucleotide templates. With poly[d(A-T)] as the template, the error frequency for dCMP incorporation was 1:1400, 1:1100, and 1:600 for Mg2+, Co2+, and Mn2+, respectively, at maximally activating concentrations. The error frequency was invariant with respect to [Mg2+] but increased with greater than activating concentrations of Co2+ and Mn2+. This increase resulted from differential rates of complementary and noncomplementary nucleotide incorporation. The enhanced error frequency was nonspecific as it occurred with all polynucleotide templates and with all noncomplementary deoxy- and ribonucleotides which were tested. Nearest neighbor analyses of the reaction products indicated that the noncomplementary deoxynucleotides were incorporated as single base substitutions. The fidelity of Ni2+-activated DNA synthesis was invariant with respect to [Ni2+] and was similar to that obtained using Mg2+. During DNA synthesis with Mg2+, the addition of Co2+, Mn2+, or Ni2+ resulted in a decrease in the fidelity of DNA synthesis. The relationship between decreases in the fidelity of DNA synthesis and metal mutagenesis, or carcinogenesis, or both, is considered.


Synthesis and secretion of rat albumin in vivo, in perfused liver, and in isolated hepatocytes. Effects of hypophysectomy and growth hormone treatment.

The effects of hypophysectomy on albumin and total protein synthesis in rat liver were investigated in vivo, in perfused liver, and in isolated hepatocytes. In all systems, hypophysectomy resulted in about a 50% decrease in the rate of total protein synthesis and a 30 to 50% decrease in the relative rate of albumin synthesis. Albumin synthesis accounted for 11 to 13% of total protein synthesis in all normal systems, but represented only 5 to 8% of the total in all systems derived from hypophysectomized rats. Growth hormone, administered subcutaneously to hypophysectomized rats for 5 days, restored the relative rate of albumin synthesis to normal in vivo; however, only partial restoration was demonstrated in the in vitro systems. Perfused livers and isolated hepatocytes exhibited linear rates of total protein and albumin secretion for 3 h. The rate of albumin secretion by normal perfused livers was 3 times that of perfused livers from hypophysectomized animals, being 0.54 and 0.17 mg/g of liver/h, respectively. Isolated hepatocytes synthesized total protein and albumin at nearly the same rate as perfused livers. The amount of albumin secreted by cells derived from normal and hypophysectomized rats was 0.38 and 0.10 mg/ml of packed cells/h, respectively. Ribosome half-transit times for albumin and total liver protein were 1.6 to 1.7 min in isolated liver cells derived from both normal and hypophysectomized rats. Analysis by polyacrylamide gel electrophoresis showed no difference in the qualitative distribution of the proteins secreted by perfused livers and isolated hepatocytes.


Purification and properties of a plasminogen activator from pig heart.

An improved procedure is described for the purification of plasminogen activator from pig heart. The initial purification steps were similar to these described previously (Bachmann, F., Fletcher, A. P., Alkjaersig, N., and Sherry, S. (1964) Biochemistry 3, 1578--1585). Use of a novel extraction medium containing EDTA, cysteine, and 2,3-dimercaptopropanol-1 facilitated the removal of large amounts of inert proteins prior to gel filtration on Bio-Gel P-150. The final product had a specific activity of 120,000 to 160,000 CTA units/mg of protein (CTA, Committee on Thrombolytic Agents of the National Heart Institute). Total purification over pig heart was 25,000 to 30,000-fold, average recovery compared to the initial extract was 6 to 8%. Polyacrylamide gel electrophoresis revealed a major and two minor components. The molecular weight of the activator determined by gel filtration was 51,500 +/- 3,400 for the major activity component and 48,000 for a minor component which was partially separated from the major peak in eight of nine chromatography runs. A gamma-globulin fraction of antiserum against purified activator neutralized the biological activity of the activator on fibrin plates. Immunoelectrophoresis of gel-filtered activator revealed only one anodic component.


Ovine submaxillary mucin. Primary structure and peptide substrates of UDP-N-acetylgalactosamine:mucin transferase.

Tryptic digests of ovine submaxillary apomucin were fractionated by gel filtration and ion exchange chromatography to give 14 peptide fractions. Three purified tryptic peptides, representing 106 of the 650 residues in apomucin, were submitted to automated sequence analysis. The NH2-terminal 50 of the 74 residues in one peptide and the entire sequence of the other two hexadecapeptides were established. These studies suggest that purified ovine submaxillary, mucin is chemically homogeneous, containing a unique primary structure without substantial repeating sequences in its polypeptide chain. The sequences adjacent to 28 known O-glycosidically substituted seryl and threonyl residues were compared. No homologies were apparent around the glycosylated seryl and threonyl residues which might define the specificity of the UDP-N-acetylgalactosaminyl:mucin polypeptide transferase that incorporates N-acetylgalactosamine into O-glycosidic linkage in glycoproteins. However, there appears to be a minimum size requirement for glycosylation, because the transferase catalyzes glycosylation of tryptic peptides efficiently, while chymotryptic and thermolytic peptides were much poorer substrates for the transferase.


Characterization of GTP-dependent Met-tRNAf binding protein.

Purified GTP-dependent Met-tRNAf binding protein (EIF2) prepared from the 0.5 M KCl eluate of reticulocyte polyribosomes was successfully resolved into its three-component subunits by isoelectric focusing in the presence of urea. The 37,000-dalton subunit focused at a pH of 5.8 and was resolved into two spots; the 48,000-dalton subunit focused at a pH of 6.6 and was resolved into three spots; and the 52,000-dalton subunit exhibited an isoelectric point of 8.9 and migrated as a single spot. When isolated 37,000- and 48,000-dalton subunit was found to possess Met-tRNAf and mRNA binding activities while the 37,000-dalton subunit was found to possess GDP binding activity. Phosphorylation of EIF2 by protein kinases present in reticulocyte lysates was demonstrated using [gamma-32P]GTP or [gamma-32P]ATP as the phosphate donor. The 37,000-dalton subunit was preferentially phosphorylated when [gamma-32P]ATP was used as substrate; the 48,000-dalton subunit was preferentially phosphorylated when the [gamma-52P]GTP was used as phosphate donor, although some phosphorylation of the 37,000-dalton subunit was also observed. The 37,000-dalton subunit of ribosome-associated EIF2 was present predominantly in a dephosphorylated form following purification.


Correlation between arginyl residue modification and benzodiazepine binding to human serum albumin.

Human serum albumin (HSA) has been chemically modified with 1,2-cyclohexanedione and N-acetylimidazole under nondenaturing conditions. Derivatives, in which 10 arginine residues (1,2-dihydroxycyclohex-1,2-ylene (DHCH)-HSA), 56 to 57 lysine and 5 tyrosine residues (acetyl-HSA), or 56 to 57 lysine residues alone (O-deacetyl-HSA) are modified, have been isolated. Their conformation has been tested by circular dichroism measurement, gel filtration on Sephadex G-150, and ultracentrifugation. From these analyses and binding studies it is concluded that only insignificant changes of conformation have occurred. The binding properties of the HSA derivatives have been tested with bilirubin, diazepam (a benzodiazepine drug), phenylbutazone, and indomethacin by circular dichroism. The binding of diazepam to DHCH-HSA is almost completely inhibited and that of phenylbutazone and indomethacin decreased, while the binding of bilirubin is essentially unaffected. In acetyl-HSA and O-deacetyl-HSA, the bilirubin binding is significantly decreased, while the binding of the drugs mentioned is less affected. It is concluded that bilirubin and the drugs bind to two separate sites which contain positive charges essential for the binding properties. The charge(s) in the benzodiazepine site from arginine.


Isolation of eukaryotic ribosomal proteins. Purification and characterization of 60 S ribosomal subunit proteins L3, L6, L7', L8, L10, L15, L17, L18, L19, L23', L25, L27', L28, L29, L31, L32, L34, L35, L36, L36', and L37'.

The proteins of the large subunit of rat liver ribosomes were separated into seven groups by stepwise elution from carboxymethylcellulose with LiCl at pH 6.5. Twenty-one proteins (L3, L6, L7', L8, L10, L15, L17, L18, L19, L23', L25, L27', L28, L29, L31, L32, L34, L35, L36, L36', and L37') were isolated from three groups (C60, E60, and F60) by ion exchange chromatography on carboxymethycellulose and by filtration through Sephadex. The amount of protein obtained varied from 0.3 to 25 mg. Nine of the proteins (L6, L8, L18, L27', L28, L29, L34, L36, and L36') had no detectable contamination: the impurities in the others were no greater than 9%. The molecular weight of the proteins was estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the amino acid composition was determined.


Interaction of bacteriophage T4 RNA and DNA ligases in joining of duplex DNA at base-paired ends.

The joining of duplex DNA at base-paired ends by bacteriophage T4 DNA ligase was confirmed using either a synthetic duplex decamer or restriction endonuclease fragments of ColE1 DNA as substrates. The reaction was not linearly dependent on enzyme concentration but increased markedly at high enzyme concentrations. Although T4 RNA ligase did not catalyze this blunt end joining, it makedly stimulated the DNA ligase reaction particularly at low DNA ligase concentrations. The apparent Km for the decamer was 50 micronM in the presence or absence of RNA ligase. In the presence of RNA ligase, T4 DNA ligase had about the same turnover number for blunt end and cohesive end joining. The joining of duplex DNA at base-paired ends was proven by several techniques including restriction endonuclease cleavage of the products. The products of the ligation reaction using restriction enzyme fragments were mostly linear oligomers but included some circular duplexes. Escherichia coli DNA ligase in the presence or absence of RNA ligase did not catalyze blunt end joining. RNA ligase only moderately affected the joining of cohesive ends by T4 DNA ligase or E. coli DNA ligase and did not itself catalyze this reaction.


Intestinal basement membrane of Ascaris suum. Characterization of carbohydrate units.

Characterization of glycopeptides obtained on alkaline hydrolysis and on extensive collagenase and pronase digestion of the intestinal basement membrane showed the existence of two distinctly different carbohydrate units. One of these is a disaccharide, composed of glucose and galactose, linked to hydroxylysine. It was shown to be identical to the unit (2-O-D-glucopyranosyl-O-D-galactopyranosylhydroxylyasine) present in vertebrate basement membranes, as determined from stability to alkaline hydrolysis, retention time on amino acid analyzer, chemical composition, graded acid hydrolysis, methylation analysis, and periodate oxidation. Direct quantitation after alkaline hydrolysis showed the presence of 9.71 disaccharide units/1000 amino acid residues, indicating that 89% of the hydroxylysine residues of the intestinal membrane are glycosylated. The other unit, consisting of the remaining monosaccharides of the membrane, was separated from the disaccharide unit by gel filtration and ion exchange chromatography of collagenase/pronase digests. Chemical analyses and molecular weight determination by thin layer gel filtration chromatography of purified glycopeptides indicated that this unit is an oligosaccharide which is composed of fucose, galactose, mannose, galactosamine, and glucosamine in a mole ratio of 1:1:1:1:2, respectively. The amount of this unit was calculated to be 2.6 units/1000 amino acid residues.


Effect of calcium on the recovery and distribution of DNA polymerase alpha from cultured human cells.

It was recently reported (Lynch, W. E., Surrey, S., and Lieberman, I. (1975) J. Biol. Chem. 250, 8179-8183) that the extraction of regenerating rat liver in solutions of isotonic sucrose containing 4 mM CaCl2 leads to almost quantitative recovery of DNA polymerase alpha (Weissbach, A., Baltimore, D., Bollum, F., Gallo, R., and Korn, D. (1975) Science 190, 401--402) activity in the purified nuclear compartment. Our application of this method to the isolation of the DNA polymerase activities activities from cultured human epithelial and lymphoblastoid cells has led to substantially different results. We have observed that the inclusion of Ca2+ in either isotonic sucrose or hypotonic aqueous extraction media leads to the irreversible inactivation of the majority, cytoplasmic fraction of DNA polymerase alpha activity and is without quantitative effect on the recovery of the nuclear fraction of this activity.


5-demethylubiquinone-9-methyltransferase from rat liver mitochondria. Characterization, localization, and solubilization.

The methyltransferase responsible for the conversion of 5-demethylubiquinone-9 to ubiquinone-9 in rat liver mitochondria has been shown to be localized in the inner membrane of rat liver mitochondria. NADH was required to generate the hydroquinone, which was the immediate substrate for methylation. The Km for 5-demethylubiquinone-9 was estimated to be in the range of 60 to 80 nM and the Km for S-adenosylmethionine was found to be 22 micronM. The methyl-transferase was solubilized by Triton X-100, a procedure which inactivated the 5-demethylubiquinone-9 reductase. Dithionite was found to partially substitute for NADH in both membranous and soluble systems. Inhibitors of catechol-O-methyltransferase were not effective inhibitors of 5-demethylubiquinone-9-methyltransferase. In addition, catechol-O-methyltransferase and 5-demethylubiquinone-9-methyltransferase were found to have reciprocal subcellular localizations. It is likely that the hydrophobic side chain of ubiquinone, added to p-hydroxybenzoate in the  first biosynthetic step, is required for attachment to the lipid bilayer. This permits subsequent metabolism of the ring system by membrane-bound enzymes, including the final methylation to form ubiquinone-9.


Chloroplast biogenesis. Net synthesis of protochlorophyllide from magnesium-protoporphyrin monoester by developing chloroplasts.

Developing chloroplasts were isolated from greening Cucumis cotyledons in a cofactor-enriched medium and were incubated in the dark with 14C-labeled and unlabeled magnesium-protoporphyrin monoester. The metabolic pools between protoporphyrin and protochlorophyllide were monitored spectrofluorometrically. The incorporation of the 14C label into protochlorophyllide was also determined. It was shown that magnesium-protoporphyrin monoester, a postulated intermediate of the chlorphyll biosynthetic pathway, was convertible into protochlorophyllide with relatively high yields. Since protochlorophyllide is the immediate precursor of chlorophyll a it was concluded that magnesium-protoporphyrin monoester was indeed an intermediate of the chlorophyll biosynthetic pathway.


Reduction of metmyoglobin derivatives by dithionite ion.

The rate constants for reduction by dithionite of a number of metmyoglobin species Mb+X(X=H2O, imidazole, OH-, F-, N3-, CNO-, SCN-, HCO2-, NO2-, and CN-) were measured at 25 degrees by stopped flow spectrophotometry. The dependence of the rate was [S2O42-]1/2, and the SO2- radical was considered to be the active reductant. Except for X=imidazole and CN-, reduction occurred through dissociation of Mb+X. Values for the dissociative rate constant obtained from dithionite reduction were in good agreement with those obtained directly. Reduction of the dissociated fragment (assumed Mb+H2O) by SO2- is 3+/-1 X 10(6) M-1S-1 at pH 8.2 for all Mb+X species examined. Reduction of Mb+ imidazole and Mb+CN- occurs directly with SO2-, and Mb0CN- (Mb0, deoxymyoglobin) is characterized as an intermediate in reduction of the latter.


Effect of hydrostatic pressure on spectra of heme compounds.

Increase in hydrostatic pressure shifts the absorption bands of oxy-, carboxy-, and deoxyhemoglobin and myoglobin toward the red by 0.4 to 0.7 nm corresponding to a change in extinction coefficient of from 4 to 8% at the peak of the difference spectrum. The pressure difference spectrum for oxyhemoglobin closely resembles the difference spectrum described by Adams and Schuster ((1974) Biochem. Biophys. Res. Commun. 58, 528-533) following addition of inositol hexaphosphate to oxyhemoglobin. A similar shift was observed for derivatives of dimethyl-deuterohemedisulfonate in both Fe2+ and Fe3+ forms indicating that the protein is not required for this effect, in contrast to earlier reports of T. L. Fabry and J. W. Hunt ((1968) Arch. Biochem. Biophys. R3, 428-429) and Q.H. Gibson and F.G. Carey ((1975) Biochem. Biophys. Res. Commun. 67, 747-571) who were unable to observe changes in aqueous solutions of protoheme derivatives.


Effect of temperature and of cytochalasin B and persantin on the nonmediated permeation of non-electrolytes into cultured Novikoff rat hepatoma cells.

The nonmediated permeation of L-glucose, cytosine, and prednisolone into Novikoff rat hepatoma cells followed first order kinetics with rate constants of 0.00404, 0.173, and 2.4 min-1, respectively. The constants were estimated from a nonlinear least squares fit of the integrated first order rate equation. The rate constants were independent of substrate concentration and correlated with the partition coefficients of the substances in octanol-balanced salt solution (0.00158, 0.0352, and 17.8, respectively) and olive oil-balanced salt solution mixtures which were between 10- and 100-fold lower. Arrhenius plots for the permeation of L-glucose, cytosine, and prednisolone were linear and indicated activation energies of 24.2, 28.0, and 19.6 kcal/mol, respectively. The permeation of L-glucose and cytosine, but not of prednisolone, was impeded in a concentration-dependent manner by the presence of cytochalasin B and Persantin, heretofore thought of as specific inhibitors of facilitated diffusion processes. The relative degree of decrease of the permeation rates of L-glucose and cytosine, however, differed for cytochalasin B and Persantin.


Effect of dietary linoleate on synthesis and degradation of fatty acid synthetase from rat liver.

The induction of fatty acid synthetase activity in rat liver is markedly reduced by feeding a diet containing a source of linoleate. Within 48 h after changing from a fat-free, high carbohydrate diet to one containing 15% by weight of safflower oil, the specific activity of rat liver fatty acid synthetase is approximately 2-fold lower than that from rats fed a fat-free diet througout. In contrast, feeding a diet containing 15% hydrogenated coconut oil, a source of saturated fatty acids, or 5% methyl oleate for the same length of time has little effect on the activity of the enzyme. The rate of synthesis of the enzyme is reduced by safflower oil feeding from the fat-free (control) level and after 48 h is approximately one-half that of the control. The rate of degradation of fatty acid synthetase is markedly increased in the safflower oil-fed animals over the control; the half-lives are 1.8 days and 3.8 days, respectively. It is suggested that polyunsaturated fatty acids, or a product derived from them, may directly or indirectly regulate the transcription or translation (or both) of fatty acid synthetase messenger RNA.


Nucleotide sequence of Thermus aquaticus ribosomal 5 S ribonucleic acid. Sequence homologies in thermophilic organisms.

The nucleotide sequence of ribosomal 5 S RNA from Thermus aquaticus grown at 75 degrees is p(A)-A-U-C-C-C-C-G-C-C-C-U-U-A-G-C-G-G-C-G-U-G-G-A-A-C-A-C-C-C-G-U-U-C-C-C-A-U-U-C-C-G-A-A-C-A-C-G-G-A-A-G-U-G-A-A-A-C-G-C-G-C-C-A-G-C-G-C-C-G-A-U-G-G-U-C-A-C-U-G-G-G-A-C-C-G-C-A-G-G-G-U-C-C-U-G-G-A-G-A-G-U-A-G-G-U-G-C-U-G-G-U-G-C-G-G-G-G-A-(U). The major molecular species is 120 nucleotides long; some molecules are one or two nucleotides shorter with one less nucleotide at either or both termini. When compared to other 5 S rRNAs, the sequence homology was greater with a thermophile bacterium (Bacillus stearothermophilus) than with mesophilic species. The comparisons further indicate that among prokaryotes, eleven of the nucleotide residues in T. aquaticus 5 S RNA may be largely restricted to thermophiles. Possible models for the secondary structure of T. aquaticus 5 S rRNA are discussed with respect to products of limited digestion and the unique nucleotides.


Photolabeling reagent for thiol enzymes. Studies on rabbit muscle creatine kinase.

A mixed disulfide reagent for photolabeling is described which reacts stoichiometrically with a cysteine sulfhydryl group of rabbit muscle creatine kinase to form a new mixed disulfide between enzyme and 2-thiobenzyl[14C]diazoacetate. When irradiated at 254 nm for 5 s in a photoreactor, the enzyme-bound diazo group is destroyed, presumably via a carbene intermediate. After photolysis, the enzyme can only be 69+/-2% reactivated by dithiothreitol, and only 67+/-1% of the radiolabel can then be removed from the protein by dialysis in the presence of dithiothreitol. Less than 3% of this permanent labeling occurs if photolysis is carried out in 6 N guanidine hydrochloride, which shows that the native enzyme structure is required for photolabeling. Identification of the tagged products after acid hydrolysis indicates that 30% of the carbene produced on photolysis reacts with the hydroxyl groups of threonine andserine with O-[14C]carboxymethylthreonine as the major product. Photochemical Wolff rearrangement is estimated to occur to less than 30%, and no S-carboxymethylcysteine was detected. The reagent employed and its isomers are proposed as bifunctional photolabeling probes to "scan" the amino acid residues near the active sites of thiol enzymes.


Further studies of mode of action of lipolytic enzymes.

Pancreatic lipase and phospholipase A2 have been shown by the monomolecular film technique to be progressively inactivated when adsorbed at the interface of their respective substrates. This inactivation is faster for lipase than for phospholipase. It is also enhanced by low film pressures and film transfer. The use of radioactive phospholipase and lipase samples offered the possibility to measure the amount of enzyme adsorbed at a monomolecular film with a reasonable accuracy. This adsorption was found to be relatively slow under the conditions of the assays. The main  conclusion drawn from these data is that the enzyme kinetics in presence of a substrate film, and probably also under bulk conditions, is controlled by an adsorption flux responsible for an initial lag period and an inactivation flux tending to decrease the reaction rate. The kinetics are linear only when both fluxes equilibrate.


Effects of colipase on hydrolysis of monomolecular films by lipase.

In a system free of bile salts we measured lipase hydrolysis of 1,3-didecanoylglycerol films in the presence or absence of colipase at different surface pressures. The strong, but not absolutely specific protective effect of colipase, most visible at low surface pressure, can account for the higher enzyme activity in the presence of colipase. This can be understood by taking into account simultaneous penetration and surface inactivation fluxes. Using radioactively labeled lipase, we have shown for the first time in a bile salt-free system that the critical surface pressure above which lipase can no longer penetrate a 1,2-didodecanoylphosphatidylglycerol monlayer is around 23 dynes/cm. Colipase increased this critical surface pressure to 30 dynes/cm indicating that it enables lipase penetration between 23 and 30 dynes/cm. The transfer experiment showed that colipase acts by first penetrating the lipid film and then serving as an anchor for lipase into the film.


Glucocorticoid control of the development of tryptophan oxygenase in the young rat.

Tryptophan oxygenase activity follows a characteristic developmental pattern in the young rat, being absent until about 2 weeks of life, then attaining adult levels by the 3rd week. We have investigated the factors which control this process and determined that the increase in enzymatic activity results from increased glucocorticoid release between the 14th and 21st days. This conclusion was based on our observation that adrenalectomy completely arrests the development of the enzymatic activity. Twenty-one-day-old animals which were adrenalectomized on the 10th day, and thus possess no demonstrable enzymatic activity, could respond to cortisol treatment with an elevation of tryptophan oxygenase activity. Administration of either cycloheximide or actinomycin D completely inhibited this response, suggesting that both protein synthesis and RNA synthesis were requisite for the development of the enzyme. Immunochemical studies revealed that the enzyme found in the young animal was identical with that extracted from the adult rat; furthermore, the increase in activity observed in the developing rat is the result of increased enzyme protein levels. Direct measurement of the synthetic and degradative rates showed the accumulation of enzyme protein to depend upon increased synthetic activity, and not upon decreased enzyme degradation.


Altered molecular species of human interferon produced in the presence of inhibitors of glycosylation.

The inhibitors of glycosylation, 2-deoxy-D-glucose or D-glucosamine, inhibit the synthesis of biologically active interferon in human FS-4 fibroblast cultures stimulated with polyinosinate-polycytidylate. Interferon synthesized in the presence of partially inhibitory concentrations of 2-deoxy-D-glucose or D-glucosamine were found to differ from interferons made in control cultures in some physical properties. Interferons synthesized in the presence of either inhibitor had a diminished charge heterogeneity demonstrable by iso-electric focusing. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, control interferon activity formed a single peak with the apparent molecular weight of 20,000, whereas interferons from cultures treated with either inhibitor could be resolved into two distinct molecular weight components, one of which was smaller than the interferon synthesized in control cultures.


Fracture of the neck and intertrochanteric region of the femur in children.

End-result evaluations after an average follow-up of seventeen years are reported in sixty-one cases (sixty patients). There were five Type-1 (transepiphyseal), twenty-seven Type-II (transcervical), twenty-two Type-III (cervicotrochanteric), and seven Type-IV (intertrochanteric) femoral fractures. The majority of Type-I, Type-II, and displaced Type-III fractures were treated with closed or open reduction and Knowles-pin fixation. The majority of undisplaced Type-III and Type-IV fractures were treated with abduction plaster spica casts. Fifty-five per cent were found to have good resultswhile 20% were fair and 25%, poor. The incidence of complications--avascular necrosis, coxa vara, premature epiphyseal closure, infection, and non-union--was compared with that in previously reported series. The use of Knowles-pin fixation appeared to reduce the complications of non-union and coxa vara. Avascular necrosis caused most of the poor results. However, younger children with avascular necrosis obtained better results than did older ones. Some children with results graded as poor roentgenographically were only mildly symptomatic.


Cup arthroplasties in teen-agers and children.

Review of cup arthroplasties performed prior to 1970 in sixty hips of fity-one consecutive patients less than twenty years old, with  follow-up on fifty-three hips, revealed that fifteen (28%) of the cup arthroplasties were followed by other salvage operations, usually total hip arthroplasty. Of the remaining thirty-eight hips, only twenty-four (63%) were rated satisfactory clinically, although most of the patients considered their cup arthroplasty an improvement over their preoperative status. Patients with bilateral hip disease did very poorly, and results were inconsistent in others. The final range of motion was frequently poor. Results were improved in hips in which acetabular reaming was not required.


Management of bleeding and associated complications of hemophilia in the hand and forearm.

Hemorrhage into the hand and forearm in hemophiliacs can be devastating if not treated early and properly. Of 200 hemophiliacs followed from 1969 to 1976, thirty-four had lesions of the hand and forearm. Hemarthrosis, the most common musculo-skeletal manifestation of hemophilia, occurred in the wrist and hand on only ten occasions. Superficial hemorrhage in the seven cases seen was not followed by significant sequelae. Bleeding into the anterior muscles and into the volar aspect of the wrist, on the other hand, was complicated by contracture, neuropathy, or both in six cases. Early diagnosis, replacement of the missing clotting factor, and immobilization are essential. Fasciotomy should also be considered, though it was not done in this series. Once a Volkmann's contracture is established, surgical reconstruction is required. The fractures in the hemophiliacs in this series, as in others, healed with normal periosteal callus formation and at a normal rate. Pseudotumors of the small bones of the hand have been described but were not seen in this series. Conservative therapy is suggested. After appropriate hematological evaluation to identify the missing factor and to exclude the presence of antibodies, it is now possible to control bleeding while proceeding with the necessary treatment to restore function and prevent deformity.


Proximal-row carpectomy for diseases of the proximal row.

Twelve patients were studied following proximal-row carpectomy. Four had a follow-up of twenty years or more. The lesions for which the operation was done included Kienböck's disease, perilunar dislocations, and fractures of the carpal scaphoid. The major complaint of pain was relieved following surgery, and a functional range of motion was obtained. Mild degenerative arthritis of the wrist was not a contraindication and progressive degenerative arthritis of the radial capitate articulation did not occur. The operation did not result in weakness and all of the patients resumed their preoperative occupations. After conservative measures fail, proximal-row carpectomy rather than arthrodesis should be considered for disorders of the proximal row of carpal bones.


Compression osteosynthesis in the hand using a small nail-plate.

A small self-compression nail-plate for stable osteosynthesis in metaphyseal fractures of metacarpals and phalanges is described. It is also used for interphalangeal arthrodesis. This type of osteosynthesis was performed twenty-five times in twenty-one patients for treatment of malunion, non-union, and the like. Bone union occurred in all instances without exception. No deleterious sequelae in terms of limitation of motion in adjacent joints were recorded subsequent to the surgical intervention.


Elongating intramedullary rods in the treatment of osteogenesis imperfecta.

Patients with osteogenesis imperfecta who have undergone multiple osteotomies with realignment and intramedullary rod fixation of a deformed bone frequently require replacement of the rod because the bone grows and angulates when the rod no longer is long enough to support the bone from metaphysis to metaphysis. The Bailey-Dubow intramedullary rod, which elongates with growth, is an attempt to solve this problem. This is a review of 153 rod-fixations in seventy-two bones (twenty patients), forty-seven being Bailey-Dubow rods. Use of the elongating rod effectively increased the average length of time between replacement operations, yielded a lower removal rate, and showed no additional adverse effects. The Bailey-Dubow rod represents a measurable improvement over non-elongating rods.


Long-term results following Lambrinudi arthrodesis.

Long-term results following triple arthrodesis by the method of Lambrinudi are reported in fifty feet. The average follow-up was eighteen years. Failures were encountered in only eight feet, and good results were obtained in twenty. Most of the patients had post-poliomyelitic paralysis. This operation allowed most braced patients to be free of the orthosis. All but three patients were able to work, and those unable to do so had other severe disabilities because of poliomyelitis. An extensive review of the literature is provided, showing the indications and contraindications for the procedure.


Transverse fractures of the sacrum. A report of six cases.

The diagnosis of transverse sacral fracture was made in six patients. All patients initially had evidence of urinary retention, a decrease in anal sphincter tone, or both. Diagnostic confirmation was established by tomography in four patients and by cystometric examination in two. All patients who had transverse sacral fractures with displacement of the fragments had resolution of their neurological deficit following posterior sacral laminectomy and decompression.


Translocation of the biceps humeri for flail shoulder.

A method of anterior translocation of the biceps humeri in the treatment of flail shoulder was undertaken in seven patients and allowed active forward flexion of 40 to 70 degrees at the shoulder joint. This gave the patients a feeling of control over the shoulder.


Extra-articular synovial chondromatosis.

Four patients with extra-articular synovial chondromatosis and one patient with combined intra-articular and extra-articular involvement are described. In all five patients the lesion was anomalously located (knee, shoulder, hip, and ankle). The lesion usually occurs in the hands and feet. Accurate diagnosis is important, because if the synovial origin of the cartilaginous proliferations is ignored, the evidence of cellular activity may lead to an erroneous diagnosis of chondrosarcoma. All five patients had a benign clinical course.


Factors affecting the mechanical stability of the cemented acetabular component in total hip replacement.

In thirty-six dissected hips from cadavera, six types of fixation of the prosthetic acetabular component to the acetabular tissue were devised, utilizing combinations of a variety of technical procedures: decorticating the cartilage, intruding the cup, reaming the bone, and perforating the residual bone tissue with anchoring holes (for cement). Mechanical tests showed that the greatest stability was achieved when the prosthetic cup was completely intruded, when all articular cartilage was removed and the socket was reamed, and when anchoring holes for cement were devised.


Effectiveness of preoperative Cotrel traction for correction of idiopathic scoliosis.

Of 206 patients less than twenty years old with idiopathic scoliosis of 40 to 90 degrees treated with Harrington distraction rods and fusion, ninety had preoperative Cotrel traction and 116 received no immediate preoperative treatment. There was no significant difference between these two groups in terms of the immediate surgical correction obtained. Cotrel traction therefore did not improve the results of treatment of idiopathic scoliosis of this degree in this age group.


Gamekeeper's thumb. Evaluation by arthrography and stress roentgenography.

We reviewed 197 previously reported cases of acute rupture of the ulnar collateral ligament of the metacarpophalangeal joint with respect to anatomy, diagnosis, surgical findings, and management. A standardized roentgenographic technique in stress abduction of the thumb was developed from thirty normal patients. The correlation between the abduction arc as recorded by our technique, the arthogram, and surgical pathology in twenty patients is reported.


Preoperative and postoperative dynamic electromyography as an aid in planning tendon transfers in children with cerebral palsy.

Electromyography was used to supplement clinical evaluation in planning tendon transfers in twenty-four children with cerebral palsy. Sixteen flexible deformities of the hind part of the foot, four internally rotated lower limbs, and four flexible deformities of the forearm and wrist were studied. When deforming muscles were active exclusively in one portion of either the gait cycle or a function of an upper extremity, appropriate tendon transfers were performed. When continuous muscle activity was noted, tendon lengthening was utilized. The desired function was obtained in all twenty-four patients six months after operation.


Prevention of hypomagnesemia and hypocalcemia in open-heart surgery.

We have studied the behavior of calcium and magnesium in the serum in dogs subjects to cardiopulmonary bypass with total hemodilution (Ringer's lactate). 5 mEq/l of calcium and magnesium was added to the filling solution. Serum levels of Ca++ and Mg++ were determined, by atomic absorption spectrophotometry, in the basal state, every thirty minutes during the perfusion, and one hour after stopping the bypass. We observed that the calcium and magnesium levels were higher than be baseline throughout the perfusion, and even 60 minutes after stopping the bypass they were still noticeably higher. Since this type of perfusion yields normal final calcium and magnesium levels, the advantages of this method become obvious, especially considering the adverse phenomena that are caused by defiencies of these cations.


Surgery for aortic dissection: 53 operated cases with 32 in the acute phase.

53 cases of aortic dissections were operated on between 1961 and 1975 in the Service of Cardiovascular Surgery at the Broussais Hospital, Paris (Prof. Ch. Dubost). The present study deals with 32 cases operated on in the acute phase. 31 originated in the ascending aorta and 1 in the descending aorta. Operative mortality was 50% (16/32), 4 patients died immediately following sternotomy. Operation consisted in replacement of the ascending aorta for the majority of type I and II dissections. When aortic insufficiency was present, a simple resuspension procedure was effective in 50% of cases. The authors prefer early operative intervention in type I and II dissections and medical treatment in type III dissections.


Intermittent claudication in premenopausal women. A correlation with the long-term use of oral contraceptives?

A group of 15 premenopausal women with intermittent claudication is presented. A uniform clinical and angiographic picture was found. A causative relationship of this early and very localized form of arteriosclerosis and the longterm use of oral contraceptive agents is suggested.


The binding sites of cytochalasin D. I. Evidence that they may be peripheral membrane proteins.

Binding sites for tritiated cytochalasin D (3H-CD) on the isolated plasma membrane from HEp-2 cells were reversibly inactivated, but not dissociated from the membrane, by dialysis in 0.6 M KCl. Activity was restored by subsequent dialysis in 0.06 M KCl. Treatment with 0.2 mM ATP at low ionic strength also inactivated these sites, apparently irreversibly. Extraction of the membrane with 6% Triton X-100 removed 75% of its protein, resulting in a two-fold increase in specific binding activity for 3H-CD. Both high and low affinity binding sites were retained by the detergent-extracted membrane; at least 60% of the high affinity sites were resistant to this treatment. Evidence is presented for the attachment to the HEp-2 plasma membrane of both actin and myosin. The results support the tentative conclusion that plasma membrane binding sites for 3H-CD are peripheral proteins on the cytoplasmic face of the membrane. They are consistent with the hypothesis that myosin may be the location of the high affinity binding site and actomyosin may be the low affinity site. Comparison of these observations with those reported for the congeneric drug, cytochalasin B, suggests that CD binding sites differ from the high affinity site for cytochalasin B.


Ultrasonic prediction of graft failure.

Forty-six femoro-popliteal by-pass grafts have been followed by serial measurements of the pressure indices in the pedal vessels. By this simple ultrasonic technique it has been possible to predict failure in eleven grafts and effect correction in eight. It is suggested that this represents a valuable method of detecting early remediable lesions and improving long term graft patency and limb survival.


The binding sites of cytochalasin D. II. Their relationship to hexose transport and to cytochalasin B.

Cytochalasin B (CB) was able to compete with tritiated cytochalasin D (3H-CD) for binding sites in HEp-2 cells. The pattern of inhibition suggested that CB associates with a low affinity class of CD binding sites. Glucose and maltose did not inhibit binding of 3H-CD to isolated HEp-2 plasma membrane. Inhibition of hexose transport by CD was negligible, but CD did not block the potent inhibition of this transport by CB. These results indicate that CD does not bind to the high affinity CB receptor reportedly associated with the hexose transport system, and that this receptor cannot mediate the morphological effects of CD. Both CD and CB induced contraction-zeoisis in HEp-2 cells; CB was less potent than CD, and their effects appeared to be additive. It was concluded that the high affinity binding sites for CD and CB are different, but that these congeners share a low affinity site. Both high and low affinity sites for CD appear to mediate its morphological effects; only the low affinity class appears to be involved for CB. Possible identification of the common low affinity binding site as actomyosin (detailed in Tannenbaum et al., '77) is further discussed.


Surgical versus medical treatment of renal artery embolism.

Seventy four case reports of acute renal arterial embolism were analyzed, to compare operative and non-operative management of this disease regarding survival and kidney salvage. The mortality was the same (25%), whatever the treatment. Surgery allows a much higher kidney salvage rate, especially whenever an embolus obstructs the main renal artery. Thus, apart from critically ill patients or segmental renal embolism, treatment should be surgical, irrespective of the time elapsed since onset. A short medical preparation will precede the operation.


The effect of elevated potassium on the time course of neuron survival in cultures of dissociated dorsal root ganglia.

The effect of potassium (K+) on the time course of neuron survival has been investigated by counting neurons over a 24-day period in live cultures of dissociated dorsal root ganglia from embryonic chick, fetal and newborn mouse, and fetal human material. In both normal K (6 mM) and in elevated K (20 mM mouse and human, 40 mM chick) there was initially a rapid exponential decrease in neuron survival. However, the magnitude of this decrease was less in the elevated K. In normal K neuron number decreased monotonically; the rate of degeneration itself decreased with time so that after 24 days neuron survival became relatively constant. In contrast, in elevated K the neuron number actually increased over a limited time interval before attaining a stable long-term value much greater than that in normal K. Thus, elevated K enhanced long-term survival by causing a lower rate of degeneration and also by causing an increase in neuron number during a limited period of the time in culture. From these observations and other evidence, it is argued that K can substitute to some extent for the trophic action normally exerted by the peripheral field of innervation of the DRG. It is further argued that K acts through its depolarizing effect on the membrane potential and that modification of intracellular ionic concentrations seems less likely to be involved.


Congenital anomalies of the iliofemoral artery.

In consequence of six cases of congenital anomalies in the area of the external iliac and femoral arteries, the embryological literature of this subject was studied. The complaints of these patients are described and some variations of the persisting sciatic artery are demonstrated. Operative treatment by means of reconstruction of the blood vessels was possible in all cases. The conclusion is that congenital anomalies in the area of the ilio-femoral artery are rare and that they may occur in different degrees. The pattern of complaints depend on the degree in which the embryonic construction has remained the same in the full-grown situation.


Axillary-to-contralateral brachial artery by-pass in the treatment of arm ischaemia.

Axillary-to-contralateral brachial artery by-pass was applied in two female patients affected by unilateral obstructive lesion of the subclavian artery with significant symptoms of arm ischemia. The result, at 10 and 12 months of follow-up, is good. The technique and its advantages are shortly outlined.


Common carotid artery stenosis due to subintimal hematoma following blunt trauma to the neck. Report of a case.

A case of cerebral ischemia secondary to carotid artery stenosis produced by a subintimal hematoma following blunt trauma to the neck is presented. A carotid endarterectomy with removal of the thrombus and intima was performed with satisfactory recovery of the patient. The various mechanisms involved in producing such stenoses and occlusions are reviewed.


A safety maneuver for closure of patent ductus arteriosus.

To avoid the risk of cut-thru during ligation or division of a patent ductus arteriosus, a simple maneuver is described to lower the pressure in the aorta and pulmonary artery by manual compression just before clamping or ligating the ductus. To gain sufficient length of the ductus for triple ligation, a technic is described utilizing the adventitia on the aortic end to prevent the linen tape or the silk ligature from slipping on the ductus.


Plasma levels of glucose and insulin during deep hypothermia with varying degrees of hemodilution in dogs.

The variations of plasma glucose and insulin levels were studied during the course of deep hypothermia with cardiocirculatory arrest of 60 minutes in 3 experimental groups of dogs using a pump mixture of homologous blood and Ringer's lactate solution at 33%, 50%, and 100% hemodilution. Insulin levels decreased in all groups during the cooling period and remained stable throughout the rest of the experiments, showing a slight significant increase only at the end of rewarming after a temperature of 30 degrees C was reached. Glucose levels reacted similarly except during rewarming, where an important increase in glucose concentration greatly preceded the rise in insulin. We stress the importance of this dissociation in view of the possible clinical implications that may exist.


Gas chromatography-mass spectrometry of metabolites in hemodialysis fluid.

An analytical method has been developed for the separation and identification of several metabolites in used hemodialysis fluid obtained during the treatment of a uremic patient on the artificial kidney. The procedure involves ion exchange, evaporation, and trimethylsilylation; the dervatized components were studied by combined gas chromatography--mass spectrometry. Twelve compounds were satisfactorily resolved; six were conclusively identified from mass spectral data. The identified components include phosphoric acid, glucopyranurono-(6 leads to 1)-lactone, citric acid, D-gluconic acid-beta-lactone, alpha-D-glucose, and beta-D-glucose. A seventh component was tentatively identified as mannonic acid.


[Determination of free cortisol in urine by high-pressure liquid chromatography (author's transl)].

The possibilities were examined for the high-pressure liquid chromatographic analysis of cortisol with methods of adsorption, distribution and reversed-phase chromatography. Free cortisol in urine can be determined by extraction with choloroform and subsequent adsorption chromatography on silica gel with a mobile phase consisting of 1.5% methanol and 0.2% water in chloroform. The time needed for this chromatographic analysis is 10--15 min; the limit of determination is 3 ng of cortisol for one injection.


Improved method for the analysis of estrogenic steroids in pregnancy urine by high-performance liquid chromatography.

The use of microparticulate packing materials and large injection volumes gives significant improvements in the analysis of complex samples, such as urine extracts by high-performance liquid chromatography. Lower detection limits and improved accuracy can now be attained. In addition, the combined use of adsorption and reversed-phase chromatography leads to reduced uncertainty in peak identification and gives more reliable quantification.


Analysis of prednisolone in plasma by high-performance liquid chromatography.

A sensitive, specific high-performance liquid chromatographic procedure for the determination of prednisolone in plasma is described. The organic solvent extract from plasma is chromatographed on a silica gel column using a mobile phase of 0.2% glacial acetic acid, 6% ethanol, 30% methylene chloride in n-hexane on a high-performance liquid chromatograph fitted with an ultraviolet dector (254 nm). Quantitation of plasma samples containing 25 ng/ml prednisolone is reported. Metabolites and endogenous hydrocortisone do not interfere with prednisolone. The determination of prednisolone concentration in plasma following administration of a 10-mg single oral dose to a human subject is described.


The use of high-performance liquid chromatography for sample clean-up in mass fragmentographic assays.

A novel high-performance liquid chromatography (HPLC) sample clean-up procedure for use in mass fragmentographic assays of (sub)-nanogram amounts of drugs in human plasma is described and compared with a conventional extraction sequence for sample purificaiton. With the assay of the new antidepressant drug mianserin hydrochloride (Org GB 94) as an example, the HPLC procedure is discussed with respect to retention time, recovery, purification, colum deterioration and convenience. It is demonstrated that HPLC sample clean-up is a useful and time-saving procedure for routine clinical analyses.


Mass fragmentographic assay of nanogram amounts of the antidepressant drug mianserin hydrochloride (Org GB 94) in human plasma.

For the assay of the antidepressant compound mianserin hydrochloride (Org GB 94) in human plasma, a mass fragmentographic method, using the deuterated analogue as internal standard and a high-performance liquid chromatographie sample clean-up procedure has been developed. The assay specifications obtained are a lower limit for reliable measurements of 1 ng/ml, and accuracy of ca. 0.01 ng/ml, a precision of 6--7% and a capacity of about 60 samples per day. The applicability of the assay method is illustrated by measurements of single-dose and steady-state plasma levels in clinical experiments, demonstrating the possibility of monitoring plasma levels during at least 24 h after a single dose of 15 mg of Org GB 94. The mean steady-state plasma levels after a daily dose of 3 X 20 mg of Org GB 94 appeared to be remarkably constant with time: 38, 36 and 34 ng/ml after 2, 4, and 6 weeks of treatment of 18 depressed patients.


Quantitative thin-layer chromatographic method for the determination of procainamide and its major metabolite in plasma.

A sensitive and accurate spectrodensitometric method was developed for the determination of procainamide and its major metabolite, N-acetylprocainamide, in plasma. The method involves extraction into organic solvent at alkine pH, separation by thin-layer chromatography and direct measurement of the absorbance of the compounds on the plate at 275 nm. Quantities as low as 10 ng could be measured and a linear relationship was obtained between peak areas and amounts of the compounds in the spots from 10 to 200 ng. The recovery of both drugs from plasma was from 95.4 to 104.8%. The method is sensitive and specific, and procainamide was well separated from N-acetylprocainamide at all investigated concentrations. The method is recommended for clinical assays and pharmacokinetics studies.


The determination of corticoids and related steroid analogs by high-performance liquid chromatography.

High-performance reverse-phase liquid chromatographic data on 28 corticoids and related steroid analogs, plus methyl and propyl parabens, are presented. Retention values, relative to acetone, are reported for each steroid on Bondapak Phenyl/Corasil, Bondapak C18/Corasil, and micro(micron) Bondapak C18 columns, and methanol/water or acetonitrile/water mobile phases. Ultraviolet detector response data for four of the steroids and calculated efficiencies for the three different reverse-phase columns are given. Chromatograms showing the separations of four steroids plus methyl and propyl parabens obtained on each of the three columns are also presented.


Trace analysis of estradiol in animal chow by electron-capture gas chromatography.

A gas chromatographic method is described for trace analysis of the natural steroidal hormone estradiol in animal chow at levels as low as 3 ppb. Salient elements of the method include extraction of the estradiol with methanol, an initial clean-up on a column of Sepadex LH-20, liquid-liquid partitioning at pH 14 and 10.2, additional cleanup on a slilica gel column, conversion of the estradiol to the pentafluoropropionly (PFP) derivative and analysis by electron-capture gas chromatography on a column of OV-25. Samples containing less than ppb of estradiol are subjected to further clean-up on silica gel after derivatization and prior to analysis.


Application of glass capillary columns to monitor petroleum-type hydrocarbons in marine sediments.

High resolution glass capillary columns coated with SE 52 liquid phase were used to resolve the indigenous hydrocarbons extracted from sediment samples collected from three outer continental shelf areas. The extracts were than spiked with small amounts of aliphatic or aromatic components isolated from a Louisiana crude oil and rechromatographed. The resolution was sufficient to separate almost all the isoprenoid, branched and cyclic alkanes associated with petroleum. Additionally, many of the key petroleum aromatics could also be resolved from naturally occurring organics. Such chromatographic procedures will aid in distinguishing between indigenous hydrocarbons of contemporary origin and those known to be associated with fossil hydrocarbon pollution of marine sediments.


Rapid gas chromatographic separation of amino acid enantiomers with a novel chiral stationary phase.

The use of novel polysiloxanes as stationary phase carrying chiral groups enables the separation of most amino acid enantiomers in a much shorter time than ever reported previously. Phases of this type exhibit very low volatility and high thermal stability and may be used in routine analysis with open tubular columns ant temperatures of at least 175 degrees C. Most protein amino acids are separated in a temperature program between 90 and 175 degrees C, thus obviating the need for multiple injections. Resolution factors are somewhat lower than those of other diamide phases containing the L-valine t-butyl-amide group, but are sufficient for resolution of almost all protein amino acid enantiomers.


Application of open-tubular gas-liquid chromatography in an investigation of the stability of pristanic and phytanic acids towards alkaline treatment of their methyl or [-] menthyl esters.

Comparison between GLC separation of the (--) menthyl and methyl esters of two diastereoisomeric isoprenoid acids of geological interest showed that a 46 m x 0.25 mm ID butanediol succinate column gives effective separation of 2S,6R,10R- and 2R,LR,10R-isomers of (--) menthyl pristanate (2,6,10,14-tetramethylpentadecanoate), but inferior separation of the corresponding methyl ester mixture. With the latter, a small proportion (2%) of 2S,6R,10R-isomer is well resolved from the major 2R,6R,10R-component on a 92 m column. Use of the methyl esters in such analyses thus avoids the cumbersome preparation of the menthyl esters. No epimerization at the 2-methyl carbon of pristanic acid and its methyl or (--) menthyl esters was detected after treatment in 1N aqueous or methanolic NaOH under reflux for 1 hr or at 120 degrees (sealed tube) for 24 hr. Similarly no 3-methyl carbon epimerization was observed inphytanic (3R,7R,11R,15-tetramethylhexadecanoic) acid and its methyl ester after the same treatments.


Selective introduction of TMS groups in tryptamines: preparation of N1-TMS derivatives of tryptamines.

Indolethylamines (tryptamines) with a primary animofunction react with Regisil under controlled conditions to yield a mono TMS derivative. Mass spectrometry to these derivatives shows that the TMS group is substituted on the indolic nitrogen (N1) and not the primary amino nitrogen. This is confirmed by converting these compounds to their isothiocyanate derivatives. The GC and GC-MS data of some typical compounds are presented.


Fetal bile salt metabolism. The intestinal absorption of bile salt.

The intestinal absorption of sodium taurocholate was studied in the near-term fetal and neonatal dog. Absorption rates were measured in vivo in isolated loops of fetal jejunum and ileum. Absorption was also measured in vitro in everted sacs and rings of fetal and neonatal jejunum and ileum. The maximal rates of taurocholate absorption observed after instillation of 1 micronmol taurocholate into closed segments of fetal jejunum and ileum with intact blood supply were not significantly different (P less than 0.2), and equalled 0.282+/-0.026 (mean+/-SEM) and 0.347+/-0.051 micronmol/h per 10-cm segment length jejunum and ileum, respectively. Similarly, the rates of absorption from open segments of jejunum and ileum perfused with 0.4 and 1.0 mM taurocholate were nearly identical (0.232+/-0.040 and 0.255+/-0.039, respectively at 0.4 mM, and 0.470+/-0.065 and 0.431+/-0.013, respectively at 1.0 mm) (P greater than 0.2). At perfusate concentrations of 4.0 mM, moreoever, jejunal absorption exceeded ileal absorption (1.490+/-0.140 and 0.922+/-0.200, respectively (P less than 0.05). As expected, concentration of taurocholate by the mucosa was readily demonstrated in adult ileal, but not in adult jejunal everted rings. In contrast, there were no significant differences in mucosal uptake of taurocholate by fetal jejunal and ileal rings. Fetal ileal mucosal concentrations were not significantly above those in the incubation medium after 1-h exposure of the mucosa to 0.003, 0.03, and 0.3 mM taurocholate. Uptake was proportional to incubation medium concentration over the full range of values. This was also true of tissues from 1-wk-old neonates. However, by 2 wk of age, ileal mucosal concentration of taurocholate was evident and adult levels were attained by 5 wk of age. It is concluded that taurocholate is absorbed by the fetal gut and that ileal absorption is no more efficient than jejunal absorption. Although active glucose transport was demonstrable in both jejunum and ileum, it was not possible to demonstrate an ileal mechanism for active transport of taurocholate in the fetus. Active ileal transport was not demonstrable in the newborn until at least 2 wk after birth.


Effect of chemotherapy and immunotherapy on tumor-specific immunity in melanoma.

The effects of chemotherapy, with nitrosoureas or dimethyl-triazeno-imidazole-carboxamide (DTIC), or immunotherapy with Bacillus Calmette-Guérin (BCG), on cell-mediated immunity (CMI), and serum blocking factor (BF) to melanoma cells were studied in 23 patients. Studies were performed with autologous or allogenic melanoma target cells obtained from recent biopsy, in 16 mm diameter plastic wells. Assays for lymphocyte-mediated cytotoxicity and BF were performed at weekly intervals over the course of 3-4 mo, with some studies extending beyond 3 yr. The specificity of cytotoxicity was good with these methods. Nine patients given nitrosoureas, predominantly methyl-chloroethyl-cyclohexyl-nitrosourea, showed a transient decline in CMI from 42.2 to 14% 3 wk after administration of a single dose of the agent, with a rapid recovery within 1 week. 10 patients given 5-day courses of DTIC at 3-wk intervals showed no decline in CMI after two courses, and 7 of the 10 had no decline even after three courses. Three of the four patients who achieved a remission lost BF previously present: BF reappeared in both patients studied during a subsequent relapse. BCG intradermally or intralesionally elevated CMI within 2 mo after initiation of therapy, but despite continuation of the injections CMI returned to base line in all but two of the nine patients studied. These results indicate that chemotherapy for melanoma with nitrosoureas or DTIC at these schedules is not profoundly immunosuppressive towards tumor-specific immunity, as measured by our procedures. Putative immunotherapy with BCG at these schedules was likewise only transiently stimulatory.


The effects of anoxia on the morphology and composite metabolism of the intact aortic intima-media preparation.

Paired samples of an intact rabbit aortic intima-media preparation were incubated for short periods under aerobic or anoxic conditions in Krebsbicarbonate buffer containing 6% albumin and 5 mM glucose. During aerobic incubation for as long as 1 h the preparation retained an electron microscopic (EM) appearance similar to that of tissue fixed in situ, and scanning EM confirmed the presence of an uninterrupted endothelial surface. After 2.5 min of anoxia there was widespread endothelial swelling, but the alterations in the EM appearance of these cells were not striking and did not progress during a subsequent 30 min aerobic incubation in fresh medium. After 10 min of anoxia there were marked and widespread alterations in endothelial cell structure, including loss of cell integrity, and numerous discrete interruptions in the endothelium were consistently observed on both transmission and scanning EM. After a subsequent 30 min aerobic incubation in fresh buffer, a major fraction of the luminal surface was denuded of endothelium. The aortic vascular smooth muscle cells did not exhibit evidence of irreversible anoxic injury after 2.5 or 10 min of anoxia or after subsequent aerobic incubation for 30 min. Exposure to anoxia for 10 min induced persistent alterations in the composite metabolism of the preparation during subsequent aerobic incubation in fresh medium; O(2) uptake was reduced, and the fraction of the glucose uptake that was accounted for by lactate production increased approximately 100%. The observations suggest that aortic endothelial cells are dependent upon respiration for the preservation of normal ultrastructure and cell integrity, and probably derive the major fraction of their energy requirements from reactions linked to respiration. Under the conditions employed in these experiments, short periods of anoxia did not induce EM evidence of irreversible anoxic injury in aortic vascular smooth muscle cells; this negative result is not incompatible with other data suggesting that these cells normally derive the major fraction of their energy requirements from respiration. Aortic intima-media does not exhibit a high rate of aerobic glycolysis under aerobic conditions which preserve a normal EM appearance of the preparation, but this pattern of metabolism can be induced by prior anoxic exposure.


Placental diffusing capacities at varied carbon monoxide tensions.

To test the hypothesis that carbon monoxide transfer across the placenta is, in part, a facilitated process, we have looked for evidence of saturation kinetics for carbon monoxide. In eight pregnant ewes, fetal to maternal carbon monoxide transfer was examined in a preparation in which the fetal side of the placenta was perfused with blood. The carboxyhemoglobin concentrations on the fetal side of the placenta were varied from 4.8 to 70% in 23 measurements. At increased carbon monoxide tensions, the transfer from fetus to mother always decreased. The slope of log rate of carbon monoxide transfer vs. log partial pressure gradient across the placenta was significantly different from 1. Placental membrane diffusing capacity was calculated separately from total placental diffusing capacity which includes hemoglobin reaction rates and erythrocyte membrane diffusion. Placental membrane diffusing capacity decreased at increased carbon monoxide tensions. Placental permeability for urea did not change with increasing carbon monoxide tensions. These results are consistent with the hypothesis that carbon monoxide diffusion in the placenta is, in part, carrier mediated.


Effects of acute bilateral ureteral obstruction on deep nephron and terminal collecting duct function in the young rat.

The effects of acute bilateral ureteral obstruction (BUO) of 18-h duration on deep nephron and collecting duct function were studied by micropuncture in 11 weanling rats. After release of BUO glomerular filtration rate was reduced (178+/-15 vs. 1,343+/-119 mul/min per g kidney weight in shams), while urine flow was increased averaging 17.5+/-1.3 vs. 6.8+/-0.72 mul/min per g kidney weight in controls. There was a marked increase in the absolute and fractional excretion of Na. Single nephron glomerular filtration rate of deep nephrons was reduced in the BUO group, mean 19.4+/-3.5 vs. 77.0+/-7.7 nl/min per g kidney weight in shams. Single nephron glomerular filtration rate of superficial nephrons fell to the same extent after relief of BUO. Mean tubular fluid to plasma inulin ratio of fluid from Henle's loop was 2.46+/-0.20 after relief of BUO vs. 8.23+/-0.85 in shams. This suggested a reduction in the reabsorption of Na and water before the bend of the loop of Henle, most likely in both the proximal tubule and descending limb. Fluid osmolality was depressed due to a decline in both Na and nonelectrolyte solute content. After release of BUO the percentage of filtered water remaining in the collecting duct (CD) at the base of the papilla was greater than in controls (13.3+/-2.0 and 1.72+/-0.01%, respectively) but fell significantly by the tip of the papilla to 7.92+/-1.12 vs. 1.17+/-0.02% in controls. These results indicate that water was reabsorbed along the terminal CD after relief of ureteral obstruction. In fact, a greater fraction was reabsorbed in this segment after release of BUO (5.37+/-1.58%) than after sham operation (0.55+/-0.15%). Similar changes were seen in Na excretion. Thus alterations in deep nephron function appear to contribute to the natriuresis and diuresis which follow release of BUO while terminal CD function in this model appears intact.


Androgenic hormones modulate autoantibody responses and improve survival in murine lupus.

Antibodies to native DNA and to polyadenylic acid (Poly A) occur spontaneously and undergo a regulated switch from IgM to IgG during the course of autoimmune disease in NZB/NZW F(1) (B/W) mice. B/W females have higher titers and earlier commitment to 7S antibodies to DNA and Poly A, whereas B/W males bind DNA and Poly A primarily by 19S antibodies. We have performed castration experiments to determine the effects of sex hormones on this switch from IgM to IgG.NZB/NZW F(1) (B/W) mice were either castrated or subjected to sham surgery at 2 wk of age and studied for immunoglobulin class of antibodies to nucleic acids at 4, 6, and 7 mo post-surgery. Prepubertal castration of males caused premature death in 60% of mice. Castrated males had a significant decline in their serum testosterone concentration, an increase in DNA and Poly A binding, and an accelerated switch from 19S to 7S antibodies to nucleic acids. Castrated females had no change in mortality. However, castrated females given maintained androgen treatment had a decreased mortality compared to castrated females receiving estrogen (14 vs. 94%). The anticipated switch to 7S antibodies to Poly A was almost eliminated in castrated females. These results suggest that sex hormones modulate immunologic regulation and that androgenic hormones are protective in murine lupus.


Inhibition of sodium transport by prostaglandin E2 across the isolated, perfused rabbit collecting tubule.

This study was designed to examine whether prostaglandin E2 can directly affect sodium transport across isolated perfused rabbit renal collecting tubules. Changes in transepithelial potential and isotopic sodium fluxes in response to peritubular prostaglandin E2 were measured. In addition, changes in transepithelial potential of the outer medullary collecting tubule in response to prostaglandin E2 were also measured. With few exceptions, all rabbits received 5 mg/day desoxycorticosterone acetate for 4-11 days before experimentation. The results of the experiments show that: (a) prostaglandin E2 inhibits the negative transepithelial potential in the cortical collecting tubule as well as the outer medullary collecting tubule; (b) prostaglandin E2 inhibits net sodium transport out of the lumen by inhibiting efflux while backflux is unaffected; (c) prostaglandin E2 produces this inhibition within 15 min, and the effects are dose dependent and reversible. These results suggest that prostaglandin E2 may modulate sodium transport in vivo and may contribute to the final regulation of sodium excretion.


Selective loss of calcium permeability on maturation of reticulocytes.

Calcium and sodium permeability of human reticulocytes have been studied and compared to mature erythrocytes. Mature erythrocytes had extremely low Ca2+ permeability which was less than 0.1% of values published for squid axon or HeLa cells. Calcium entry was markedly increased in reticulocyte-rich suspensions and the uptake was linearly related to the percentage of reticulocytes present. The data suggest that reticulocytes are 43-fold more permeable to Ca2+ than mature cells although their Ca2+ concentration is not increased. Sodium influx into reticulocyte-rich suspensions was also increased in direct proportion to the percent of reticulocytes present. Reticulocytes are sixfold more permeable to Na+ than mature cells so the ratio of Ca2+:Na+ permeability falls by sevenfold as the reticulocyte changes to an erythrocyte. [3H]Ouabain binding was increased in reticulocyte-rich cell suspensions and the correlation suggested a value of about 4,000 sites per reticulocyte compared with 362+/-69 per mature cell. Maturation of the human reticulocyte produces disproportionate changes in cation permeability and in particular a selective loss of Ca2+ permeability.


Exaggerated prostaglandin biosynthesis and its influence on renal resistance in the isolated hydronephrotic rabbit kidney.

Basal and hormone-stimulated prostaglandin biosynthesis was compared in isolated perfused rabbit kidneys with and without ureteral obstruction. At 72 h there was enhanced responsiveness to bradykinin in the ureter-obstructed hydronephrotic kidney. The amount of prostaglandin-like substance released from the perfused kidneys by 25 ng of bradykinin was 533+/-163 ng from the ureter-obstructed, 28+/-4 ng from the contralateral, and 26+/-3 ng from the normal kidney. The enhanced response was also noted with angiotensin II and with norepinephrine. This exaggerated responsiveness by the ureter-obstructed kidney could not be explained by decreased prostaglandin (PG) destruction or by decreased renal peptide inactivation (bradykinin or angiotensin). There was no enhanced PG biosynthesis with exogenous arachidonate, suggesting there was no increase in cyclo-oxygenase activity in the ureter-obstructed kidney. Renal tubular transport of PG from medulla to cortex was apparently not essential for the enhanced PG biosynthesis to hormone stimulation since the same exaggerated responses were noted during perfusion with the ureter ligated. The cyclo-oxygenase inhibitor, indomethacin, increased basal perfusion pressure in the obstructed kidney and enhanced the magnitude and duration of the renal vasoconstriction produced by angiotensin II in the hydronephrotic kidney. These results suggest that the local exaggerated biosynthesis of PG may be occurring in the cortical resistance vessels and may be important to the alteration in blood flow and excretory function that occur in ureteral obstruction.


The presence of protein-bound gamma-carboxyglutamic acid in calcium-containing renal calculi.

The amino acid gamma-carboxyglutamic acid (Gla) is found in four blood-clotting proteins, in a bone protein, in kidney protein, and in the protein present in various ectopic calcifications. This paper reports the presence of Gla in the EDTA-soluble, nondialyzable proteins of calcium-containing renal calculi including calcium oxalate, hydroxyapatite, and mixed stores of apatite and struvite (MgNH4PO4). Calculi composed of pure struvite and those composed of only uric acid or cystine do not contain Gla. From calcium oxalate and hydroxyapatite stontes, a protein of about 17,000 daltons was obtained which contained about 40 residues of Gla per 1,000 amino acids. The amino acid composition of this protein had no apparent relationship to the Gla-containing bone protein or to the similarly-sized F1 fragment of prothrombin which contains about 64 residues of Gla per 1,000 amino acid residues. The Gla-rich protein in calcium-containing renal stones thus may be a different Gla-containing protein. These data as well as other studies demonstrating the presence of Gla in pathologically calcified tissues not normally containing Gla suggest that the Gla-containing proteins may be of considerable pathophysiological significance.


The interdigestive motor complex of normal subjects and patients with bacterial overgrowth of the small intestine.

Intraluminal pressures were measured in the gastric antrum and at different levels of the upper small intestine in 18 normal subjects to investigate whether or not the interdigestive motor complex, identified in several animal species, occurs in man and, if so, to determine its characteristics. In all normal subjects, the activity front of the interdigestive motor complex was readily identified as an uninterrupted burst of rhythmic contraction waves that progressed down the intestine and that was followed by a period of quiescence. Quantitative analysis of various parameters of the complex and simultaneous radiological and manometrical observations revealed that it resembled closely the canine interdigestive motor complex. To test the hypothesis that disorders of this motor complex may lead to bacterial overgrowth in the small intestine, similar studies were performed in 18 patients with a positive (14)CO(2) bile acid breath test and in an additional control group of 9 patients with a normal (14)CO(2) breath test. All but five patients had normal interdigestive motor complexes. The five patients in whom the motor complex was absent or greatly disordered had bacterial overgrowth as evidenced by (14)CO(2) bile acid breath tests before and after antibiotics. These studies establish the presence and define the characteristics of the normal interdigestive motor complex in man. They also suggest that bacterial overgrowth may be due to a specific motility disorder i.e., complete or almost complete absence of the interdigestive motor complex.


Rubella virus serology:detection of residual lipoprotein inhibitors of haemagglutination using sensitive indicator arboviruses.

Immune and non-immune rubella sera were examined in the haemagglutination inhibition (HAI) test to evaluate the removal of lipoprotein non-specific inhibitors (NSI) of haemagglutination after kaolin and manganous chloride/heparin treatments. The sera were titrated fresh or after lipoprotein deterioration brought about by ageing the samples at 4 degrees C for various periods of time and by freezing at -20 degrees C with subsequent thawing. Deterioration was seen as altered electrophoretic mobility while the lipoproteins in treated sera were detected by indicator arboviruses whose haemagglutination is known to be strongly inhibited by the native macromolecules. After both treatments, notably manganous chloride/heparin, residual NSI activity was detected in deteriorated samples, particularly with group B arboviruses such as Japanese encephalitis and west Nile viruses but generally less so or not at all with the group A arboviruses employed. Absolutely fresh sera are considered highly desirable for rubella virus HAI assay, and it is suggested that the efficiency of lipoprotein NSI removal regardless of treatment protocol could be monitored in parallel HAI tests using carefully chosen indicator arboviruses. This could be done in conjunction with density gradient centrifugation of doubtful sera should ultracentrifuging facilities be available. The suitability of the monitoring procedure would be dependent to some extent on whether certain arboviruses are known to be endemic in a particular area.


The binding and cleavage characteristics of human Hageman factor during contact activation. A comparison of normal plasma with plasmas deficient in factor XI, prekallikrein, or high molecular weight kininogen.

The ability of human Hageman factor (coagulation factor XII) to bind to a glass surface and its susceptibility to limited proteolytic cleavage during the contact activation of plasma have been studied using normal human plasma and plasmas genetically deficient in factor XI, prekallikrein, or high molecular weight kininogen (HMWK). When diluted normal plasma containing (125)I-Hageman factor was exposed to a glass surface for varying times, the Hageman factor was found to bind to the surface, and within 5 min became maximally cleaved from its native 80,000 mol wt to yield fragments of 52,000 and 28,000 mol wt. Hageman factor in factor XI-deficient plasma behaved similarly. In prekallikrein-deficient plasma, the binding of Hageman factor to the glass surface occurred at the same rate as in normal plasma but the cleavage was significantly slower, and did not reach maximum until 60 min of incubation. Cleavage of Hageman factor in HMWK-deficient plasma occurred at an even slower rate, with greater than 110 min of incubation required for maximal cleavage, although the rate of binding to the glass was again the same as in normal plasma. Normal rates of cleavage of Hageman factor were observed for the deficient plasmas after reconstitution with purified human prekallikrein or HMWK, respectively. These observations suggest that normal contact activation in plasma is associated with proteolytic activation of surfacebound Hageman factor. The cleavage of the surface-bound Hageman factor molecule responsible for the formation of the 52,000-and 28,000-mol wt fragments occurred at two closely situated sites, one of which was within a disulfide loop. Cleavage at the site external to the disulfide bond resulted in the release from the surface of the 28,000-mol wt fragment. Cleavage at the site within the disulfide loop resulted in the formation of a 28,000-mol wt fragment which remained surface bound, presumably by virtue of the disulfide linkage to the larger fragment.


Staphylococci as urinary pathogens.

During the course of one year all (382) strains of staphylococci isolated in significant culture from urine specimens were typed by the Baird-Parker method. Staphylococcus aureus accounted for only 63 (16%) of the infections. Novobiocin-resistant micrococcal infection occurred predominantly in young women but also in children of both sexes; it was not restricted to M3. To try to detect possible sources of micrococcal infection other than faeces the normal flora of the throat, urinary tract, and vagina of young women was studied. Novobiocin-resistant micrococci were rarely found. Previous reports that micrococci are the second commonest urinary pathogens in young women in domiciliary practice were confirmed. The laboratory records of patients with these infections suggested that they respond well to treatment and that recurrences are usually due to a different organism.


A simple discriminant function for hepatic disease.

Tests of hepatic function do not correlate highly and this, together with their skew distribution, makes the information provided by them difficult to interpret. By means of an appropriate transformation it is possible to normalise their distributions so that the information from them can be combined in the form of a discriminant function which is not only easy to interpret but gives an index of severity of disease which is more reliable than any one single test. In this study nine tests were examined, and after normalisation it was found that only four tests were required to present almost all the information available in the nine. A formula is given for calculating the index in T-score form, which is particularly easy to interpret. A discriminant function of the type described retains the maximum information with the minimum number of biochemical tests. It therefore contributes to the solution of a problem which is causing increasing concern--the exponential increase in the number of investigations which the hospital services will soon find it impossible to bear.


Skeletal muscle pathology in chronic heart block.

Skeletal muscle samples, mainly from the deltoid, were studied morphologically and histochemically in 35 patients with chronic heart block and from nine elderly control subject. The average age of the first group was 67-7 (range 11-94) years. Abnormalities were present in 20 cases, no difference being found between patients with idiopathic and secondary heart block. In 15 samples there was increased oxidative enzyme activity in some muscle fibres, and in six there was fibre type grouping. Six had unexplained type 11 fibre atrophy and two had a predominance of type 1 fibres. Tubular aggregates were conspicuous in one biopsy specimen, and in another, rod bodies were found. Minor abnormalities were also seen in the control group. In the absence of any consistent pattern many of these changes were attributed to ageing.


Mucin producing carcinoid tumours of the vermiform appendix.

Six cases of an unusual mucoid variant of carcinoid tumour of the appendix are reported. These lesions are small, detected incidentally, and carry a good prognosis. Their histological features are distinctive and differ from those of ordinary carcinoid tumours and adenocarcinoma. They have been confused with adenocarcinoma, sometimes resulting in radical surgery. Surgery beyond appendicectomy is probably unwarranted in these cases. The mucin secreted by goblet cells of the normal mucosal epithelium and by the cells of these carcinoid tumours have certain histochemical similarities.


Apparent hypoxic changes in pulmonary arterioles and small arteries in infancy.

The pulmonary arterioles and small arteries were studied and their musculature and its nuclei were quantified in 90 neonates, infants, and young children who had suffered from a variety of clinical and hypoxic conditions immediately before death. Among the 90 cases investigated in this study, 30 were of sudden infant death syndrome (SIDS). No evidence was found to support the view that cases of SIDS are subjected to chronic hypoxia before death as significantly more medial muscle tissue in the pulmonary arterioles and small arteries was found in the chronic hypoxic group compared to the SIDS, non-hypoxic, and acute hypoxic groups. Furthermore, there was no statistically significant difference in the amount of medial muscle tissue of the pulmonary vessels as between the SIDS, non-hypoxic, and acute hypoxic groups. With other signs of acute hypoxia found at the necropsy of SIDS, the results of this study could be considered to support the view that cases of SIDS succumb as a result of an acute episode of hypoxia, or possibly repeated short-duration episodes of acute hypoxia which do not produce pulmonary vascular changes.


Telencephalic efferents of the tiger salamander Ambystoma tigrinum tigrinum (Green).

The efferent projections of the telencephalon in the tiger salamander were examined by the Nauta and Fink-Heimer methods following unilateral hemispherectomies, rostral hemispheric ablations and pallial lesions. The cerebral hemisphere connects with most areas of the contralateral hemisphere via the pallial, anterior and habenular commissures. The descending fibers travel in the medial and lateral forebrain bundles and in the tracts comprising the stria medullaris. Degenerating fibers and terminals were present throughout the diencephalon but were more abundant ipsilaterally. Fibers reach the pretectum and optic tectum via dorsal and ventral pathways. There is a heavy projection to the midbrain tegmentum and a sparse projection to the tectum via the ipsilateral lateral forebrain bundle. This tract continues into the medulla oblongata and the cervical spinal cord. Rostral and dorsal hemispheric ablations revealed that the majority of fibers forming the olfacto-peduncular tract originate in the ventral, rostral one-third of the hemisphere. It was also determined that the majority of the descending efferent fibers located in the lateral forebrain bundle originate from the caudal lateral hemispheric wall, and that these fibers form connections characteristic of mammalian corticofugal and striatofugal systems. The cytoarchitecture and connections of the caudal lateral hemispheric wall suggest that it is homologous to parts of motor isocortex and amygdala of amniotes.


The distribution of acetylcholinesterase in the central nervous system of jumping spiders and wolf spiders (Arachnida, Araneida: Salticidae et Lycosidae).

The distribution and activity pattern of acetylcholinesterase in the central nervous system of salticid and lycosid spiders has been studied. Enzyme activity was limited to the neuropile mass. The salticid and lycosid species investigated showed different intensities of enzyme reactions in the protocerebrum. The differences observed may be related to the somewhat contrasting habits of these spider families. Reactions were strong especially in the optic ganglia (lamina glomerularis, corpora pedunculata) and in the cerebral ganglion, while the central body showed only weak to moderate activity. In the cheliceral ganglia, as in the pedipalpal and the leg ganglia, including the fibre tracts of the ventral cord the demonstration of acetylcholinesterase was of moderate to strong intensity.


The second somatic sensory area (SmII) of opossum neocortex.

Organization of the neocortical second somatic sensory area (SmII) of anesthetized Virginia opossums has been examined utilizing micro-electrode recording techniques. SmII is situated between the first somatic sensory area (SmI) medially, and the rhinal fissure laterally. The head representation is located anteromedially within SmII, and the hindlimb representation posterolaterally, with the forelimb representation in between. Approximately 49% of SmII is devoted to representation of the head, 36% to forelimb representation, and 15% to trunk and hindlimb representation. All peripheral receptive fields (RF's) were either contralateral or bilateral. Approximately 63% of head RF's, 25% of forelimb RF's and 100% of hindlimb RF's were bilateral. For a given body locus, SmII RF's are larger than those for SmI. SmII is contained entirely within an area yielding evoked potentials responses to auditory click stimuli.


Visual discriminations of cats with cortical and tectal lesions.

Fourteen cats were trained on three visual discrimination tasks: light vs. dark, horizontal vs. vertical stripes, and upright vs. inverted triangles. Four of the cats then underwent large, bilateral occipito-temporal cortex ablations; postoperatively, they demonstrated little or no visually guided orienting behavior and solve only the brightness task and not the preoperatively learned pattern discriminations. Six other cats underwent the same cortical ablations plus a transection of the commissure of the superior colliculus; postoperatively, they demonstrated good visually guided orienting behavior (i.e., the Sprague effect) but still could solve only the brightness task. The final four cats were controls and underwent no surgery; they demonstrated good retention of the pattern task despite an extensive idle period corresponding to the postoperative period before retesting the above ten cats. These data indicate that, while a transection of the collicular commissure after visual decortication dramatically improves visual orienting, it does not obviously improve visual discrimination abilities.


The effect of cortical lesions upon visual discriminations in binocularly deprived cats.

Four cats were raised with binocular eyelid suture and, after their eyes were opened, were trained on a series of discrimination tasks. They performed at normal rates on the brightness task but indicated some difficulty with the pattern tasks. They then received large, bilateral occipito-temporal cortex ablations. Postoperatively, this in no observable way affected their visually guided orienting behavior, but it did destroy their capacity to perform the preoperatively learned pattern tasks. Postoperative performance on the brightness task remained good. These data indicate that, in these deprived cats, there is little or no cortical development for visual orienting, but cortex is necessary for visual discrimination learning.


Acetylcholinesterase staining in subdivisions of the cat's inferior olive.

The present paper describes a unique distribution of true AChE activity in the IO. In the dorsal accessory olive three areas with high AChE activity can be distinguished. The medial accessory olive can be subdivided into a caudal part which shows rostro-caudally directed bands with different enzymatic activity, and a rostral part which shows a more uniform, medium activity. In the nucleus beta and the dorso-medial cell column, AChE activity is low. The ventral and dorsal lamellae of the principal olive contain areas with high, medium, and low activity. The dorsal cap is strongly positive, while the ventrolateral outgrowth is negative for AChE. Enzyme distribution cannot be fully explained on the base of the known afferent and efferent connections with the IO. However, histochemical results provide evidence that generally supports a subdivision of the IO that mirrors these connections (Brodal, '40; Armstrong et al., '74' Boesten and Voogd, '75; Groenewegen et al., '75).


Fiber counts of regenerating peripheral nerves in axolotls and the effect of metamorphosis.

Counts have been made of myelinated and unmyelinated fibers in the sixteenth nerve root of the axolotl, before, during and after regeneration following nerve section. There is no loss of myelinated fibers. Many collateral sprouts are formed and eventually withdrawn. Regeneration during metamorphosis is similar but there is more sprouting. There is no fiber loss from cut nerves in metamorphosis. This suggests that rising thyroid hormone levels are not in themselves detrimental to nerves without peripheral connections. Other factors that may govern cell loss in limb development are discussed.