Biosynthesis of bacterial menaquinones. Menaquinone mutants of Escherichia coli. The isolation of six menaquinone mutants of Escherichia coli is described. It was shown that the mutants fall into two genetic classes. The first class carries mutations in a gene designated menA, which was located at minute 78 on the E. coli chromosome by cotransduction with the glpK and metB genes. The second class carries mutations in a gene designated menB. It was shown that this gene was not cotransducible with the menA gene. The biosynthesis of menaquinone in E. coli was studied using a variety of mutants blocked in aromatic biosynthesis together with the two classes of menaquinone mutants. It was demonstrated that chorismate is the branch point compound leading to menaquinone, and that 2-succinylbenzoic acid and 1,4-dihydroxy-2-naphthoic acid can serve as menaquinoone precursors in E. coli. It was also shown that menA- and menB- strains accumulate 1,4-dihydroxy-2-naphthoic acid and 2-succinylbenzoic acid, respectively, in their culture supernatants. The accumulation of the two compounds by the mutants together with their activity as menaquinone precursors provide strong evidence that they ar true intermediates in menaquinone biosynthesis. A pathway is proposed for the biosynthesis of bacterial menaquinones in which each intermediate has been adequately characterized.


Endonuclease II of Escherichia coli: degradation of gammairradiated DNA. Irradiation of DNA in a nitrogen atmosphere with 60Co gamma-radiation produces at least two types of damage. The first type leads to single strand breaks in the DNA observed after exposure to alkali. This type of alkali-labile bond will be designated a spontaneous break. The second type of damage to DNA is an alteration which makes the DNA susceptible to phosphodiester bond hydrolysis by a 1600-fold purified preparation of endonuclease II of Escherichia coli and is designated an enzyme-sensitive site. This site is not alkali-labile. After irradiation, preincubation of the DNA either for days at 0 degrees or for 4 hr at 37 degrees increases both the spontaneous breaks and the enzyme sensitive sites. There is a greater increase of spontaneous breaks when the preincubation is in O2 compared to N2. The increase of enzyme sensitive sites due to the preincubation is not altered significantly by O2. The increase of spontaneous breaks during the preincubation is almost completely prevented by addition of either NaBH4 or NH2OH after the irradiation. The treatment can be before or after the preincubation. This effect indicates that these breaks are due to alkali-labile bonds possibly produced by depurination or depyrimidination reactions. That the spontaneous breaks are due primarily to alkali-labile bonds is supported by an experiment in which formamide gradients were used. Neither NaBH4 nor NH2OH has any effect on the enzyme sensitive sites. Addition of beta-mercaptoethanol (0.5 M) at the start of the preincubation prevents in part the appearance of both spontaneous breaks and enzyme-sensitive sites. It has no effect when added at the end of the preincubation. Catalase added before the preincubation has no effect on either type of damage. It is postulated that the spontaneous breaks occur because purine or pyrimidine radicals are formed (possibly hydroxyl radicals) which can then interact with oxygen to produce unstable intermediates. The intermediates then undergo either depurination or depyrimidination. The subsequent alkali catalyzed beta-elimination reaction of depurinated or depyriminidinated DNA is prevented by NaBH4 or NH2OH. An alternative hypothesis would involve damage to the sugar rather than to bases. The enzyme-sensitive sites represent another form of base damage which is not oxygen dependent. The chemical nature of either form of primary damage is not known.


Transcription of bacteriophage T4 genome in vitro. Heterogeneity of RNA polymerase in crude extracts of normal and T4-infected Escherichia coli B. In order to obtain RNA polymerase preparations carrying the necessary specificity determinants to transcribe the delayed-early genes of bacteriophage T4, crude extracts of uninfected and T4-infected Escherichia coli were fractionated in glycerol gradients of low ionic strength. In contrast to the reported sedimentation behavior of the purified enzyme, the RNA polymerase activity in crude extracts of normal and infected cells sedimented heterogeneously over a wide range of sedimentation coefficients. When the "heavy" (24-33 S) and "light" (14-20 S) regions of the gradient were precipitated with ammonium sulfate and recentrifuged, the former split into two subfractions, one again sedimenting heavy and the other sedimenting light. The latter did not split under the same conditions. The resulting subfractions from uninfected cell extracts had different thermal thermal stabilities at 50 degrees (half-lives ranging from 2-3 to 25 min) while those from T4-infected cell extracts were very thermolabile (half-life of 1-2 min). All the subfractions were more active on T4 DNA than on calf-thymus DNA. They also formed rifampicin-resistant, RNA chain initiation complexes with T4 DNA. Based on the kinetics of heat inactivation with T4 and calf thymus DNAs as templates and preferential transcription of T4 DNA, it is proposed that the T4-infected cell enzymes prepared as described here harbor heat-labile initiation factor(s). During infection the heavy sedimenting RNA polymerase activity disappears after 2.5 min at 37 degrees. This appears to require phage-specific protein synthesis because (a) it does not happen in the presence of chloramphenicol and (b) it does not happen in T4 ghost-infected cells.


The outer membrane of Proteus mirabilis. I. Isolation and characterization of the outer and cytoplasmic membrane fractions. 1. The crude envelope preparation obtained by sonication of Proteus mirabilis cells in the presence of lysozyme was separated into outer and cytoplasmic membrane fractions by sucrose density gradient centrifugation. The outer membrane fraction accounted for about two thirds of the dry weight of the envelope preparation. 2. In thin sections, the outer and cytoplasmic membrane fractions were shown to consist of vesicles bounded by a single trilaminar membrane, but those of the outer membrane were considerably smaller and were frequently open, forming C-shaped structures. The cytoplasmic membrane vesicles were cleaved by freeze fracturing to expose fracture faces studded with particles, while the outer membrane fragments resisted cleavage. 3. The outer membrane fraction consisted of protein (similar to 40%), lipopolysaccharide (similar to 36%) and lipid (similar to 18%) and had a density of about 1.22 g/cm3. The cytoplasmic membrane fraction consisted mostly of protein (similar to 56%) and lipid (similar to 38%), had a density of about 1.16 g/cm3, and contained almost all the NADH oxidase, succinate and D-lactate dehydrogenase activities of the crude envelope preparation. 4. Electrophoresis in polyacrylamide gels containing sodium dodecylsulfate revealed over 20 polypeptide bands in the cytoplasmic membrane fraction and only 6-7 in the outer membrane fraction. The outer membrane electrophorogram was dominated by a major band (mol. wt 40 000) which was resolved into two bands when electrophoresed in an acidic gel system. Amino acid analysis revealed a higher content of polar amino acids in the protein moiety of the outer membrane.


Relationships between non-extractable DNA and the bacterial growth cycle. Escherichia coli DNA has been fractionated into extractable and non-extractable DNA after deproteinization of detergent-lysed cell preparations with chloroform-isoamyl alcohol. The former was extracted with dilut buffered saline whereas the latter remained in the interphase layer associated with residual cellular debris from which almost 40 percent could be released by incubating with pronase. About 20-25 percent more amino acid residues were bound to the pronase-released DNA than to the extractable DNA, but the relative distribution of the residues in the two DNA samples was virtually identical. The specific activities and the relative amounts of denser (1.709g-cm-3) and lighter (on the surface of CsCl gradients) DNA fractions from E. coli, grown in the presence of labeled thymidine, indicated that these two corresponded to extractable and non-extractable DNA, respectively. The relative amounts of the two fractions varied with the growth phase primarily as a function of the growth rate. Age and metabolic state of cells in the culture or those used as inocula could modify this relative distribution. When growth rate was maximal, the ratio of the two remained at about 1. During lag phase when no appreciable net synthesis of DNA could be detected, there was a rapid and preferential incorporation of labeled thymidine into non-extractable DNA. A disproportionate increase in the fraction of the total DNA which was extractable, was also abserved but only when stationary phase cultures were used as inocula. Complete equilibration of the label in the two DNA fractions was attained only after cultures had reached mid-log phase of growth. Similar results were obtained when prelabeled cells were used. These data have been interpreted as suggesting that the rate of cell growth and DNA synthesis are related to the number or size of sites of attachment of DNA to some cellular structure. Newly synthesized DNA would be attached to different sites on this structure and initiation of DNA replication in lag phase would require reorientation of the two kinds of DNA. Small peptides which are firmly bound to the DNA and which vary quantitatively with the rate of DNA synthesis could perhaps be involved in the attachment to the sites.


Aminoacylation of tRNA-Leu species from Escherichia coli and from the cytoplasm, chloroplasts and mitochondria of Phaseolus vulgaris by homologous and heterologous enzymes. Leucyl-tRNA synthetase from Phaseolus vulgaris chloroplasts could be separated from its cytoplasmic counterpart upon chromatography on hydroxyapatite, but the cytoplasmic and mitochondrial leucyl-tRNA synthetases could not be distinguished. The tRNALeu species from the various plant cell compartments and from Escherichia coli were aminoacylated using either homologous or heterologous enzymes; the levels of aminoacylation and the profiles of the leucyl-tRNAs upon reverse-phase chromatography were studied. Cytoplasmic tRNALeu species could be aminoacylated by the cytoplasmic or by the mitochondrial enzymes and in both cases yielded two peaks upon reverse-phase chromatography (RPC-5). But they could not be charged by the chloroplast-specific or by the E. coli enzynes. Mitochondrial tRNALeu species could be charged by the mitochondrial or by the cytoplasmic enzymes and in both cases yielded four peaks upon reverse phase (RPC-5) chromatography. But they could not be aminoacylated using the chloroplast-specific or the E. coli leucyl-tRNA synthetases. Chloroplastic tRNALeu species can be divided into two classes: the first class contains four isoacceptor species which can be charged by the cytoplasmic or mitochondrial enzymes, but not by the chloroplast-specific or the E. coli enzymes; the second class contains three chloroplast-specific tRNALeu species which can be charged by the chloroplast-specific or the E. coli enzymes but not by the cytoplasmic or the mitochondrial enzymes. There are five isoacceptor tRNALeu species in E. coli; all are charged by the E. coli or the chloroplast-specific enzymes, while only one is aminoacylated by the plant cytoplasmic or mitochondrial enzymes.


Escherichia coli mutants deficient in RNA accumulation at high temperature. A selection technique is described which has permitted isolation of 10 mutants that continue to form protein, but are deficient in the accumulation of rRNA at 42 degrees C. DNA-RNA hybridization experiments have demonstrated that most of these mutants are specifically defective in their ability to synthesize rRNA at the restrictive temperature. The bulk of the pulse-labeled RNA formed in such mutants at 42 degrees C appears to be mRNA, with normal instability as measured in the presence of rifampicin. The remaining mutants appear to synthesize normal levels of rRNA, but that rRNA does not accumulate in a stable form. Since all of these mutants continue to form protein, and most do not accumulate significant levels of ppGpp at 42 degrees C, it appears likely that the shut-off of rRNA synthesis at 42 degrees C does not act through a lesion in the rel locus. Thus, these mutants may reveal another element(s) required to promote ribosomal RNA formation.


Contractile proteins. Major components of nuclear and chromosome non-histone proteins. Two of the major non-histone proteins from Physarum polycephalum have been isolated under nondenaturing conditions and identified as actin and myosin. A third protein has been purified from crude nuclear actomyosin and from residual nonhistone fractions and found to bind actomyosin in the presence of Mg2+. In Physarum these proteins are not components of the nuclear membrane. Based on sodium dodecylsulfate-polyacrylamide gel electrophoresis, similar proteins are also present in nuclei of HeLa cells and mouse embryo fibroblasts. Isolated metaphase chromosomes from Physarum show a several-fold enrichment in myosin and an altered ratio of actin to the Mg2+-dependent actomyosin binding protein as compared to interphase nuclei. When non-proliferative states are induced in any of these cells, the Mg2+-dependent actomyosin binding protein decreases while actin increases several fold in intranuclear concentration; concomitantly, there is a generalized condensation and inactivation of chromatin. Experiments with added purified radioactive nuclear actomyosin; comparative studies on nuclear protein during stepwise nuclear purification; and studies on isolated metaphase chromosomes indicate that these proteins exist in nuclei in vivo. These observations suggest that contractile proteins may function in the structural interconversions of chromatin and in the regulation of cell proliferation;


13C-enriched phosphatidylethanolamines from Escherichia coli. Escherichia coli have been grown using [1-13C]acetate and [2-13C]acetate as the sole carbon source. The 13C NMR spectra of the whole cells and spheroplasts can be readily obtained but give limited information. The 13C NMR spectra of t-e isolated 13C-enriched phosphatidylethanolamines are assigned and analysed to give the biosynthetic pathway for acetate incorporation. This method provides a ready source of 13C-enriched phospholipids.


Uridine diphosphate 2-deoxyglucose. Chemical synthesis, enzymic oxidation and epimerization. The paper describes chemical synthesis of uridine diphosphate 2-deocyglucose (UDPdGLc) through reaction of uridine 5'-phosphomorpholidate with 2-deoxy-a-D-glucopyranosyl phosphate. The prepared analog of uridine diphosphate glucose (UDPGlc) served as a substrate for calf liver UDPGlc dehydrogenases (EC 1.1.1.22), the reaction product was identified as nucleotide deoxyhexuronic acid derivative. The apparent Km for UDPdGlc was found to be 60 times that of UDPGlc, and the relative V value for the analog was 0.09. The peculiar lag-eriod in reaction kinetics has been observed for the analog and is presumably connected with the slow rate of the initial stages of the reaction. UDPdGlc was found to be quite an efficient substrate for UDPGlc 4-epimerases (EC 5.13.2) from yeast, calf liver and mung bean seedlings.


Alteration of regulation of arginine biosynthesis in Escherichia coli W by mutation to rifampin resistance. The synthesis of acetylornithine delta-transaminase is induced by arginine and by rifampin in an arginine-inducible mutant of Escherichia coli W. A mutant of the arginine-inducible strain was isolated which is resistant to rifampin. The mutation which has brought about rifampin resistance has altered RNA polymerase and has simultaneously altered the regulation of arginine biosynthesis. Three of the enzymes of arginine biosynthesis, acetylornithinase, ornithine transcarbamylase, and argininosuccinase, show a greater rate of derepression and a 2--12-fold higher level of enzyme activity in the rifampin-resistant mutant than in the parent arginine-inducible strain. Acetylornithine delta-transaminase is no longer inducible by rifampin alone, and the level of inducibility by arginine plus rifampin has been reduced by 70%. The results indicate that RNA polymerase is involved in regulation of arginine biosynthesis in E. coli W.


Purification of an inhibitor of DNA photolyase with fluorescent spectra similar to those of the enzyme. 1. The binding of DNA photolyase, the enzyme which uncouples cyclobutadipyrimidines in DNA upon illumination, to its substrate was enhanced by a substance isolated from acidified autolysates of baker's yeast. 2. This substance, referred to as activator, was partially purified by chloroform extraction, ion-exchange chromatography on Dowex 50 and DEAE-cellulose, and gel filtration on Sephadex G-10. Upon lyophilization and storage at -20 degrees C, it was converted to a potent inhibitor of enzyme binding to substrate. 3. The inhibitor appeared as a single band in two thin-layer chromatography systems and was detected by ultraviolet absorbance, fluorescence, and ninhydrin staining. 4. The similarity between the fluorescence spectra of the inhibitor and the enzyme suggested that the inhibitor was structurally analogous to a chromophore in the enzyme and that the activator from which the inhibitor was derived may be the active chromophore of the enzyme.


Action of exonuclease V (the recBC enzyme) on ultraviolet-irradiated DNA. Exonuclease V (the recBC enzyme) of Escherichia coli can release pyrimidine dimers from ultraviolet-irradiated linear duplex DNA though it acts more slowly on irradiated DNA than on non-irradiated DAN. However, close circular lambda-dv DNA or phi X174 replicative form I DNA is not attacked by exonuclease V even though the DNA has been irradiated and treated with T4 endonuclease V to produce single-stranded breaks at the 5'-side of pyrimidine dimers. When irradiated circular DNA, previously nicked by T4 endonuclease V, is briefly exposed to elevated temperature, the DAN becomes susceptible to the action of exonuclease V, and pyrimidine dimers are selectively released. The increased susceptibility to exonuclease V may be resulted from locarized denaturation, or "fraying" of the 5'-termini at the nicks. The preferential release of pyrimidine dimers was observed when irradiated DNA, treated with T4 endonuclease V, was incubated with crude extracts of Escherichia coli. The activity was found in various strains defective in exonuclease V and/or DNA polymerase I.


Excision of pyrimidine dimers in normal and T4-infected Escherichia coli: effect of polA and other mutations. Strains carrying both polA1 and recBts1 mutations, which are defective in DNA polymerase I and have thermolabile exonuclease V (the recBC enzyme), are viable at 30 degrees C but not at 42 degrees C. These mutants exhibit almost normal rate of dimer excision in vivo even at the restrictive temperature. Similar results were obtained with other polA minus strains. We have also investigated effect of host and phage mutations on excision of dimers in T4-infected cells. Only a small amount of dimer is excised in T4v-1-infected cells whereas an extensive and selective release of dimers takes place in T4D-infected cells. Other phage mutations, including mutations in gene 43 and gene 30, do not affect excision of dimers in infected cells.


Cell division in Agmenellium quadruplicatum: evidence for the negative control by a protein. A high temperature conditional snake mutant, strain D1, of Agmenellum quadruplicatum was isolated which immediately stopped dividing following a shift to 41 degrees C following treatment with nitrosoguanidine. This mutant was stimulated to divide at 41 degrees C by the addition of inhibitors of RNA or protein synthesis: rifampicin, streptomycin, puromycin and chloramphenicol. Each of these inhibitors exhibited a discrete concentration optimum. The optimal concentration of chloramphenicol for cell division corresponded to the minimal concentrations necessary for the rapid inhibition of protein synthesis. The ability of chloramphenicol and other inhibitors to induce cell division in filaments decayed rapidly upon shifting to 30 degrees C. These results are interpreted as evidence for a protein acting as a negative regulator late in the cell cycle. At 41 degrees C, DNA was found distributed as a continuous zone throughout the length of the filaments. The addition of inhibitors of protein or RNA synthesis resulted in a rapid condensation of this nuclear material into multiple discrete nuclear regions suggesting that the negative control may be at the level of nuclear compartmentalization.


A double-blind study of electrosleep for anxiety and insomnia. Despite largely negative findings, several subjects reported a remarkable improvement in their symptoms some two to three weeks after electrosleep (ES) treatment was concluded, so that it remains unclear whether or not ES may be an effective treatment. The clinical experience reported suggests that five half-hour ES treatments may not be sufficient to produce significant changes in the patient's anxiety and insomnia. Further investigations are required to examine the effects of varying durations of treatment.


Light-scattering study of the temperature dependence of Escherichia coli motility. Two light-scattering techniques are used to study the temperature dependence of translational and rotational motility in Escherichia coli. The method of number fluctuation spectroscopy is developed theoretically and experimentally to measure the translational swimming speed of a smooth swimming strain of E. coli. Interference fluctuation techniques are used to study the rotational component of the motion. The results demonstrate that the thrust remains proportional the the torque generated by the flagella throughout the range studied and also show that relative changes in translational swimming speed may be inferred from the dynamics of rotational motion.


Adverse effects of intrathecal methotrexate in children with acute leukemia in remission. A toxic syndrome characterized by fever, headache, and vomiting, lasting 2-5 days, occurred in 61% of 39 children with acute leukemia in complete remission, receiving central nervous system prophylaxis with intrathecal methotrexate, and in 14% of 34 children receiving the same plus cranial radiation. The syndrome was accompanied by pleocytosis with lymphocytes, monocytoid cells, and neutrophils. There was evidence of cumulative Mtx toxicity, since the toxic syndrome occurred mostly after the third and fourth dose and did not recur with longer intervals between doses. The incidence of the syndrome was significantly reduced by the use of Elliott's B solution as Mtx diluent, rather than water or normal saline. The occurrence of pleocytosis and toxic clinical syndrome was also significantly reduced in patients receiving concomitant cranial radiation, probably due to the lympholytic action of radiotherapy and the depressed cellular response of irradiated tissues. The use of Elliott's B solution as diluent for IT Mtx and an appropriate interval between Mtx doses are suggested for prevention of this toxic syndrome.


Effect of bleeding on hematopoiesis following irradiation and marrow transplantation. In previous studies, bleeding after irradiation did not affect the rate of regeneration of endogenous spleen colony-forming cells, but induced an early (4-6 days after irradiation) appearance of erythrocytic colonies which differentiated and disappeared by days 7-8. This "abortive" wave was associated with a similarly abortive wave of splenic 59Fe uptake. The present experiments were done to determine whether or not an abortive wave of erythropoiesis could be induced in the transplanted, exogenous stem cell system. Lethally irradiated mice were given normal bone marrow cells and one-half of the group were bled of about one-third their blood volume within 4 hr of irradiation. Groups were killed on days 3-10 after irradiation. Seventeen to twenty hours prior to killing, 59Fe was injected. Hematocrits, spleen weights, colony numbers, and per cent 59Fe uptake were determined. Hematocrits of bled mice averaged about 70% of those of cell-injected controls. Spleen weights, colony counts, and per cent 59Fe uptake per spleen began to increase about 1 day earlier in bled mice (days 4-5 as compared to days 5-6), and rates of increase were the same as those of controls. However, no abortive wave of erythropoiesis was detected. A large cell dose resulted in earlier increases in all parameters than a small dose. Thus, bleeding after injection of cells produced results similar to those obtained by increasing the cell dose. The inability of bleeding to induce an early abortive wave of erythropoiesis in transplanted as compared to endogenous colony-forming systems may reflect differences in the cell cycling characteristics of these systems.


The role of granulocytes in the activation of intravascular coagulation and the precipitation of soluble fibrin by endotoxin. This study examines the role of neutrophils (PMN) in the pathogenesis of endotoxin-induced microclot formation. It is intended to clarify whether granulocytes are involved in endotoxin-induced activation of intravascular coagulation (generation of soluble fibrin) and/or in endotoxin-induced precipitation of soluble fibrin. Precipitation of soluble fibrin was achieved by injection of endotoxin into ancrod-infused rabbits with circulating soluble fibrin (first model). Activation of intravascular coagulation was elicited by two intravenous injections of endotoxin into rabbits (second model). Seventy-two and ninety-six hours after injection of nitrogen mustard, leukopenic rabbits had PMN counts between 0 and 50 cells per mul. Neutropenia did not prevent the occurrence of glomerular microclots after infusion of ancrod and injection of endotoxin (first model). Neutropenia influenced neither the decrease in mean fibrinogen concentrations nor the drop in mean platelet counts after ancrod and endotoxin administration. In contrast to the first model, neutropenia prevented the occurrence of glomerular microclots and of circulating soluble fibrin after two injections of endotoxin (second model). It did not, however, protect rabbits from the decrease in mean platelet counts after endotoxin administration. These data indicate that granulocytes are involved in endotoxin-induced activation of intravascular coagulation and the production of soluble fibrin but are not essential to endotoxin-induced precipitation of soluble fibrin.


[Nitroblue-tetrazolium test in isolated human monocytes]. Depending upon the NBT charge 50 to 80% of isolated non stimulated human monocytes reduce NBT ro morphologically demonstrable Formazan. Similar to neutrophils two different patterns of reaction are exhibited: discrete load with finely distributed Formazan granula surrounding the nucleus; massive reaction with large distributed Formazan granula surrounding the nucleus; massive reaction with large Formazan deposits throughout the cytoplasma. In a low percentage morphologically desintegrated, massive loaden cells appear. After an additional incubation in medium without NBT the number of these necrotic cells increases. The phagocytosis of IgG-coated red cells is impaired after incubation of the monocytes in NBT. In the presence of an alkylating agent not only the NBT-reaction is inhibited; the number of necrotic cells is also diminished. Apparently the Formazan is cytotoxic. The significance of these results in particular in cases of enhanced NBT reduction is discussed. Compared with the findings of other authors concerning the neutrophils the monocytes possess a markedly stronger NBT reducing activity. Considering the lower bactericidal capacity of the monocytes NBT reduction does not in every case parallel bactericidal activity.


Assessing psychological conflict.

Assessing psychological conflict. A method was constructed to assess cognitive conflict. It uses a large number of a subject's beliefs and attitudes which may be connected with his personal problems. The subject rates the direction and the degree of these beliefs and attitudes and the proportion of conflict involved in them is then calculated by computer according to a model basically derived from Heider's cognitive balance theory.


Single layer anastomosis after rectosigmoid resection. Anastomosis after resection of the rectosigmoid colon is often followed by leakage at the suture line. When occult leakage discovered on routine contrast study is included incidence rates of over 50 per cent have recently been reported. A single layer interrupted seromuscular inverting technique was used for the anastomosis after elective rectosigmoid resection in 52 consecutive patients. The total incidence of suture line leakage was 6 per cent. Therefore a high incidence of anastomotic dehiscence in the rectum can be avoided and it is concluded that a single layer technique is sound.


Selective labelling ot the methyl carboxylate substituents found in the anticodon sequences of some species of yeast transfer RNA. (1) By incubation in 0.1 M NaOH for 10 min at room temperature, it is possible to "saponify" some of the methyl carboxylate linkages in bulk yeast tRNA. By incubation with S-adenosyl(Me-14-C)methionine and either homologous (yeast) or heterologous (wheat-embryo) enzymes, it is then possible to "re-esterify" the "saponified" tRNA and thereby effect selective labelling at 5-carboxymethyluridine (Me-14-C)methyl ester residues. (2) There is also selective labelling at 2-thio-5-carboxymethyluridine (Me-14-C)methyl ester residues when "saponified" yeast tRNA is incubated with S-adenosyl(Me-14-C)methionine and homologous (but not heterologous) enzymes. (3) When selectively labelled yeast tRNA is hydrolyzed by RNase T-1, both 5-carboxymethyluridine (Me-14-C)methyl ester and its 2-thio-analogue are released as part of large oligonucleotides, each of which contains roughly 10 nucleotide residues. (4) There are at least three, and possibly four (Me-14-C)methyl ester-containing oligonucleotides released by RNase T-1 digestion of selectively labelled "saponified" yeast tRNA. A comparison of the chromatographic properties of the different (Me-14-C)oligonucleotides suggests that the same 5-carboxymethyluridine residues are probably targets for both homologous and heterologous enzymes. (5) The properties of the selectively labelled oligonucleotides are consistent with the view that some of them probably are derived from yeast tRNA-3-Glu, tRNA-2-Lys, and tRNA-3-Arg, all of which are known to contain 5-carboxymethyl methyl esters as part of their anticodon sequences.


An improved method for the isolation of surface membranes from cells in monolayer culture. A modification of the procedure reported by Scher and Barland (Biochim. Biophys. Acta 255 (1972) 580-588) for the isolation of the surface membranes of cells grown in monolayer culture is described. The modification involves addition of a purification step utilizing the two-phase polymer system described by Brunette and Till (J. Membr. Biol. 5 (1971) 215-224). The modified method permits the isolation of surface membranes from cells not yet at confluence, when they may easily become detached from the substratum.


Degradation of RNA in nuclei from Ehrlich ascites cells. Nuclei were prepared from Ehrlich ascites cells in 80% yield by homogenization of the cells in an aqueous solution containing Triton N-101 and washing of the nuclear fraction by centrifugation and resuspension. Compared to the enzyme activities present in cell extracts, approximately 47% exo-RNase I, 15% alkaline RNase II, 9% acid RNase II and 7% acid phosphatase were associated with the nuclear fraction after isolation. Exo-RNase I and alkaline RNase II were rapidly lost from nuclei during incubation at 37 degrees C. The degradation of newly synthesized RNA in nuclei incubated at 37 degrees C was followed by polyacrylamide gel electrophoresis and by characterization of acid-soluble degradation products. The rate of hydrolysis of the nuclear RNA was rapid during the initial stages of incubation and then proceeded at a much reduced rate. Nucleoside 5'-phosphates were the major acid-soluble degradation products, in agreement with the presence of exo-RNase I. Although a considerable amount of alkaline RNase II was associated with the nuclear fraction, extensive endonucleolytic cleavage of the nuclear RNA was not apparent. Compared to the processing of nuclear RNA in whole cells, however, the degradation in isolated nuclei was relatively non-specific.


An experimentally pathogenic Bacillus species. I. Description and characterization of the organism. A previously underscribed Bacillus species has been characterized. The organism was isolated from a blood transfusion bottle implicated in a fatal bacteriogenic transfusion reaction and has been examined from a number of different aspects to characterize it as completely as necessary to provide identification. It was placed into group 3 of the genus Bacillus on the basis of its cellular morphology, the morphology of the sporangium, and the location of the spore within the cell. There are only three other species described in this group. Since the organism differed markedly from each of these, it appears to represent a new species.


Fungal fimbriae. III. The effect on flocculation in Saccharomyces. Flocculent strains of Saccharomyces cerevisiae and S. carlsbergensis (S. uvarum) produced many short (0.5 mum) hairs (fimbriae) on the outer cell walls. Non-flocculent strains produce few fimbriae. Cells that are flocculent in wort but not in defined medium produce fimbriae only in the former medium. Cells treated with pronase lose both their fimbriae and the ability to flocculate. Cells treated with alpha-amylase retain some fimbriae but lose the ability to flocculate. It is suggested that the fimbriae may be the surface mannan-protein complexes known to be involved in flocculation and also in protein secretion. Haploid cells of both mating types also produced fimbriae, some of which apparently have a terminal knob.


Evaluation of polyglycolic aicd sutures in colon anastomoses. An experimental study was carried out in dogs to compare the healing characteristics of colon anastomoses constructed with silk and catgut, with those in which Dexon sutures of the same gauge were used. Anastomoses constructed with Dexon sutures showed minimal suture reaction on the serosal surfaces, and excellent healing strength as determined by bursting-pressure studies. Inspection of the mucosal surfaces in these anastomoses revealed flawless, smooth, healing suture lines. The flexibility of tissues at the anastomotic line was found to be good, and friability of tissue minimal.


Cystic adventitial disease of the popliteal artery. The first case of cystic adventitial disease of the popliteal artery recorded in Canada is reported and discussed in conjunction with a review of the world literature of 45 reported cases. This rare cause of arterial insufficiency occurs usually in young men and should be considered in the differential diagnosis of claudication. Angiographic findings are diagnostic and surgery is usually corrective. The theories of etiology and pathogenesis are discussed and the gross and microscopic pathology is demonstrated.


Isolation of dermatophytes, Candida species and systemic fungi from dermatologic specimens in Montréal, 1963 to 1973. Of 10 057 specimens of scrapings from skin, nails and scalp examined for dermatophytes, yeasts, pityriasis versicolor and systemic mycoses between 1963 and 1973, 30.4 percent were positive for fungi. Skin produced the highest proportion (68.6 percent) of positive scrapings, scalp the lowest (4.2 percent). Trichophyton rubrum was the predominant species (23.6 percent); of lesser prevalence were Microsporum canis (9.3 percent), T. mentagrophytes (8.4 percent) and Epidermophyton floccosum (4.8 percent). Double infections were encountered on 102 occasions; T. rubrum and Candida parapsilosis were the most frequent cohabiting species. The introduction in 1966 of periodic acid-Schiff staining for routine examination of scrapings resulted in better diagnostic results, particularly in the case of culturally nonproductive specimens and cases of pityriasis versicolor. Blastomyces dermatitidis and Cryptococcus neoformans were isolated from two patients in the course of routine investigation for dermatophytes.


Winter illness and vitamin C: the effect of relatively low doses. After their random -llocation to one of three treatment aroups, 622 volunteers received either vitamin C or placebo in a maintenance dose of 500 mg once weekly and a therapeutic dose of 1500 mg daily on the 1st day and 1000 mg on the next 4 days of any illness. Two forms of vitamin C were employed: a sustained-release capsule containing ascorbic acid and a regular tabet containing a mixture of sodium and calcium ascorbate. In the 448 subjects who completed an average of 15 weeks in the study of total of 635 episodes of illness were recroded. Respiratory symptoms were recorded on at least 1 day in 92 per cent of these episodes. There were no consistent or significant differences in the sickness experience of the subjects receiving the sustained-release vitamin capsules compared to those receiving the vitamin tablets, but subjects in both vitamin groups experienced less severe illness than subjects in the placebo group, with approximately 25 per cent fewer days spent indoors because of the illness (P smaller than 0.05). These results are  compatible with the belief that supplementary vitamin C can reduce the burden of winter illness, but the intake need not be as high as has sometimes been claimed.


Extensive intraglomerular fibrin deposition after renal transplantation: recovery without anticoagulation. Acute renal failure developed in a 55-year-old man 6 days after he had received a cadaver renal allograft. This was associated with thrombocytopenia. Extensive intraglomerular fibrin deposition was seen in a renal biopsy specimen. He was treated with corticosteroids, azathioprine, cyclophosphamide and hemodialysis with regional heparinization but not with systemic anticoagulation. This was followed by complete recovery of both renal function and histologic damage despite the fact that he did not receive anticoagulant therapy. This suggests that treatment with anticoagulants may not be necessary for all patients with intraglomerular deposits of fibrin.


Reticulum cell sarcoma of the brain. A review of the literature and a study of 19 cases. Reticulum cell sarcoma of the brain is a rare neoplasm. However, patients who have been recipients of transplants have had a much higher than expected incidence of this tumor; reticulum cell sarcomas of the brain may be seen more often in the future. Most of the information available in the literature deals with the pathology and clinical course of the disease. Although several authors have commented on the radioresponsiveness of the tumor, little detail as to the exact treatment methods has been provided. Furthermore, it has been assumed that the prognosis of this tumor, after some form of surgery and postoperative radiotherapy, may be fairly good. This paper presents 19 patients who have had surgery followed by postoperative radiotherapy. Although a few patients have had good relief of symptoms for 3 to 4 years, only 1 patient is alive and free of disease over 5 years. This patient is the sixth reported long-term survival in the literature. Suggestions for the management of this disease are provided. If possible, limited surgery to remove circumscribed tumors should be done, but extensive resection should be avoided. Postoperative irradiation should include the whole brain to at least 4500 rads at 1000 rads per week, with a boost to residual tumor, if present, of 500 to 1000 rads in two to five treatments.


Antipyretic effect of cycloheximide, and inhibitor of protein synthesis, in patients with Hodgkin's disease or other malignant neoplasms. Infusion of cycloheximide i.v., an antibiotic known to inhibit synthesis of protein, at a rate of 0.2 mg/kg/hr, reliably caused lysis of fever in 15 chronically febrile patients with Hodgkin's disease who did not have detectable bacterial, fungal, or viral infection. Antipyretic effects were also seen in some patients with reticulum cell sarcoma, lymphosarcoma, acute leukemia, histiocytic medullary reticulosis, plasma cell myeloma, carcinoma of the lung, and carcinoma of the cervix. The drug failed to produce defervescence in four patients with normal granulocyte reserves, who were febrile due to bacterial infection. When infused at a rate of 0.2 mg/kg/hr, the drug apparently caused an acute alteration of protein metabolism in man in that plasma amino acid nitrogen rose acutely while plasma levels of muramidase and ribonuclease fell during the period of the infusion. The data suggest that continuing synthesis of protein may be involved in nonbacterial fever of neoplastic disease. Mammalian granulocytes and monocytes are known to elaborate a pyrogenic protein following appropriate stimulation; it is suggested that in some types of neoplastic disease, particularly Hodgkin's disease, tumor cells may produce and release a pyrogenic protein and that drug-induced inhibition of its synthesis is responsible for the observed lysis of fever.


Effect of Acinetobacter glutaminase-asparaginase treatment on free amino acids in mouse tissues. Acinetobacter glutaminase-asparaginase (AGA) and Escherichia coli asparaginase were compared for their effects on plasma and tissue levels of amino acids, ammonia, and glutamyl transferase activity in the mouse. Free asparagine was depleted similarly in plasma and tissues by both enzymes. AGA treatment produced partial depletion of glutamine concentrations in muscle, spleen, small intestine, and liver. Brain and kidney glutamine concentrations actually rose with treatment. Despite over 100-fold increase in plasma glutamate, only the kidney showed a substantial increase in free glutamate levels during AGA treatment. Glutamine biosynthesis measured by glutamyl transferase activity showed an appreciable increase only in the kidney. Ammonia levels in tissues and plasma rose 1.3- to 4.3-fold. In general, E. coli asparaginase treatment had much less effect on these measurements than did AGA. The changes in these levels are discussed in relation to sites of possible toxicity and antitumor effects.


Electrophysiological effects of the antiarrhythmic agent QX-572 in the human heart with special reference to rate-induced changes in effective refractory periods. The antiarrhythmic agent, N,N-bis(phenylcarbamoylmethyl)dimethylammonium chloride (QX-572) 8 mg/kg body weight produced a decreased conduction time through the atrioventricular node but did not influence intra-atrial or intraventricular conduction. Effective refractory periods of the atrium, the atrioventricular conduction system, and the ventricle were shortened by the drug. The observed effects were present at the end of drug infusion but had disapnerbd 30 min later. The probable explanation is an increased sympathetic tone induced by QX-572.


Immunohistochemical localization of human pancreatic polypeptide (HPP) to a population of islet cells. HPP is the human counterpart of APP--a polypeptide that has been isolated from avian pancreas and shown to exert pronounced biological effects. The availability of anti-HPP serum enabled us to use immunohistochemical procedures for the localization of HPP in human pancreas. A small population of pancreatic islet cells showed strong immunofluorescence after staining with anti-HPP serum. The HPP cells were mainly localized at the periphery of the islets. Sometimes they were also seen scattered in the exocrine parenchyma as well as within the epithelium of small to medium-sized ducts. While rare in the pancreas of adult man, HPP cells were abundant in certain parts of the pancreas of foetuses (18-20 weeks gestational age). Their staining properties showed them to be distinct from the A, Band D cells of the pancreatic islets.


Fluorescence histochemical and elec-ron microscopical observations on sympathetic ganglia of the chick embryo cultured with and without hydrocortisone. Lumbar sympathetic ganglia of 12-day-old chick embryos were cultured in organ  cultures for 14 days with 1, 10 or 100 mg/l of hydrocortisone or without it. Catecholamines were demonstrated by the formaldehyde-induced fluorescence method. For electron microscopy, the cultures were fixed with glutarialdehyde and osmium tetroxide. Two types of cells with catecholamine fluoresecence were observed in the control cultures: (1) weakly fluorescent sympathetic neurons and sympathicoblasts with long nerve fibres, which were the most common cell type in the explant, and (2) brightly fluorescent cells with or without fluorescent processes, which were less common and were scattered in the explant. Hydrocortisone caused a great increase in the number of the brightly fluorescent cells. With 10 mg/l of hydrocortisone the increase was about ten-fold as compared with the control cultures. There was no change in the morphology of the cells, nor could any change be observed in the fluorescence intensity by eye. Electron microscopically the mature neurons were the most common cell type on the surface of the culture, while more immature sympathicoblasts were seen in the deeper layers. Cells were also found which contained large numbers of catecholamine-strong granular vesicles 105-275 nm in diameter. These cells were infrequent. They had round vesicular nuclei and resembled also in other respects sympathicoblasts or young nerve cells. One such cell was found in mitotic division by electron microscopy. Hydrocortisone caused a marked increase in the number of these granule-containing cells and their processes. Cells which could have been classified as the small intensely fluorescent cells of the mammalian ganglion type or their electron microscopic equivalent, the granule-containing cells were found neither in the control cultures nor in the hydrocortisone-containing cultures. It is concluded that most brightly fluorescent cells in cultured sympathetic ganglia of the chick are nerve cells or sympathicoblasts rich in amine-storing granular vesicles.


Electron microscope study on the occurrence of electron-dense deposits at the cell membrane of chicken erythrocytes. Electron-dense deposits were found along the inner side of the cell membrane of chicken erythrocytes when fixed in a CaCl2-containing glutaraldehyde solution. Osteoblasts and osteoclasts were studied in an attempt to determine whether the deposits are related to calcium metabolism. No electron-dense deposits adjacent to the cell membrane were found in either these cells or in rat erythrocytes. Therefore, it is assumed that the deposits are not calcium-binding protein necessary for the cellular calcium uptake.


Electron microscopie and enzyme cytochemical studies on the guinia pig metaphysis with special reference to the lysosomal system of different cell types. A transmission electron microscopic study of demineralized, methaphyseal bone of the young guinea pig is presented. Special attention is paid to the lysosomal system of the different cell types. Visualization of acid phosphatase and aryl sulfatase activity was used to identify tissue components as belonging thereto. The distribution of alkaline phosphatase activity, a plasma membrane marker, was also examined. Osteoblasts were distinguished by a marked development of the granular endoplasmic reticulum and the Golgi complex. Perivascular cells type A, morphologically resembled the osteoblasts, and are believed to represent an early stage in the specialization of the latter. A few lysosomes were normally found in the osteoblasts; they were less common in the type A cells. In contrasts to their regular occurrence in guinea pig epiphyseal cartilage, dense bodies of lysosomal nature ("type I vesicles") were only rarely seen in the bone matrix. Structures analogous to the type II vesicles in cartilage were, however, normally present. Their membrane showed activity of alkaline phosphatase. Possible functions of lysosomes and matrix vesicles in osteogenesis are discussed. Perivascular cells type B and chondroclasts both contained a prominent Golgi complex and large numbers of free ribosomes, mitochondria and lysosomes. In the type B cells, inclusion material of varying appearance often occurred in the lysosomes and in endocytic vesicles. The chondroclasts sometimes presented a ruffled border, with associated vacuoles and lysosomes in the subjacent cytoplasm. It is suggested that both cell types participate in the resorption of the epiphyseal cartilage. Chondroclasts presumably arise by fusion of type B cells and/or monocytic precursors from the peripheral blood.


The fate of injected human IgG in the mouse liver: Uptake, immunological inactivation, and lysosomal reactions. 1. Human IgG is stored in the form of remarkable droplets in the cytoplasm of mouse hepatocytes following intravenous injection. 2. The earliest protein uptake occurs within 1 minute after injection and droplets can be found up to 32 hours thereafter; 64 hours p.i. the foreign protein is no longer visible in hepatocytes. 3. Electron microscopy reveals that the uptake occurs by a process of "macropinocytosis". The resulting protein droplets fuse with primary lysosomes and are transformed into phagolysosomes. 4. At the same time as the phagolysosomes are formed the IgG-droplets loose their immunological activity--as early as 2 hours after injection.


Subcellular localization of calcium in the mouse hypophysis. I. Calcium distribution in the adeno- and neurohypophysis under normal conditions. Application of the K-pyroantimonate technique combined with glutaraldehydeosmium fixation results in a reproducible intracellular distribution of mineral precipitates in the mouse hypophysis. Control experiments--with chelators and electron probe microanalysis--reveal that these precipitates consist mainly of calcium. Regularly present in the mitochondria, Ca also seems to be stored in the Golgi apparatus of the glandular cells and in the axoplasmic reticulum and the "synaptic" vesicles of the neurosecretory fibres. These structures thus appear able to control intracytoplasmic calcium movements. These observations agree with physiological data showing the existence of an intracellular Ca pool that can be mobilized by specific stimulation. The presence of diffuse precipitates in the pituicytes, together with the existence of gap junctions between them, suggest that these cells regulate the ionic environment of the neurosecretory nerve fibres; in this way, they too might participate in neurohypophysial hormonal release.


Replicating circular DNA molecules in yeast. The yeast Saccharomyces cerevisiae contains a class of small circular DNA molecules, approximately 2 mum in contour length (Sinclair et al., 1967). In this report, it is shown that these molecules replicate as double-branched circles, similar to those observed during replication of the bacteriophage lambda and Escherichia coli chromosomes. A normal rate of replication of these DNA circles requires the function of a nuclear gene, cdc 8.


Properties of membrane-associated folded chromosomes of E. coli related to initiation and termination of DNA replication. Analysis of folded chromosomes prepared from amino acid-starved E. coli cells of from a dnaC initiation mutant indicates that a unique structure is associated with completion or near completion of rounds of chromosome replication in E. coli. Chromosomes remain associated with portions of the bacterial cell envelope throughout the DNA replication cycle, but become more rapidly sedimenting as replication proceeds in the absence of reinitiation. Before reinitiation of chromosome replication occurs after restoring required amino acids to amino acid-starved cells or after lowering the temperature in a thermosensitive dnaC mutant, sedimentation velocities of the membrane-associated folded chromosomes decrease substantially. The decrease in sedimentation velocity does not depend on renewed DNA synthesis, but does require the activity of at least the dnaC gene product.


The effects of unsubstituted polycyclic aromatic hydrocarbons on the growth of Escherichia coli. The effect on growth of E. coli of a series of 3-, 4-, and 5-ring unsubstituted polycyclic aromatic hydrocarbons (PAH) is determined at concentrations of 10-5M, 10-6M, and 10-7M. The angular hydrocarbon configurations of 1,2-benzanthracene, 1,2,5,6-dibenzanthracene (DIBA), and 3,4-benzpyrene (BP) promote growth; tetracene and pyrene have little or no effect on growth while at most concentrations anthracene, phenanthrene chrysene, 1,2,3,4-dibenzanthracene, and pentacene inhibit bacterial growth. No universal pattern of concentration effect is manifest, but based on the results an angular acene configuration is a necessary condition for growth stimulation.


Neutral lipids in the cells and cell envelope fractions of aerobic baker's yeast and anaerobic brewer's yeast. The neutral lipids from whole cells and cell envelopes of aerobic Saccharomyces cerevisiae and anaerobic Sacch. carlsbergensis and the cell walls isolated from the cell envelopes were analysed. The effect of anaerobiosis was particularly clear on the neutral lipid composition of the plasma membrane. Compared to the anaerobic membrane, the aerobic membrane contained more C16:1, C18:1 and other unsaturated fatty acids, more total sterol, more than ten times as much ergosterol and less than one tenth as much squalene, reflecting differences between the aerobic and anaerobic whole cellmthe main sterol in the aerobic membrane ergosterol, was mainly in the free form, whereas zymosterol, 24(28)-dehydroergosterol, epi- or fecosterol and lanosterol were predominantly esterified. In contrast, the anerobic membrane contained small amounts of biosynthetic sterol precursors of ergosterol (mainly esterified), and was clearly richer in saturated fatty acids having a greater variation in chain length and in 18:2 acid. Both plasma membranes contained a considerable amount of triacyglycerols, while the amount of lower acylglycerols was clearly higher in the anaerobic plasma membrane. The lipid composition of both cell walls were relatively similar, consisting mainly of triacylglycerols and lower acylglycerols.


A new method of measuring propagation coefficients and characteristic impedance in blood vessels. True propagation coefficients of pulse wave harmonics in an artery can be determined in vivo by measuring pulsatile blood pressure and flow at each of two points along the length of the vessel. These coefficients, which are complex numbers that describe the attenuation and the phase shift imposed on a traveling wave, are independent of the reflected waves in the circulation and thus provide information about the viscoelastic state and other properties of an artery. The equations involved are implicit in standard transmission-line theory, but they have not previously been applied in this particular way to blood vessels. The femoral artery, exposed in situ, was studied in 11 anesthetized dogs. At 1.5 Hz, true attenuation constants averaged 0.0151 nepers/cm, and true phase constants averaged 0.0155 radians/cm. As frequency increased, the apparent phase velocity of flow, in contrast, was relatively low at the first harmonic and rose as frequency increased. True phase velocities lay between the apparent pressure and flow values. Characteristic impedance at 1.5 Hz had an average modulus of 1.76 times 10-4 dyne sec/cm5 and a phase of minus 0.31 radians. The modulus diminished as frequency increased, and the phase became less negative. These results show that true phase constants and characteristic impedances determined by this method are consistent with data reported by others and provide information not previously available about flow wave propagation.


Quantitative studies of lipoprotein-X in familial lecithin: cholesterol acyltransferase deficiency and during cholesterol esterification. In eight patients with familial lecithin: cholesterol acyltransferase (LCAT) deficiency the plasma concentration of the abnormal lipoprotein LP-X ranged from 43 mg/100 ml to 251 mg/100 ml with a mean of 127 mg/100 ml. This is above the mean level of LP-X found in a group of patients with intrahepatic cholestasis (49 mg/100 ml) and below the mean level found in patients with extrahepatic cholestasis (341 mg/100 ml). Following blood transfusions, an increase in LCAT activity and a decrease in the plasma concentration of LP-X were observed. This decrease was not a simple dilution phenomenon. Quantitation of LP-X during long-term incubation of a whole plasma system demonstrated that LP-X was a source of free cholesterol (FC) in the LCAT reaction. More free cholesterol was derived from LP-X during cholesterol esterification than corresponding to the percentage of the plasma free cholesterol found in the LP-X fraction. The present investigation did not answer the question whether LP-X only was an easily accessible source of free cholesterol for cholesterol esterification or whether LP-X acted directly as a substrate for the LCAT reaction.


Neonatal and childhood gonococcal infections. Maternal, fetal, and neonatal morbidity and mortality can be significantly decreased by selective screening of prenatal patients for gonorrhea, early treatment, and follow-up cultures after treatment for gonorrhea. Ophthalmia is the most significant neonatal gonococcal infection. Administration of silver nitrate at delivery is the best prophylaxis against gonococcal ophthalmia. Treatment of gonococcal ophthalmia, however, requires parenteral penicillin, conjunctival antimicrobial therapy, and hospitalization. Treatment of the gonococci-infected mother is also indicated. Childhood gonorrhea is most commonly manifested as vulvovaginitis in girls or urethritis in boys, usually without associated disseminated gonococcal infection. Transmission can occur by indirect contact with an infected parent or involuntary or voluntary sexual activity: in children over age 10 years transmission of gonococci most commonly involves voluntary sexual activity. Emphasis should be placed on early treatment of the infected child, follow-up cultures, and contact tracing. Diagnosis of gonococcal infection requires adequate specimens, gram stains, and cultures for N. gonorrhoeae. In disseminated neonatal gonococcal disease, gram strains and cultures of the conjunctiva, oropharynx, orogastric aspirates, anogenital area, umbilicus, and external ear canal frequently aid in the diagnosis. Frequently, a gram strain of the urethral discharge in boys is sufficient for diagnosis of gonorrhea; in girls cultures are necessary. Since the incidence of gonorrhea in children has increased, the importance of epidemiologic analysis and follow-up after treatment can not be overemphasized.


Meperidine kinetics in man. Intravenous injection in surgical patients and volunteers. The plasma concentration-time profiles of meperidine following intravenous injection in surgical patients and volunteers were investigated by reference to a classical two-compartment open model. Physiologic characteristics of the subject and variables associated with the surgery and anesthesia were screened as determinants of the kinetic patterns observed. When meperidine administration preceded induction of anesthesia, induction was consistently followed by an increase in venous plasma concentrations that prevented classical kinetic analysis. To facilitate calculations in subsequent studies in patients, meperidine injections were made following induction of anesthesia. Type of anesthesia or premedication, patients' sex, or cigarette smoking did not appear to be important factors in this evaluation. Increasing alcohol consumption was associated with increasing volumes of distribution. Increasing age was associated with increasing fraction of drug unbound in plasma. These factors may relate directly to clinical observations that heavy alcohol consumers tend to be more refractory to central nervous system (CNS) depressants and that elderly patients are more susceptible to respiratory depression from narcotics.


Long-term treatment of hypercholesterolemia with colestipol hydrochloride. Colestipol hydroxhloride (15 gm/day) (an anion exchange resin that binds bile acids) of placebo was administered to 92 patients with hypercholesterolemia who were followed for periods up to 36 months. There was a prompt (1 month), significant (p smaller than 0.05 minus 0.001), and sustained (36 months) lowering of serum cholesterol in the colestipol HCl-treated group, but no significant change in the placebo group. Serum triglycerides increased in both treatment groups in parallel; the reason was not apparent. Side effects were equally distributed between colestipol HCl and placebo and were primarily gastrointestinal (upper abdominal distress, constipation). Colestipol HCl appears to be a safe and effective treatment for hypercholesterolemia; tolerance does not seem to develop.


Interaction of physostigmine and delta-9-tetrahydrocannabinol in man. To investigate the hypothesis that delta-9-tetrahydrocannabinol (THC), the major psychoactive ingredient of marihuana, acts by interfering with cholinergic brain mechanisms, 0.75 to 1.25 mg of physostigmine, a centrally active cholinergic drug, was given intravenously to 5 normal volunteers who had ingested 20 to 40 mg of THC 2 hours earlier. Physostigmine decreased the degree of tachycardia and conjunctival injection produced by THC. The major psychologic effects of physostigmine were amplification of the lethargy and somnolence which occur late in the course of THC intoxication. We interpret the lack of physostigmine counteraction of the peak psychologic effects of THC as evidence against the hypothesis that THC acts predominantly by an anticholinergic mechanism.


Hypnotic efficacy of diphenhydramine, methapyrilene, and pentobarbital. The antihistamines diphenhydramine and methapyrilene were compared with pentobarbital for hypnotic effect in two Veterans Administration Hospital populations using subjective-response methods. In the first part of the study, 60 mg and 180 mg of pentobarbital were compared with 50 mg and 150 mg of diphenhydramine. A positive dose-response relationship was obtained only for pentobarbital; neither dose of diphenhydramine was significantly different from 60 mg of pentobarbital for any response variable. In the second part of the study, 100 mg of pentobarbital, 50 mg of diphenhydramine, and 50 mg of diphenhydramine, and 50 mg of methapyrilene were compared with placebo. One hundred mg of pentobarbital and 50 mg of diphenhydramine were significantly different from placebo, but 50 mg of methapyrilene was not.


Commentary. The normal volunteer in clinical investigation: how rigid should selection criteria be? Twenty-nine healthy young male volunteers were screened by history, physical examination, urinalysis, 24 different blood tests, and electrocardiography for admission to a Phase I drug study. The protocol submitted to, and approved by, the FDA listed the normal ranges for the local laboratories and stipulated that the test results for any volunteers accepted should all be within these ranges. During screening 46 abnormal values were found in the laboratory tests. Only 4 subjects had all their test data within "normal" range, a result that could have been anticipated simply form the number of tests performed. These findings indicate the need for deciding on realistic protocol criteria for studies involving healthy volunteers.


Etorphine in man. I. Subjective effects and suppression of morphine abstinence. The effects of etorphine, a potent morphine-like drug, were qualitatively and quantitatively compared to those of morphine. In nondependent subjects, etorphine in doses of 0.025, 0.050, and 0.100 mg produced pupillary constriction and morphine-like subjective effects and euphoria. Etorphine was 500 times as potent as morphine, with a very rapid onset and short duration of action. In morphine-dependent subjects, etorphine suppressed abstinence but for a shorter period than morphine. These studies indicate that in man etorphine is a morphine-like drug with a high abuse potential.


Bioavailability of different lots of digoxin tablets from the same manufacturer. The bioavailability of single doses from 4 randomly selected lots of digoxin from a single manufacturer that were recently marketed in the United States was compared. In a crossover study in 6 normal volunteers, serum digoxin concentrations for 0 to 6 hr and urinary glycoside excretion for 24 hr after 0.5-mg doses were measured by radioimmunoassay. The variation in bioavailability between the 4 lots was no greater than that seen when one of them was taken twice. Intersubject variation in areas under the serum concentration-time curves and in the 24-hr urinary digoxin measurements was more than twice that of intrasubject variation. The variability of the urinary digoxin determinations was somewhat less than that of the areas under the serum concentration-time curves.


Effect of foodstuffs on the absorption of zinc sulfate. Single doses of zinc sulfate were given to healthy young volunteers, either in the fasting state or with various types of meals. Dairy products (milk and cheese) and brown bread decreased zinc absorption, as indicated by a significant drop in peak serum zinc levels. Zinc absorption was decreased when zinc was given in the fasting state with the same amounts of purified phosphate or phytate as those found in foods above. Experiments in vitro have shown that zinc is precipitated by phosphate and phytate at pH values close to that of the intestinal lumen. Coffee also seems to inhibit zinc absorption.


Cryptogenic fibrosing alveolitis.

Cryptogenic fibrosing alveolitis. Clinical, radiographic, and histologic data, together with pulmonary function features, in eleven patients with cryptogenic fibrosing alveolitis, are described in the following study. All these patients presented with a history of exertional dyspnea. Eight of the eleven had radiographic evidence of pleural involvement as manifested by blunting of one or both costophrenic angles. A restrictive defect was present in eight patients, all of whom had x-ray changes showing diffuse involvement. One of the patients presented showed marked improvement in all parameters including a repeat lung biopsy. Immunofluorescent studies of the lung biopsies from six patients revealed no evidence of immunologic damage. Based upon the histologic pattern of pulmonary reaction in the biopsies and the intervals between the onset of symptoms and the time of biopsy, a tenative scheme of temporal evolution of this disease is presented. Lung injury may lead either directly to diffuse interstitial pulmonary fibrosis (DIPF) or lungs may react to injury by an initial alveolar, mural or mixed pattern, all of which might ultimately progress to DIPF. An initial alveolar or mural pattern might change to a mixed pattern.


Diphenidol treatment of arrhythmias.

Diphenidol treatment of arrhythmias. The antiarrhythmic activity of diphenidol, an antiemetic, has been demonstrated both in electrophysiologic studies of patiens and in experimental arrhythmias in animals. Accordingly, 18 patients with tachyarrhythmias were treated with intravenous diphenidol in doses of 0.5 to 1.5 mg/kg. In six patients with atrial arrhythmias, there was no notable effect. Similarly, 12 patients with premature ventricular contractions were treated and studied. In six of them, ectopic beats were abolished, at least transiently; in three the number of ventricular premature contractions decreased; in two there was no effect; and in one, the number of premature beats was increased. Of the total number of 18 patients, 14 suffered adverse effects related to the central nervous system. These adverse effects were of such severity as to suggest that further studies with diphenidol as an antiarrhythmic are not warranted.


Suture-line recurrence in carcinoma of the colon and rectum. Seventy-three patients with adenocarcinoma of the sigmoid, rectosigmoid and rectum were reviewed to determine the incidence of suture-line recurrence. Four patients (5.5 per cent) developed suture-line recurrence despite the use of a number of techniques to reduce this problem. All four recurrences were in patients who had Dukes' C lesions of the rectum. It would appear that such recurrences are not related to implantation factors alone.


[Coagulation tests in von Willebrand's disease(author's transl)]. The diagnosis of von Willebrand's disease can be made by a selected series of easily performed clotting tests. A STudy of 36 patients with the disease (all but one with a mild form) revealed a significantly increased reaction time in the thrombelastogram from 210 plus or minus 33 percent to 270 plus or minus 69 percent (P less than 0.001), and of the partial thromboplastin time from 43 plus or minus 3s to 54 plus or minus 9s (P less than 0.001 than 0.001). Factor VIII activity was corresponding significantly reduced from normal (127 plus or minus 51 percent) to 51 plus or minus 12 percent (P less than 0.001). Other plasma factors and platelet counts were within normal limits. A platelet defect was indicated by an abnormal change in the speed of clot formation (k-time) from 162 plus or minus 39 percent to 341 plus or minus 121 percent (P less than 0.001), maximal thrombelasticity from 121 plus or minus 16 percent to 75 plus or minus 22 percent (P less than 0.001), and marked reduction in platelet-factor 3 liberation from 122 plus or minus 59 percent to 23 plus or minus 16 percent (P less than 0.001). An increase in bleeding time, from 162 plus or minus 41s to 204 plus or minus 128s (P less than 0.05), was not always present. Recently introduced immunological methods for determining factor VIII and factor VIII-associated protein, as well as special platelet-function tests, give further information on the pathogenesis of the bleeding disease, but their complexity restricts them to special laboratories.


[Glossopharyngeal neuralgia with cardiac arrhythmias (author's transl)]. In a 68-year-old female patient glossopharyngeal neuralgia of unknown origin occurred accompained by cardiac arrhythmias (bradycardia to asystole) and loss of consciousness. Subcutaneous atropine injections improved bradycardia but did not influence attacks of pain. Complete and permanent freedom from symptoms was established only after treatment with carbamazepine (Tegretal) 200 mg t.d.s.


[Studies on the bio-availability of tolbutamide (author's transl)]. Two different batches of Rastinon 1,0 Hoechst were administered, a year apart, to two groups of healthy subjects (ten and six men, respectively) without any difference in effect on blood sugar being found. The blood sugar concentration was measured in six healthy men before and after oral administration of 1,000 mg tolbutamide (as Rastinon 1,0 Hoechst or tolbutamid tablets Ratiopharm), in a blind test. After tolbutamide Ratiopharm, the area under the blood sugar concentration-time curves was only 29 percent (0-4 hafter medication) and 32 percent (4-8 h after medication) of that after Rastinon 1,0 (P 2alpha less than 0.01), with marked scatter between individuals. Maximal serum concentration was 80 percent below that after Rastinon. The first measurable value was reached 0.8 plus or minus 0.2 h after medication of Rastinon and 3.6 plus or minus 0.8 h after tolbutamide Ratiopharm. The areas under the serum concentration under the curves after tolbutamide Ratiopharm were only 16 percent (0-4 h after medication) and 19 percent (0-8 h after medication) of those after Rastinon (P 2alpha less than 0.05 and 0.01, respectively). The differences demonstrate that tolbutamide Ratiopharm tablets and Rastinon 1,0 Hoechst are not equivalent biologically and therapeutically.


[Abnormal hypothalamic-hypophyseal regulation as a new endocrine sign in the testicular feminisation syndrome (author's transl)]. In a 38-year-old woman with the testicular feminisation syndrome (TFS) there were increased serum levels of LH and FSH, despite normal testosterone levels, ranging from 808 to 1330 ng/dl. The patient exhibited complete end-organ resistance to androgens, as well as decreased free testosterone fraction of 1.5 percent. On stimulation with LH-RH there was enhanced LH reaction to 15 ng/ml, while FSH levels remained unchanged. Fluoxymesterone did not suppress LH and testosterone, as it did in normal controls. But the functional Leydig-cell reserve could be normally stimulated with HCG. Our results apparently confirm the suspected reduction in sensitivity of the hypothalamic-pituitary axis to androgens and suggest that this plays an important part in the pathogenesis of TES.


[The cast structure after vacuum-pressure casting and centrifugal casting]. Evaluation of polished sections revealed the following: 1. Grain size in marginal areas of the crown is, in general, smaller than it is in the occlusal region. 2. Grain size varies depending on the casting method applied. Differences in hardness were found only to a limited extent. They were not always in line with the grain structure. This phenomenon deviating from the rule cannot satisfactorily be explained. The piping distribution was in accordance with observations made so far.


[Effect of thermal expansion on the accuracy of fit of cast crowns]. Extent and direction of dimensional changes in the finished cast crown resulting from the thermic expansion of the wax model are determined as a function of the time the crown was subjected to tempering as a function of tempering temperature. Expansion of the wax model does not come up to the expectation of preventing a reduction of the cast object.


[Gingival hyperplasia in bar joint prostheses]. The occurrence of mucosal hyperplasias under 57 bars (according to Dolder) was examined. It was found that apart from the time factor, cleaning habits of the patient and the construction of the dentures played an important role. Short bars are more difficult to clean than those with wide distances between the abutments. The influence of additional factors can only be demonstrated in combination with other factors, this suggests rather complex connections.


[Bacteriological studies of removable splints and dentures]. The risk of germ contamination through already used dental replacement cannot or only with difficulties be solved by disinfection. As to new dental replacements, which may constitute a germ vehicle between the dental laboratory and the dental office, specific consequences can be drawn. It is tried to explain where contamination may start so that such contamination can be prevented.


[Prosthetic rehabilitation of dysgnathic patients]. There are only few reports on the prosthetical care of patients with dysgnathias. Thus, it seems appropriate first to delimit the field by well-defined terms from maxillo-facial surgery and orthodontics. Reichenbach, Köle, and Brückl introduced the term "principal symptom" ("Leitsymptom") for the most significant dysgnathic finding. With a view to prosthetical aspects, this term is broadened to include primary and secondary dysgnathias, after several proposals of nomenclature were made. It is reported on the number of dysgnathic patients needing treatment. The description of some of the goals aimed at by prosthetical care concludes the paper.


[Special problems in the prosthetic rehabilitation of deep bite]. In cases of deep bite where the lower front teeth contact the palatal gingiva or where the upper teeth contact the labial gingiva of the lower jaw, the attempt to improve the situation by grinding-in the teeth was always crowned with temporary success only. The question as to whether the front teeth may be maintained determines further measures to be taken. If they can and if they are to be kept it is necessary to change the vertical relation of practically all teeth by safeguarding the antagonistic contract also in the front area. Therapeutical means and risks involved are considerable. If the teeth concerned have to be removed, plastic correction of the alveolar ridge is necessary before bridges and removable dentures are fitted in. This solution is even less satisfactory and disregards the significance of natural teeth for the well-being of the patient.


[Control studies on periodontal and periodontal-gingival retention of partial prosthesis]. Follow-ups of 722 removable partial dentures that have been used for an average 5.2 years reveal that the way they were used and the type of balancing as well as the construction of the denture have an influence on the inflammation of the denture-bearing area and the loosening of the abutment teeth. In this investigation it was not possible to demonstrate the influence of daily personal oral hygience.


[Follow-up studies on dentures in patients with teeth in both jaws which do not occlude (Group C-1 according to Eichner)]. Follow-ups in class C 1 patients who underwent prosthetical treatment seemed indicated since little was known about the chances of success. It was possible to examine 20 patients of this groups mainly with regard to objective functional efficiency of the dental replacement worn from 1 to 8 years. Furthermore, articulation occlusion, relationship of upper and lower jaws, and frequency of required repairs were checked. Purely gingivally borne prostheses as well as periodontally supported and gingivally borne dentures had the same rate of success. The mixed form, i.e. gingivally borne in the upper jaw and periodontally supported and gingivally borne in the lower jaw, seemed to be less favorable due to differing load tolerances.


[Current studies on shape changes of total prostheses in relation to the method of their construction]. It was shown that marginal gaps occurring after the preparation of upper complete dentures vary depending on the material or method applied and that in all tested materials the gaps become smaller again during subsequent swelling, but are not fully compensated.


[Shape retention of plastic prostheses manufactured by the pouning method]. In investigations was shown that PMMA dentures fabricated by injection molding have a better dimensional stability than polycarbonate dentures (Andoran) and are slightly better than packed Paladon 65 dentures. When using the injection molding technique a slight increase in occlusion could not be prevented in any of the dentures.


[Studies on the retention of total lower dentures in relation to the time of wearing]. Clinical experience in the prosthetical care of edentulous patients shows that primarily patients wearing dentures for the first time express the desire, after a few months, to have original denture adhesion restored. Especially for complete lower dentures diminishing adhesion is unfavorable. Measurements regarding the adhesive strength of complete lower dentures of 34 patients showed that adhesive strength is decreasing. This was measured and statistically evaluated in this investigation. 33 percent of the original adhesive strength is lost within 6 months. With originally strong adhesion (2, 000 p) this reduction is more drastic than with originally small adhesion (200 p). It was not possible to establish that adhesive strength is declining preferably as a result of a specific anatomic situation.


Synthesis of DNA in isolated pancreatic islets maintained in tissue culture. The DNA synthesis of isolated pancreatic islets has been determined after culture in vitro of the islets for 5 days in media containing either various glucose concentrations or tolbutamide or cortisone. Only glucose stimulated the replication of DNA, while cortisone was inhibitory and tolbutamide without effect. It is concluded that glucose by itself stimulates the mitotic rate of the islet cells and that the corresponding effect of steroids in vivo may be mediated through the ensuing hyperglycemia.


Failure to detect luteinizing hormone-releasing hormone in third ventricle cerebral spinal fluid under a variety of experimental conditions. Cerebral spinal fluid (CSF) was examined for the presence of luteinizing hormone-releasing hormone (LH-RH) at times when copious amounts of LH were being secreted by the adenohypophysis. A "push-pull" apparatus was designed which permitted the perfusion of artificial CSF through the 3rd ventricle from the region of the medial preoptic area (MPOA) to the site of recollection adjacent to the median eminence (ME). It was observed on radioimmunoassay that LH-RH concentrations were low in CSF and peripheral plasma of Nembutal-blocked proestrous rats. Intraarterial injections of LH-RH did not increase CSF concentrations of this decapeptide, although a significant release of pituitary LH occurred. Further, electrochemical stimulation of the MPOA also elevated plasma LH without altering CSF-LH-RH levels. When synthetic LH-RH, dissolved in artificial CSF, was perfused through the 3rd ventricle, plasma LH levels rose dramatically, and a significant decrease in the concentration of LH-RH in the CSF efflux was observed on collection and assay. CSF collected from MPOA-stimulated rats also was tested for biologically active LH-RH by its reinjection into Nembutal-blocked proestrous recipients. While such injections failed to induce pituitary LH release, it was observed that LH-RH, when mixed with artificial CSF, and allowed to stand at room temperature (24 C), also lost all biological potency even though immunoreactivity was retained. The results suggest that CSF does not serve as a vehicle for transport of LH-RH to the median eminence under physiological conditions.


Effects of porcine stalk median eminence and prostaglandin E2 on rat growth hormone secretion in vivo and their inhibition by somatostatin. The studies described were performed to develop a technically simple, yet sensitive in vivo model for growth hormone (GH) releasing activity in porcine stalk median eminence (pSME) extracts, to compare the GH releasing effects of pSME with those of prostaglandin E2 (PGE2), and to study the effect of somatostatin (SRIF) on the above stimuli. The use of the one day estrogen-primed male rat in conjunction with intracarotid injection of test materials provided a model sensitive to the injection of one pSME. Neither increasing the duration of estrogen pre-treatment nor reserpine resulted in a greater response. The GH releasing effects of pSME were directly related to the preinjection GH level. Two successive injections of pSME at 30 minute intervals evoked similar responses. In contrast, PGE2 effects were not potentiated by estrogen pre-treatment and were independent of the preinjection GH level. The GH releasing effect of pSME was not related to its content of TRH or K+. Extracts of porcine cerebral cortex also contained GH releasing activity, although at a lower concentration than in pSME. Somatostatin inhibited the GH releasing effects of pSME, PGE2, and cerebral cortex extract. These results provide evidence for the direct inhibitory effect of SRIF on GH secretion in vivo and suggest that SRIF is capable of blocking a variety of different stimuli to GH release.


Augmentation of pituitary responsiveness to luteinizing hormone/follicle stimulating hormone-releasing factor (LH-RF) as a result of acute ovariectomy in the four-day cyclic rat. Within 6 h of ovariectomy in adult rats with 4-day estrous cycles, plasma FSH titers were significantly elevated above those of sham-operated controls at all stages of the estrous cycle, whereas plasma LH concentrations were raised by ovariectomy only during diestrus day 2, proestrus or estrus. A single intravenous injection of a partially purified extract of ovine stalk-median eminence tissue (LH-RF) into sham-operated control rats or incubation of pituitaries from similar rats with the same releasing factor extract elevated LH and FSH output at all stages of the cycle. This response to LH-RF was related to the stage of the estrous cycle, maximal pituitary responsiveness occurring on proestrus. Ovariectomy 6 h before administration of LH-RF significantly augmented pituitary LH and FSH responsiveness both in vivo and in vitro. This elevation in hypophyseal responsiveness observed in vitro followed a cyclic pattern which could be superimposed on the normal cyclic changes in pituitary responsiveness, whereas the cyclic increase in pituitary LH responsiveness to LH-RF in vivo following acute ovariectomy was first detectable 16 to 18 h earlier than in intact controls, that is, on late diestrus day 2 rather than on the morning of proestrus. These results indicate that acute ovariectomy can augment pituitary responsiveness to LH-RF and can also advance the onset of the cyclic augmentation of pituitary responsiveness to LH-RF during the 24 h immediately preceding the ovulatory gonadotropin surge. Possible explanations for these effects are discussed.


An in vivo assay for anti-LH-RH and anti-FSH-RH activity of inhibitory analogues of LH-RH. A convenient and sensitive method for testing anti-LH- and FSH-releasing activities of antagonists of LH-RH was devised. The peptides to be tested were injected subcutaneously together with synthetic LH-releasing hormone (LH-RH) in immature male rats. Serum LH and FSH concentrations were measured 30 min later by radioimmunoassay. In this system, [DesHis2]-LH-RH failed to block the pituitary response to LH-RH in agreement with previous data. The administration of [DesHis2, desGly10]-LH-RH ethylamide, [Des-His2, Leu3, des Gly10]-LH-RH ethylamide, [Des-His2, D-Ala6, des-Gly10]-LH-RH ethylamide, and [DESHis2, D-Ala6]-LH-RH in doses of 200 mug/rat, and [DesHis2, Leu3, D-Ala, desGly10]-LH-RH ethylamide in doses ranging from 100-400 mug/rat caused a significant inhibition of LH-RH-induced release of LH and FSH. On the other hand, [DesHis2, desGly10]-LH-RH propylamide, and [Leu2, Leu3, D-Ala6, desGly10]-LH-RH ethylamide failed to block the response to LH-RH. Among the peptides tested, [DesHis2, d-ala6, desGly10]-LH-RH etylamide, [Leu2, Leu3, D-Ala, desGly10]-LH-RH ethylamide in doses of 200 mug and [DesHis2, Leu3, D-Ala6, desGly10]-LH-RH ethylamide in a dose of 400 mug/rat showed some intrinsic LH and FSH-releasing activities. The method described here is simple and convenient for studying the anti-LH-RH release activity of inhibitory analogues of LH-RH, and also enabled us to demonstrate for the first time an in vivo inhibition of FSH release by these analogues.


In vitro release of lutenizing hormone from anterior pituitary fragments superfused with constant or pulsatile amounts of luteinizing hormone-releasing factor. Anterior pituitary fragments from ovariectomized rats were superfused with medium to which lutenising hormone-releasing factor (LH-RF) was added for either prolonged or short intervals. Samples of superfusate were collected at 2-min intervals before and after the onset of LH-RF administration. In control experiments without anterior pituitary fragments, infusions of constant amounts of NIAMDD-Rat LH were made into the superfusion chamber. The superfusate was collected in all experiments and luteinizing hormone (LH) levels measured by radioimmunoassay. LH release was increased 100-200% following superfusion with concentrations of LH-RF ranging from 10-9 to 10-5M. The percent coefficient of variation (%CV) was determined for LH to quantitate fluctuations in hormone release. A nonepisodic pattern of LH release was obtained prior to LH-RF administration and during superfusion with constant levels of LH-RF. There was no significant difference in the %CV in these periods as compared to that obtained during infusion of constant amounts of LH into the superfusion chamber. However stimulation by LH-RF for only 2-5 min caused episodes of LH release lasting 30-40 min. The %CV of superfusate LH during these pulsatile stimulation studies was significantly increased compared to that obtained during superfusion with constant levels of LH-RF. These results suggest that the pituitary is not the site of origin of the mechanism underlying episodic release of LH, but can release LH in a pulsatile manner when driven by pulsatile LH-RF stimulation.


Insulin biosynthesis: the monoaminergic mechanisms and the specificity of "glucoreceptor". Islets of Langerhans, isolated from the rat, did not synthesize insulin in the presence of L-glucose, fructose, galactose, 3-0-methyl glucose or sorbitol. A small amount of insulin was synthesized in the presence of glucosamine. Addition of caffeine to these compounds did not enhance the biosynthesis of insulin. It is suggested that the specificity of the membrane surface "glucoreceptor", if it exists, may be rather narrow, at least with respect to insulin biosynthesis. Serotonin, dopamine and isoproterenol did not inhibit or enhance insulin biosynthesis induced by glucose. Propranolol also failed to modify insulin synthesis in the absence or presence of isoproterenol. It is concluded that the monoaminergic mechanisms do not affect insulin biosynthesis in spite of their significant regulatory influence on insulin release. Methysergide, at the concentration reported to potentiate insulin release induced by glucose and tolbutamide in rabbit, strongly inhibited insulin biosynthesis.


Chromatographic evidence for the existence of another species of luteinizing hormone-releasing factor (LRF). Fractionation of ultrafiltrates of rat hypothalamic extracts has been performed on Sephadex LH-20 in dimethylformamide/water. The fractions were examined for their content of luteinizing hormone-releasing activity by bioassay in vitro utilizing LH radioimmunoassay, and also for their content of LRF decapeptide-like material by direct radioimmunoassay. Each method revealed the same pattern of activity. This consisted of a major peak whose elution position coincided with that of authentic decapeptide, and a minor region which closely preceded it, often taking the form of a shoulder on the leading edge of the main peak. The active component in the minor peak could be completely resolved by repeated filtration in the same system thus indicating that it represented another gonadotropin-releasing species rather than a chromatographic artefact although its biological and immunological activities indicate that its structure must resemble closely that of the decapeptide.


Relationship of thyrotropin to exophthalmos-producing substance: formation of an exophthalmos-producing factor by pepsin digestion of mouse pituitary tumor and human thyrotropin preparations. The present report demonstrates that mouse tumor thyrotropin preprarations have exophthalmogenic activity. In addition it shows that the exophthalmogenic activity of mouse tumor thyrotropin can be increased by partial pepsin digestion, whereas the thyroid stimulating activity of mouse tumor thyrotropin is rapidly destroyed by such treatment. Thus, after 30 min of pepsin digestion, mouse tumor thyrotropin has 130% of its initial exophthalmogenic action but only 10% of its thyroid stimulating activity. Preparations of human thyrotropin are similarly sensitive to partial pepsin digestion, i.e., there is a rapid destruction of thyroid stimulating activity but a very much slower destruction of exophthalmogenic activity. Thus, after 30 min of pepsin digestion, preparations of human thyrotropin retain 80% of their exophthalmogenic activity but only 20-30% of their thyroid stimulating action. Since these results are analogous to those obtained in studies of the partial pepsin digestion of bovine thyrotropin (1), partial pepsin digestion of both purified human and purified mouse tumor thyrotropin preparations should yield an exophthalmogenic fragment of the TSH molecule devoid of thyroid stimulating action.


Entomopathogens as insecticides.

Entomopathogens as insecticides. Entomopathogens, diseases of insects, are suggested as a possible new generation of safe, selective insecticides. Over a thousand pathogens have been isolated from insects. Many of these, associated with major insect pests, are potential candidates for development into microbial insecticides. Phases in the development of a microbial insecticide are discussed as well as factors (production, safety, efficacy, registration, non-technical) which may influence this development.


Manipulating sex pheromones for insect suppression. A review is given of different methods of using sex pheromones in pest control programs. Survey and monitoring traps have been used extensively for determining the presence and abundance of pest populations are commercially available. Pheromone trapping studies for insect suppression have shown success with some species and could be a useful tool in some situations. The technique of attracting insects to the host for insect removal or exposure to insecticide has also proved to be useful in some programs. Mating disruption studies using widely-separated pheromone evaporators or encapsulated pheromone distributed throughout an area have indicated a great potential for this technique in insect suppression programs.


Control of insects by sexual sterilization. The control of insect populations by restricting their birth rate offers several advantages over methods based on increasing the death rate. The sterile-male technique scored practical success in the control of the screwworm, Cochliomyia hominivorax (Coquerel), but more general applications of the sterility procedure may be expected in conjunction with the development of integrated procedures for pest insect management.


Development of morphogenetic agents in insect control. Chemicals which interfere with the growth and development of insects (morphogenetic agents) have been receiving major attention as potential means of selective insect control. Major advances in this field resulted from the identification of Juvenile Hormones -1, -2, and -3, and the discovery that various terpenoid and sesquiterpenoid derivatives were more potent morphogenetic agents than the three known Juvenile Hormones. Several highly active compounds have emerged from these research programs. Their field performance, problems, and prospects in selective insect control are considered here.


Two-dimensional polyacrylamide-gel electrophoresis for purification of small RNAs specified by virulent coliphages T4, T5, T7 and BF23. RNAs synthesized in Escherichia coli infected with virulent phages T4, T5, T7 and BF23 were labelled with 32PO4 3- after phage infection. [32P]RNAs of low molecular weight were separated by two-dimensional polyacrylamide gel electrophoresis, in which electrophoresis was carried out in two dimensions at different concentrations of acrylamide. The fractionated RNAs were characterized by RNA-fingerprint patterns made after T1 ribonuclease digestion. The two-dimensional gel of 10% yields 20% acrylamide was suitable for RNA of less than 200 nucleotides, while that of 5% yields 10% was preferred for RNAs of about 150--400 nucleotides. With T4 phage, 16 RNA species were separable on a single slab gel. Among those, 11 were identified as the known RNA species, including eight T4 tRNAs, one tRNA precursor and two non-tRNA molecules. In the case of T5 and BF23, more than 20 RNA species were separated on a slab gel; 15 or more RNAs were found in the 4-S RNA region, and several in 5-S and 6-S region. The RNA-fingerprint patterns of many BF23 RNAs were very similar to those of corresponding RNAs of T5. Pseudouridine and ribosylthymidine, minor nucleosides generally present in tRNA, were found in several BF23 4-S RNAs tested. Possibility of those BF23 4-S RNAs as tRNAs is discussed. With phage T7, three RNAs were detected, two of which were much smaller than tRNAs.


Comparative studies on the properties of tryptophanase and tyrosine phenol-lyase immobilized directly on Sepharose or by use of Sepharose-bound pyridoxal 5'-phosphate. Tryptophanase from Escherichia coli B/qt 7-A and tyrosine phenol-lyase (beta-tyrosinase) from Escherichia intermedia were immobilized on Sepharose 4B by several direct coupling reactions or through pyridoxal 5'-phosphate previously bound to Sepharose. The most active preparation of immobilized tryptophanase was obtained by coupling tetrameric apoenzyme to pyridoxal-P bound on Sepharose at the 6-position through a diazo linkage. This immobilization procedure involves the formation to Schiff base linkage between 4-formyl group of Sepharose-bound pyridoxal-P and the epsilon-amino group of the lysine residue at the active center of one subunit of tetrameric apo-tryptophanase, followed by the fixation of the Schiff base linkage by reduction with NaBH4. In the case of beta-tyrosinase having two catalytic centers, however, this method was not so suitable as the case of tryptophanase. Direct coupling of the apoenzyme to CNBr-activated Sepharose or to a bromoacetyl derivative of Sepharose gave better results. In each case, the affinity for substrate or coenzyme was scarcely influenced by the immobilization. When used repeatedly in a batch system or continuously in a flow system in the absence of added pyridoxal-P, immobilized holo-tryptophanase of holo-beta-tyrosinase gradually lost its original activity; however, supplement of pyridoxal-P to the reaction system restored its initial activity. From the kinetic analyses of these phenomena, the rate constants of coenzyme dissociation from immobilized tryptophanase and beta-tyrosinase were calculated. Upon immobilization, the pH optima of both enzymes shifted 0.5 to 1.0 pH unit to the alkaline side. Both immobilized enzymes showed higher thermal stability and resistance to a denaturing agent such as guinidine-HCl than their free counterpart. Furthermore, the reactivity of sulfhydryl group of beta-tyrosinase, in connection with its coenzyme-binding property, was conveniently studied by use of the immobilized enzyme.


An investigation of the 16-S RNA binding sites of ribosomal proteins S4, S8, S15, and S20 FROM Escherichia coli. The RNA binding sites of four 30-S ribosomal subunit proteins from Escherichia coli, namely S4, S8, S15, and S20 were prepared from reconstituted single protein - 16-S-RNA complexes by mild enzymic digestion of non-protected RNA regions. Oligonucleotide fingerprints of the protected RNA regions were obtained and their positions were located within the 16-S-RNA sequence. They were not completely contiguous regions of RNA; oligonucleotides had been excised from each of them. The binding sites of S4 and S20, and those of S8 and S15 showed overlapping. The specificity of the RNA binding sites was confirmed by a reconstitution method.