Membrane-associated nucleotide sugar reactions: influence of mutations affecting lipopolysaccharide on the first enzyme of O-antigen synthesis. Both the synthesis of lipopolysaccharide O-antigen and the synthesis of peptidoglycan in Salmonella typhimurium proceed via membrane-bound glycosylated lipid intermediates. The first enzyme of each pathway transfers a sugar phosphate from a nucleotide sugar to the glycosyl carrier lipid (P-GCL). Each enzyme catalyzes an exchange reaction between the reaction product urine monophosphate, and the nucleotide sugar substrate. Several strains of S. typhimurium defective in lipopolysaccharide synthesis accumulate glycosylated lipid intermediates under appropriate conditions. In addition, strains lysogenic for phage P22 synthesize a glucose derivative of the carrier lipid. These strains were used to demonstrate the P/GCL requirement of the exchange reaction catalyzed by galactose-diphosphoglycosyl carrier lipid (GCL-PP-Gal) synthetase, the first enzyme of O-antigen synthesis. Enzyme activity is greatly reduced when glycosylated P-GCL accumulates on the cytoplasmic membrane. The exchange reaction catalyzed by the first enzyme of peptidoglycan synthesis is unaffected by the accumulation of O-antigen fragments on the carrier lipid and may interact with a different pool of P-GCL within the membrane. GCL-PP-Gal synthetase activity cannot be detected in the membranes of two rfa mutants that synthesize incomplete lipopolysaccharide core. Either the synthesis of GCL-PP-Gal synthetase or the stable integration of the enzyme into the membrane structure may be disrupted in the rfa mutants. Peptidoglycan synthesis is unaffected by the mutations affecting the core glycosyltransferases.


Effect of temperature on ergosterol biosynthesis in yeast. Anaerobically grown Saccharomyces cerevisiae was aerated for 7 hr at 20 degrees, 30 degrees, or 40 degrees, in a phosphate buffer containing 2% glucose. At elevated temperature (40 degrees), de novo synthesis of squalene and sterois in the aerated yeast was only 32-35% of that at lower temperature (20 degrees or 30 degrees), and this decrease was attributed to the repression of the enzymes involved in the synthesis of mevalonate from acetyl-COA. In addition, at elevated temperature, the metabolic flux from squalene to ergosterol was blocked at squalene epoxidation, lanosterol demethylation, and ergosta-5, 7, 22, 24(28)-tetraene-3beta-ol reduction.


Conformational transitions of polypeptide chain elongation factor Tu. I. Studies with hydrophobic probes. The conformational difference between polypeptide chain elongation factor Tu (EF-Tu)-GTP and EF-Tu-GDP has been studied using hydrophobic and fluorescent probes. The interaction of EF-Tu-GDP with 1-anilino-8-naphthalenesulfonate (ANS) was measured in terms of the enhancement of the fluorescence intensity at the emission maximum of 475 nm. When EF-Tu-GDP-ANS complex was converted to EF-Tu-GTP-ANS complex by incubation with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40], there was a roughly 2-fold increase in fluorescence intensity and a blue shift of the emission maximum from 475 to 467 nm, indicating a conformational transition of the protein. The conformational change was found to be reversible and the spectrum promptly returned to that of EF-Tu-GDP-ANS complex upon addition of excess GDP. A similar change in the spectrum was also observed when aminoacyl-tRNA, but not deacylated tRNA, was added to EF-Tu-GDP-ANS complex. Measurement of the number of binding sites by gel filtration indicated that EF-Tu-GTP and EF-Tu-GDP bind 2.9 and 1.7 molecules of ANS, respectively. These results suggest that in EF-Tu-GTP the conformation was altered and one additional binding site for ANS was created at or near the site interacting with aminoacyl-tRNA. Another reagent, N-(1-anilinonaphthyl-4) maleimide (ANM) was covalently bound to the sulfhydryl group in EF-Tu-GDP which is essential for interaction with aminoacyl-tRNA. The binding could be determined spectrofluorometrically, since the reagent, which is nonfluorescent in aqueous solution, emitted a strong fluorescence upon binding with the sulfhydryl group, indicating a marked hydrophobicity of the local environment. Measurements of the kinetics of the binding revealed that ANM reacted rapidly with the sulfhydryl group in EF-Tu-GTP, while the reaction with that in EF-Tu-GDP proceeded more sluggishly. The difference in the reactivity of the sulfhydryl group essential for aminoacyl-tRNA binding between EF-Tu-GTP and EF-Tu-GDP probably reflects a conformational transition of the protein near the active site. These results, together with those on spin-label studies previously published (Arai, Kawakita, Kaziro, Maeda, & Onishi (1974) J. Biol. Chem. 249, 3311), demonstrate that reversible conformational transition does occur in EF-Tu on changing the ligand from GDP to GTP.


Crystallographic studies on L-asparaginase from Proteus vulgaris. II. Symmetry and location of the tetrameric molecule. Analyses of the x-ray diffraction intensity data by the Patterson synthesis and rotation function techniques show that the true space group of the monoclinic crystals of L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) from Proteus vulgaris is P21, that the molecular centers lie at x = 0.054, y = 0, z = 0.256, and its symmetry related positions, and that the tetramer molecules possess three approximate, mutually perpendicular 2-fold rotational symmetries, the axes of which run along the directions of the crystallographic a*-, b-, and c-axes. In addition, an investigation of the molecular packing arrangement in the crystal indicates that the tetramer molecules possess an approximately regular tetrahedral subunit structure.


In vitro synthesis of transfer RNA. I. Purification of required components. We have described an in vitro system in which active su+III tRNATyr is synthesized from a phi80psu++III DNA template. Using this system, we have identified four essential components that are required for synthesis of tRNA. The first of these is DNA-dependent RNA polymerase. It has been shown that a crude preparation of DNA-dependent RNA polymerase synthesizes su++III tRNATyr precursor similar to that which has been isolated in vivo, and that this preparation is capable of supporting high levels of tRNA synthesis. With purified DNA-dependent RNA polymerase, the su++III tRNATyr precursor was not observed as a transcription product and tRNA synthesis was below detetable levels. On this basis, a second essential component for tRNA synthesis was identified. This fraction, designated Fraction V, in combination with purified RNA polymerase, catalyzes the synthesis of precursor tRNA. The third component is a ribonuclease (RNase P III), which specifically catalyzes the removal of the extra nucleotides present at the 3' terminus of the tRNA precursor. In the absence of this fraction, the in vitro synthesized su++III tRNATyr is slightly larger than 4 S and contains additional nucleotides beyond the normal --CCAOH 3 terminus of the mature tRNA. The fourth essential component required is a fraction containing RNase P, a previously identified endonuclease which specifically catalyzes the removal of the 5' extra nucleotides present on tRNA precursors.


In vitro synthesis of transfer RNA. II. Identification of required enzymatic activities. We have shown that the synthesis of active su+III tRNATyr from a phi80psu+III DNA template requires the action of four distinct enzymatic activities. The first of these, DNA-dependent RNA polymerase, catalyzes the formation of a large molecular weight transcript, initiating synthesis at a specific site 41 nucleotides proximal to the 5' end of the su+III tRNATyr structural gene and continuing at least 100 nucleotides beyond the 3' terminus of the su+III tRNATyr sequence. The second required component, designated Fraction V, allows purified DNA-DEPENDENT RNA polymerase to function in tRNA synthesis. We have shown that this fraction contains an endonuclease that together with DNA-dependent RNA polymerase is responsible for the synthesis of su+III tRNATyr "precursor". Thus, su+III tRNATyr precursor is not itself the primary transcription product of the su+III tRNATyr gene, but rather, it arises as a result of post-transcriptional cleavage of a much larger transcript by the action of the nuclease present in Fraction V. The third enzymatic activity required for synthesis of active su+III tRNATyr is a ribonuclease (RNase P III) that specifically catalyzes the removal of the 3' extra nucleotides from the su+III tRNATyr precursor. The fourth activity required for synthesis of tRNA is a previously identified endonuclease, RNase P, that specifically catalyzes the removal of the 5' extra nucleotides from tRNA precursors. The properties of RNase P purified according to the procedure developed in this laboratory have been compared with those of the enzyme purified from ribosomes according to the procedure described by Robertson et al. (Robertson, H.D., Altman, S., and Smith, F.D. (1972) J.Biol. Chem. 247, 5243-5251.).


Structure of an exophthalmos-producing factor derived from thyrotropin by partial pepsin digestion. Previously reported experiments (Winand, R.J., and Kohn, L.D. (1970) J. Biol. Chem. 245, 967-975; Kohn, L.D., and Winand, R.J. (1971) J. Biol. Chem 246, 6570-6575) have demonstrated that partial pepsin digestion of bovine thyrotropin preparation yields a fragment of the thyrotropin molecule which is exophthalmogenic but has negligible or no thyroid-stimulating activity. In the present report this exophthalmogenic derivative of the thyrotropin molecule is shown to contain two major polypeptide components with approximate molecular weights of 14,000 and 6,000. Amino acid analyses, carbohydrate analyses, and tryptic digestion experiments indicate that this exophthalmogenic factor is composed of an intact or nearly intact beta subunit of thyrotropin and an NH2-terminal fragment of the alpha subunit of thyrotropin. Neither polypeptide component of the exophthalmogenic factor has the in vivo exophthalmogenic activity of the intact structure. In vitro the intact exophthalmogenic derivative of the thyrotropin molecule can bind to the thyrotropin receptor on thyroid membranes less efficiently than thyrotropin but significantly better than either its own polypeptide components or the alpha or beta subunits of thyrotropin. The exophthalmogenic factor and its parent thyrotropin molecule can stimulate adenylate cyclase activity in retro-orbital tissue membranes from guinea pigs, a mammalian model of exophthalmos; its polypeptide components have little or no such activity.


Enhancement of migration inhibitory factor activity by plasma esterase inhibitors. The plasma esterase inhibitors alpha2-macroglobulin, alpha1-antitrypsin, C1-inhibitor, antithrombin-heparin cofactor, and, as previously described, soybean trypsin inhibitor (Kunitz) and diisopropylphosphorofluoridate (9) enhance the response of guinea pig macrophages to migration inhibitory factor. To obtain this effect, macrophages are incubated with inhibitors prior to assay. The data suggest that (a) the enhancement of migration inhibitory factor response is due to the inhibition of esterases associated with the macrophage through a distinct active site on the inhibitors, and (b) that the active sites of antithrombin-heparin cofactor and soybean trypsin inhibitor, which interact with the macrophage enzymes, are different from the active sites of these inhibitors which interact with thrombin and trypsin respectively. Chemical modification of the active site of antithrombin-heparin cofactor for thrombin and of soybean trypsin inhibitor for trypsin does not affect their capacity to enhance the migration inhibitory factor response. From studies with thrombin, it was known that antithrombin-heparin cofactor has a heparin binding site. Addition of heparin was found to prevent the migration inhibitory factor-enhancing effect of antithrombin-heparin cofactor. The present results suggest that plasma esterase inhibitors may play a regulatory role in the response of macrophages to mediators of cellular immunity.


The purification and properties of formate dehydrogenase and nitrate reductase from Escherichia coli. The membrane-bound formate dehydrogenase of Escherichia coli grown anaerobically in the presence of nitrate was solubilized with deoxycholate and purified to near homogeneity. The purification procedure included ammonium sulfate fractionation and chromatography on Bio-Gel A-1.5m and DEAE Bio-Gel A in the presence of the nonionic detergent, Triton X-100. This detergent caused a significant decrease in the molecular weight of the soluble formate dehydrogenase complex and allowed the enzyme then to be resolved from other membrane components. Anaerobic conditions were required throughout due to the sensitivity of the enzyme to oxygen inactivation. Formate dehydrogenase was judged to be at least 93 to 99% pure by the following procedures: polyacrylamide gel electrophoresis in the presence of Triton X-100 and sodium dodecyl sulfate, gel filtration, and sedimentation velocity studies. The purified enzyme exists as a detergent-protein complex (0.20 +/- 0.03 g of Triton X-100/g of protein) which has an S20,w of 18.1 S and a Stokes radius of 76 A. This corresponds to a molecular weight of 590,000 +/- 59,000. The enzyme had an absorbance spectrum of a b-type cytochrome which could be completely reduced by formate. The heme content corresponds to an equivalent weight of 154,000 which suggests a tetrameric structure for the enzyme. Formate dehydrogenase was found to contain (in relative molar amounts): 1.0 heme, 0.95 molybdenum, 0.96 selenium, 14 non-heme iron, and 13 acid-labile sulfide. Neither FAD nor FMN could be detected. The enzyme contains three polypeptides, designated alpha, beta, and gamma, whose molecular weights were estimated by gel electrophoresis in the presence of sodium dodecyl sulfate to be 110,000, 32,000, and 20,000, respectively. After separation of the polypeptides by gel filtration in the presence of sodium dodecyl sulfate alpha, beta, and gamma were found in 1:1.2:0.55 molar ratios. A study of the enzyme obtained from cells grown with [75Se]selenite showed that only the alpha polypeptide contained significant amounts of selenium. The enzyme will catalyze the formate-dependent reduction of phenazine methosulfate, dichlorophenolindophenol, methylene blue, nitroblue tetrazolium, benzyl viologen, methyl viologen, ferricyanide, and coenzyme Q6. Cyanide, azide, p-hydroxymercuribenzoate, iodoacetamide, and oxygen inhibit the enzyme. The procedure which was designed for the purification of formate dehydrogenase also yields a highly purified preparation of nitrate reductase. This nitrate reductase has been shown to contain significant amounts of heme (Enoch, H. G., and Lester, R. L. (1974) Biochem. Biophys. Res Commun. 61,1234-1241). The enzyme contains three polypeptides with molecular weights of 155,000, 63,000, and 19,000. When measured in the presence of Trition X-100 the Stokes radius of nitrate reductase is 75 A and the S20,w is 16 S which corresponds to a molecular weight of 498,000.


Anaerobic transport in Escherichia coli membrane vesicles. Anaerobic lactose and/or amino acid transport by membrane vesicles prepared from Escherichia coli ML 308-225 can be coupled to at least four electron transfer systems: alpha-glycerol-P-dehydrogenase:nitrate reductase, formate dehydrogenase:nitrate reductase, alpha-glycerol-P dehydrogenase:fumarate reductase, and formate dehydrogenase:fumarate reductase. Vesicles contain one or more of these electron transfer systems depending on the growth conditions of the parent cells. alpha-Glycerol-P dehydrogenase and fumarate reductase are present only in vesicles prepared from cells grown in the presence of glycerol or fumarate, respectively. Formate dehydrogenase and nitrate reductase activities, on the other hand, are present in vesicles from cells grown on a variety of media. alpha-Glycerol-P and formate are able to drive aerobic transport in vesicles prepared from anaerobically grown cells, indicating coupling between aerobic and anaerobic electron transfer systems.


Photoinactivation of the beta-galactoside transport system in Escherichia coli membrane vesicles with an impermeant azidophenylgalactoside. 2'-N-(2-Nitro-4-azidophenyl) aminoethyl-1-thio-beta-D-galactopyranoside (APG2) is a competitive inhibitor of lactose transport in membrane vesicles isolated from Escherichia coli ML 308-225, exhibiting an apparent Ki of 30 to 40 muM, but is not transported. When irradiated with visible light in the presence of D-lactate, APG2 irreversibly inactivates the lac transport system. Imposition of a membrane potential (positive outside) by valinomycin-induced potassium efflux also causes APG2 photoinactivation. Strikingly, photoinactivation is not observed in the absence of D-lactate or a potassium diffusion gradient. Kinetic studies of the inactivation process yield a KD of 35 muM. Since lactose protects against the inactivation, it is apparent that these effects are specific for the lac transport system. The results show that APG2 inactivates from the outer surface of the vesicle membrane and support the previous hypothesis that the lac carrier protein is unable to bind external substrate in the absence of energy coupling.


Aspartate transcarbamylase of Escherichia coli. Mechanisms of inhibition and activation by dicarboxylic acids and other anions. The interactions of several dicarboxylic acids and monoanions with Escherichia coli aspartate transcarbamylase and with its catalytic subunit have been studied by ultraviolet difference spectroscopy and steady state kinetics, with the following major findings. 1. A variety of dicarboxylic acids compete with carbamyl-P for the active sites of unliganded catalytic subunit, with steric requirements very different from those important for competition with L-aspartate for the subunit/carbamyl-P complex. Competition with carbamyl-P is much reduced if the dicarboxylic acid has a positively charged amino group. Acetate and chloride also compete. 2. At pH 7, equal concentrations of lysine acetate and L-aspartate are equally effective in displacing the transition state analog N-(phosphonacetyl)-L-aspartate (PALA) from the active sites of the concentrations of L-aspartate and lysine acetate is constant, increasing the concentration of L-aspartate does not relieve inhibition of the enzyme by PALA (Collins, K.C., and Stark, G. R. (1971) J. Biol. Chem. 246, 6599-6605). Therefore, the L-aspartate/subunit complex, like the acetate/subunit complex, must be incapable of participating in the catalytic reaction. We conclude that the kinetic mechanism is ordered, in agreement with the recent findings of Wedler and Gasser (Wedler, F.C., and Gasser, F.J. (1974), Arch. Biochem. Biophys. 163, 57-68) and in disagreement with the interpretation of Heyde et al. (Heyde, E., Nagabhushanam, A., And Morrison, J.F. (1973) Biochemistry 12, 4718-4726)...


Binding of Met-tRNAf to native 40 S ribosomal subunits in Ehrlich ascites tumor cells. Two forms of native 40 S ribosomal subunits, distinguishable by their buoyant densities, are recovered from Ehrlich ascites cells. In this communication, we describe experiments designed to test whether Met-tRNAf is associated with either form. Our results indicate that (a) in the cell, Met-tRNAf is bound to the native 40 S subunit, and in particular, to the subunit of density 1.40 g x cm-3; (b) under the growth conditions used, less than 10% of the low density native subunits have bound Met-tRNAf; (c) the majority of the Met-tRNAf containing native 40 S subunits, isolated from sucrose gradient analyses of cell extracts, join with 60 S subunits in vitro to form 80 S monosomes only if additional ribosomal wash factors are provided; and (d) the 80 S monosomes so formed are completed initiation complexes that can form peptide bonds. These results support the hypothesis that various forms of the native 40 S subunit represent different stages in the formation of a complete initiation complex.


Evidence against a covalent intermediate in the adenosine triphosphate phosphoribosyltransferase reaction of histidine biosynthesis. 14C-Labeled 5-phospho-alpha-D-ribose-1-diphosphate (PRibPP) was synthesized and its interaction with adenosine triphosphate phosphoribosyltransferase was examined by gel filtration in a search for a form of this substrate covalently bound to the enzyme. Wide variation in solvent conditions gave little labeling of the enzyme. Heavy labeling was found only in the presence of the second substrate, ATP, and this was shown to arise from tightly but noncovalently bound product. Previous reports of a covalent intermediate in this enzymatic reaction probably were due to contaminating ATP in 5-phospho-alpha-D-ribose-1-diphosphate. Feedback inhibition of the enzyme by histidine was shown to occur at the step giving product or at some earlier step in the mechanism.


Relation of growth and protein synthesis to the adenylate energy charge in an adenine-requiring mutant of Escherichia coli. When Escherichia coli K-12 strain PC 0294, which is unable to synthesize adenine nucleotides, thereonine, proline, and leucine, was starved for adenine, the concentrations of ATP, ADP, and AMP fell rapidly. The adenylate energy charge was not affected until the adenine nucleotide pool fell to about 30% of its normal value. Similarly, cells in an adenine-limited chemostat grew at intracellular adenine nucleotide pool values as low as 30% of normal, but the energy charge in all cases was approximately 0.90. Incorporation of [14C]leucine into protein in adenine-starved cells continued rapidly as long as the energy charge was in or near its normal range, even when the concentration of ATP was 30% to 10% of its normal value. When glucose was added to cells that had been resuspended in medium lacking both glucose and adenine, the energy charge rose rapidly but the concentration of ATP fell, presumably because of nucleic acid synthesis. The rate of [14C]leucine incorporation into protein rose rapidly while adenine nucleotide concentrations fell, and then declined roughly in parallel with the energy charge. At a given value of energy and of total concentration of adenine nucleotides, the rate of protein synthesis may, of course, vary with the concentrations of precursors and modifiers; thus, the rate cannot be predicted from knowledge only of the energy charge. Our results suggest, however, that the rate of protein synthesis and the capacity for growth are much more sensitive to changes in the value of the energy charge than to changes in the concentration of ATP. Growth occurs when the ATP concentration is reduced to one-third of its normal value, but has not been observed when the energy charge has fallen by as much as 10%.


"Cytoplasmic" deoxyribonucleic acid polymerase. Structure and properties of the highly purified enzyme from human KB cells. The freshly prepared crude cytoplasmic fraction of aqueously extracted KB cells contains a single major species of DNA polymerase activity (DNA polymerase C) that sediments homogeneously in low ionic strength sucrose gradients with a peak at 10.8 S. The enzyme activity from frozen crude extracts sediments heterogeneously under these conditions with peaks at 8.4 and 10 S. In 0.45 M salt-containing gradients all of the polymerase activity is recovered as a single 6.4 S species. When purified to a specific activity of 7,300, DNA polymerase C sediments in low ionic strength gradients as a single species of 6.5 S. From combined sedimentation and gel filtration analysis, we estimate the molecular weight of the active protomeric species of the polymerase to be about 170,000. Under no conditions of ionic strength does the enzyme disaggregate to active species smaller than 6.4 to 6.5 S. Sodium dodecyl sulfate-polyacrylamide gel analysis of the most highly purified enzyme fractions reveals two major protein bands of 87,000 and 175,000 daltons, respectively. These data suggest that DNA polymerase C contains an 87,000-dalton component and permit the interpretation that the active protomer of Mr equal 170,000 may be a dimer. The purified enzyme shows maximal activity with gapped duplex DNA and has an absolute requirement for 3'-hydroxyl termini. It utilizes initiated polydeoxynucleotide templates poorly and initiated polyribonucleotide templates not at all. Although the polymerase is inhibited by PPi it has only minimal ability to promote PPi exchange (0.8% of the polymerase activity). The purified enzyme is free of endonuclease and exonuclease activities (less than or equal to 0.003% of the polymerase activity) and demonstrates no primer-template-dependent conversion of substrate dNTP to free dNMP during the polymerization reaction. Finally, DNA polymerase C does not excise misparied primer termini from a synthetic homopolymer primer-template but can utilize such termini as initiation sites, although at a very slow rate.


Thermal regulation of the membrane lipid composition of Escherichia coli. Evidence for the direct control of fatty acid synthesis. Starvation of strains of Escherichia coli which are glycerol auxotrophs and are also defective in beta oxidation results in the accumulation of large amounts of free fatty acid (Cronan, J. E., Jr., Weisberg, L. W., and Allen, R. G. (1975) J. Biol. Chem. 250, 5835-5840). We now report that the ratio of saturated to unsaturated species appearing in the free fatty acid fraction depends on the incubation temperature at the time of synthesis of these acids. This result indicates that fatty acid synthesis is one site of the thermal control of phospholipid fatty acid composition. We also report experiments on the incorporation of exogenously supplied fatty acids into membrane phospholipids. The effect of temperature on this incorporation supports the hypothesis that a second site of thermal regulation acts at the level of phosphatidic acid synthesis.


Fractures and anatomical variations of the proximal portion of the fifth metatarsal. Fractures of the tuberosity of the fifth metatarsal are frequent and with conservative treatment uniformly heal well. In contrast, fractures of the metatarsal shaft distal to the tuberosity heal slowly. Of 100 fractures of the tuberosity, almost all were clinically united within three weeks and roentgenographically united within two months. Of twenty fractures in the proximal part of the shaft, five required bone grafts and the time elapsed before roentgenographic and clinical evidence of union was variable and often prolonged.


Repair of experimental autologous grafts of cortical bone. Fresh autologous segmental grafts of fibula, four centimeters long, were obtained from thirteen adult dogs. Holes were drilled in each before replantation. The amount of repair was determined after intervals of from four to twenty-four weeks. Torsional stress testing showed that the six drill holes 500 micrometers in diameter confined within an expanse of two centimeters did not mechanically weaken the transplants at the time of surgery. The drill holes were rapidly filled with cancellous bone which matured to cortical bone within twelve weeks. Microradiography and tetracycline labeling of drilled and non-drilled grafts demonstrated similar biological reparative patterns.


Actin in erythrocyte ghosts and its association with spectrin.  Evidence for a nonfilamentous form of these two molecules in situ. Actin was isolated from erythrocyte ghosts.  It is identical to muscle actin in its molecular weight, net charge, ability to polymerize into filaments with the double helical morphology, and its decoration with heavy meromyosin (HMM).  when erythrocyte ghosts are incubated in 0.1 mM EDTA, actin and spectrin are solubilized.  Spectrin has a larger molecular weight than muscle myosin.  When salt is added to the EDTA extract, a branching filamentous polymer is formed.  However, when muscle actin and the EDTA extract are mixed together in the presence of salt, the viscosity achieved is less than the viscosity of the solution if spectrin is omitted.  Thus, spectrin seems to inhibit the polymerization of actin.  If the actin is already polymerized, the addition of spectrin increases the viscosity of the solution, presumably by cross-linking the actin filaments.  The addition of HMM of trypsin to erythrocyte ghosts results in filament formation in situ.  These agents apparently act by detaching erythrocyte actin from spectrin, thereby allowing the polmerization of one or both proteins to occur.  Since filaments are not present in untreated erythrocyte ghosts, we conclude that erythrocyte actin and spectrin associate to form an anastomosing network beneath the erythrocyte membrane.  This network presumably functions in restricting the lateral movement of membrane-penetrating particles.


Observations on the sorting-out of embryonic cells in monolayer culture. Two problems are raised concerning the movement of cells during tissue-specific sorting-out of chick embryo cells in mixed aggregates. (i) A possible expectation from the hypothesis of 'contact inhibition' is that cells which are entirely surrounded by other cells in monolayer should be held stationary. Cells within solid aggregates, being totally surrounded by others, might also not be expected to move. How is it then that cell movement takes place within solid aggregates during sorting-out? (ii) Are the movements of cells within sorting aggregates 'passive', being driven by adhesive differentials or 'active', being merely guided by such differentials? In order to study these questions, sorting out experiments with chick embryonic limb bud mesenchyme and liver cells were carried out in monolayer culture, permitting direct observation of cell movements. Cell behavior was observed by time-lapse cinematography. Sorting-out of these cells in monolayer began before and continued after the cells had spread to confluency. During sorting, liver cells showed ruffing activity even when they appeared to be totally surrounded by other cells. Both cell types showed contact inhibition as judged by the criterion of monolayering, for they did not move over each other but remained attached to the substratum. Yet the cells in the confluent monolayer were not immobilized. Because of this, we suggest that the observed restraint against overlapping did not result from an inhibition of movement. Several considerations, detailed in the text, suggest that cell movement during sorting-out involve active locomotion. Previous work suggest that sorting-out configurations are determined by the relative intensities of intercellular adhesive strengths, the more cohesive of 2 cell populations tending to adopt the internal position. While limb bud cells form internal islands surrounded by liver cells in solid aggregates, the reverse was found to be the case in these monolayers. This suggests that, in the monolayer, limb bud cohesiveness is depressed relative to liver cell cohesiveness. This is consistent with the observation that the limb bud cells flattened themselves markedly against the substratum, significantly decreasing their area of mutual apposition.


Cell locomotion within a contact-inhibited monolayer of chick embryonic liver parenchyma cells. Using time-lapse filming, the relative movement of cells (nuclei) within a contact-inhibited monolayer of chick embryonic liver parenchyma cells has been studied. Two techniques were employed to determine the amount of relative cell movement during a culture period of 6 h. Firstly, the number of neighbours lost or gained by each nucleus was counted. Secondly, the relative distance moved by each nucleus in relation to other nucleus in the monolayer was measured. (The numerical results obtained from these analyses and details of the methods used are given in the text). A considerable amount of relative movement of nuclei within the monolayer was found during this period of culture. Although some gaps were occasionally seen between the cells in the monolayer, it was observed that cells able both to "ruffle" and to translocate when no gap was detectable; i.e. the cells appeared able to move while entirely surrounded by other cells. Because of this, we suggest that the monolayering of these epitheloid cells on a surface may be due to restriction of overlapping between them rather than to inhibition of movement by mutual contact. We argue that the term "contact inhibition of overlapping" relates to this behaviour better than the term "contact inhibition of movement".


New methods of quantitation in thin-layer chromatography: tubular thin-layer chromatography [TTLC]. Techniques used in the quantitative evaluation of thin-layer chromatograms are compared. The quantitation of tubular thin-layer chromatograms by means of vapor-phase detectors is emphasized, and typical examples of the use of this method are outlined. Applications of tubular thin-layer chromatography (TTLC) in the quantitative analysis of fish liver oils are described.


A review of the analysis of free fatty acids [C2-C6]. A large number of the techniques and methods used to analyze FFA[C2-C6] from the advent of GLC to the present are reviewed. Possible reasons as to why it has taken such a long time to establish an unequivocal quantitative method are suggested. The results of analyzing standard solutions of FFA by three different methods are compared, and that based on the addition of formic acid to the carrier gas is recommended.


Response to LH-RH and clomiphene citrate in two women with the Prader-Labhart-Willi syndrome. Two women with the Prader-Labhart-Willi syndrome are presented. The gonadotropin response to LH-RH administration was studied prior to, immediately following, and 6 months after a 6-week trial of clomiphene citrate, 200 mg per day for 21 days in divided doses, followed by 100 mg for  21 days in divided doses, followed by 100 mg per day for 14 days in divided doses, and followed by 50 mg per day in a single dose for an additional 14 days. During therapy, the basal gonadotropin and estradiol concentrations rose from prepubertal levels to those of mature women in midmenstrual cycle. However, 6 months after cessation of treatment, the basal gondadotropin and estradiol levels had returned to the prepubertal range. The initial response to LH-RH in the 2 patients differed in that one was clearly prepubertal and the other indistinguishable from the broad range of the adult normal response. The LY and FSH responses to LH-RH administration was greater after 6 weeks of clomiphene citrate therapy than they were either before (both patients) or 6 months after treatment (1 patient). We conclude that there is heterogeneity in the response to LH-RH administration in the Prader-Labhart-Willi Syndrome, just as there is in other syndromes of hypogonadotropic hypogonadism. A normal adult response of gonadotropins to the administration of LH-RH was acheived during clomiphene citrate therapy.


A computer program that evaluates patients with hypercalcemia. A computer program has been developed to assist the physician in managing patients with hypercalcemia. The program requests appropriate information, such as the patient's serum calcium concentration, age and x-ray results. Based on the abnormalities detected, the program asks additional questions needed to suggest the most helpful course of action for evaluating or treating the patient's hypercalcemia. As soon as the data have been entered, the program generates an evaluation note that includes diagnostic possibilities, suggestions for additional laboratory studies, therapeutic recommendations and references to the medical literature. The program can be used from any general purpose teletype-compatible terminal (including cathode ray tube displays) connected to the Bell Telephone System. The time required to enter the data and obtain the evaluation note is approximately 4 minutes with a teletypewriter, and less if a faster terminal is used. The cost for computer time is comparable to that of a laboratory test. Comparison of diagnoses produced by the program with those obtained from pathologic examination of from consulting endocrinolgists shows that the program is sufficiently accurate for clinical use. Finnally, the explanation of the pathophysiology contained in the evaluation note may be useful to the student.


Secretion of alpha subunits of luteinizing hormone (LH) by the anterior pituitary. Free alpha subunit chains of the glycopeptide pituitary hormones have been found in the sera of normal subjects and postmenopausal women. To ascertain whether the alpha subunit of LH is directly secreted by the pituitary or formed as a result of degradation of intact LY in the periphery, alpha subunits and intact LH were measured by radioimmunoassay in human volunteers after LRF stimulation and purified LH infusion. In 4 subjects a loading dose of 90 IU, followed by the infusion of 22.5 IJ of purified human LH over 30 min, produced peak serum LH levels of 41 mIU/ml but no change in alpha subunit levels of 35 IU of purified human LH to an additional 4 subjects, produced peak LH levels of 8* mIU/ml, but again, no change in alpha subunits. In the same two groups of subjects 100 mug of LRF produced peak LH levels of 25 mIU/ml and 75 mIU/ml, respectively, with significant alpha subunit elevations at 20 min of 1.7 ng/ml and 2.7 ng/ml, respectively. In separate groups of men LRF was administered over a wide dose range of 1 to 3,000 mug and LH and the alpha subunit measured. A dose-response curve existed over the entire LRF dose range for blood LH; no minimum or maximum plateaus were observed over the range studied. However, the alpha chain response appeared to reach a maximal plateau at a dose of 100 mug of LRF. The results are compativle with the hypothesis that the alpha subunits appearing in the peripheral circulation in response to LRF are due to secretion by the anterior pituitary and not due to peripheral degradation of intact secreted LH.


Secondary drug failure occurring during chronic treatment with LHRH: appearance of an antibody. Secondary drug failure with regard to subjective and hormonal responses occurred in a patient treated chronically with LHRH. This failure of response to hormone therapy was correlated with increased binding of 125I-LHRH by the patient's sera. Also, competitive displacement of 125I-LHRH by unlabelled LHRH was demonstrated. Affinity constants and titers of order appropriate for antibody were obtained. These data support strongly a report of antibody formation to exogenous LHRH in man.


Circulating polyribophosphate in Hemophilus influenzae, type b meningitis. Correlation with clinical course and antibody response. In systemic infections caused by Hemophilus influenzae, type b, the capsular polysaccharide, polyribophosphate, is released into the circulation. Polyribophosphate was quantitated in serial serum and cerebrospinal fluid samples from 45 children with H. influenzae, type b meningitis by means of a radiolabeled antigen-binding inhibition assay. Polyribophosphate was regularly found in acute serum and cerebrospinal fluid samples and could be detected in unbound form for periods of 1-30 days after initiation of effective therapy. Complexes of polyribophosphate dissociable with acid and pepsin were detected in serum samples from 17 patients, in one case for a period of 145 days after hospitalization. Polyribophosphate levels and patterns of clearance were studied in relation to hospital course and antibody response. Patients with prolonged antigenemia had protracted fevers and severe neurological symptoms during hospitalization, frequently with focal complications.Antipolyribophosphate antibody responses were detected during the first 100 days of convalescence by radioimmunoassay in 79% of the patients studied, including 60% of the children 1 yr or less in age. The intensity of antibody response although clearly related to the age of the patient, was more reliably predicted by the efficiency of antigen clearance. Antibody responses were uniformly of low magnitude in patients with prolonged antigenemia, irrespective of age. Paients who failed to develop antibody to polyribophosphate after meningitis also exhibited impaired antigen clearance. These studies suggest that mechanisms necessary for clearance of polyribophosphate may influence the development and intensity of the humoral immune response and raise the possibility of developmental deficiencies in the clearance system in infants and children.


A morphological basis for intercellular communication between alpha- and beta-cells in the endocrine pancreas. By degranulating beta-cells in the islets of Langerhans of the rat with sulfonylurea, it has been possible to distinguish unambiguously alpha-cells from beta-cells in freeze-fracture replicas. In such preparations, we found morphologically typical tight and gap junctions occurring between alpha- and beta-cells. The presence of gap junctions offers indirect evidence that these cells are coupled with one another; coupling may influence the secretory behavior of alpha- and beta-cells maintaining glucose homeostasis within tightly constricted limits.


Functional role of antibody against "core" glycolipid of Enterobacteriaceae. Antibodies against the "core" glycolipid of Enterobacteriaceae (2-keto, 3-deoxyoctonate-Lipid A) have been associated with protection against the sequelae of gram-negative rod bacteremia. To investigate the nature of this protection, dogs and rabbits were immunized with purified glycolipid prepared by phenol-chloroform-petroleum ether extraction of the "Re" mutant of Salmonella minnesota 595 and opsonophagocytic and bactericidal tests were carried out using lapine peritoneal granulocytes and serum factors. Whereas 1-4 mug/kg of glycolipid i.v. produced hypotension in dogs and 8 mug/kg i.v. was lethal, a rising dosage schedule of immunization with an average total dose of 1 mg/kg produced striking protection against shock and death against challenge with heterologous organisms. 20 control dogs, given approximately 10(10) live, serum-resistant Escherichia coli 0.85:H9 or Serratia marcescens 03 during a continuous intra-arterial pressure transducer recording, showed a drop in mean systolic pressure from 186 (+/- 6 SE) to 101 (+/- 12 SE) MM Hg and a fall in mean diastolic pressure from 118 (+/- 3 SE) to 64 (+/- 8 SE) mm Hg within 60-120 min. Minor pressure changes (average less than 12% of prechallenge levels) were seen in the same number of immunized dogs. In contrast, no significant difference was noted in the bloodstream clearance of these serum-resistant organisms over a period of 4-6 h between immunized and control dogs. Intravascular clearance was greater in animals immunized with the challenged strain or in glycolipid-immunized animals challenged with highly serum-sensitive E. coli 0.14:K7. Antibody against core glycolipid protected against the hemodynamic sequelae of bacillemia, augmented intravascular clearance of serum-sensitive organisms, and abrogated the pyrogenic response to enteric bacilli, but did not enhance clearance of serum-resistant organisms. Although canine and lapine antiserum against core glycolipid passively protected mice against a heterologous challenge, opsonophagocytic and bactericidal activity was at least 100-fold less than type-specific antiserum.


Behavior of eosinophil leukocytes in acute inflammation. II. Eosinophil dynamics during acute inflammation. The marked diminution in the number of circulating eosinophils, which has been shown to occur during acute bacterial infections, is a distinctive aspect of eosinophil physiology and of the host response to acute infection. The mouse rendered eosinophilic by infection with trichinosis provides a suitable model for study of the eosinopenic response induced by acute inflammation. The alterations in eosinophil dynamics associated with acute inflammatory reactions in trichinous mice were studied with pneumococcal abscesses, with Escherichia coli pyelonephritis, with Coxsackie viral pancreatitis, and with acute subcutaneous inflammation due to turpentine. Each of these stimuli of acute inflammation markedly suppressed the eosinophilia of trichinosis. This suggests that the eosinopenia is a response to the acute inflammatory process rather than the response to a specific type of pathogen. These studies apply quantitative techniques to ascertain the effects of acute inflammation on eosinophil production in bone marrow and on distribution of eosinophils in the peripheral tissues. From these observations, it is apparent that the initial response to acute inflammation includes a rapid drop in numbers of circulating eosinophils, a rapid accumulation of eosinophils at the periphery of the inflammatory site, and an inhibition of egress of eosinophils from the bone marrow. With prolongation of the inflammatory process, inhibition of eosinopoiesis occurs.


Hereditary deficiency of the seventh component of complement. Deficiency of the seventh component of complement has been found in the serum of a 42-yr-old Caucasian woman who has Raynaud's phenomenon, sclerodactyly, and telangiectasia. Partial deficiency was found in the serum of the patient's parents and children, indicating a pattern of inheritance of autosomal codominance. Transfusion experiments indicated that exogenous C7 had a 91-h halk-life in the patient. There was no evidence for C7 synthesis after transfusion. No C7 inhibitors were detected in the patient's serum. The patient's serum was found to support the activation of complement by both the classical and properdin pathways to the C7 stage. The addition of C7 to the patient's serum permitted it to support hemolytic reactions initiated by either pathway. No defects could be detected in plasma or whole blood coagulation. The patient's serum was deficient in opsonizing unsensitized yeast particles in serum and in the generation of chemotactic factor by antigen-antibody complexes and endotoxin. Both deficiencies were corrected by the addition of C7. These observations suggest a key role for C7 for in vitro yeast phagocytosis and chemotaxis generation. However, the patient's lack of infections indicates a relatively minor role for C7 in human resistance to infection.


An animal model of hypersensitivity pneumonitis in the rabbit. This study was devised to produce an animal model of hypersensitivity pneumonitis in order to study both the induction and the elicitation of the disease. Rabbits exposed by aerosol to large quantities of pigeon antigens developed a humoral, but not cellular, immunologic response. Moreover, their lungs were essentially normal histologically. A single i.v. injection of killed BCG in oil permitted the induction of pulmonary cell-medid hypersensitivity to the inhaled antigen, as well as the development of pulmonary lesions which were more severe than that caused by the administration of BCG alone. The humoral immunologic response to the inhaled antigen was not increased after BCG injection. Since many individuals are exposed to the etiologic agents of hypersensitivity pneumonitis for extended periods without developing the disease, these findings in animals suggest that some event may occur to induce cell mediated hypersensitivity in order to initiate the disease process. In addition, we have shown that animals with normal lung histology and circulating complement-fixing antibodies undergo serum complement (CH50) depression after an aerosol challenge with the specific antigen. Animals with circulating, complement-fixing antibodies, and inflamed lungs (BCG-induced failed to undergo a complement depression subsequent to an aerosol challenge with specific antigens. These results re consistent with those seen in symptomatic and asymptomatic pigeon breeders and suggest that antigen distribution through the lung is important in the pathogenesis of hypersensitivity pneumonitis.


Lactational responses to postruminal administration of proteins and amino acids. Rumen by-pass of nutrients has improved performance of lactating dairy cows, growing stepers, and sheep. Postruminal supplementation of casein has increased milk production 1 to 4 kg per cow per day and milk protein yield by 10 to 15%. Component(s) in casein eliciting the improved performance have not been determined adequately. Glucose, amino acids, hormone secretion, or a combination of these factors may be responsible for the improved performance. Future research to delineate limiting nutrients for postruminal supplementation should prove fruitful and should make a major contribution to improved production by ruminant animals.


Rumen bypass and protection of proteins and amino acids. Potent rumen microbial proteases and deaminases rapidly degrade protein and amino acids which are soluble in the rumen liquid phase. Because protein sources vary in their solubility, the degree of degradation in the rumen is variable. Methods of decreasing protein and amino acid degradation in the rumen include heat treatment, chemical treatment, encapsulation, use of amino acid analogs, selective manipulation of balances of rumen metabolic pathways, and esophageal groove closure. It is important that procedures do not interfere with ruminal metabolism or post-ruminal digestion. Bypassing the rumen changes sites in the digestive tract of nutrient digestion and absorption and provides a mechanism for supplementing outflow of nutrients from the rumen. A feasible approach to production of animal protein from ruminants would be utilization of nonprotein nitrogen for rumen protein production, maximization of rumen bypass of dietary protein, and supplementation with rumen nondegradable amino acids.


Nitrogen requirement and utilization in dairy cattle. Formulation of dairy cow rations should consider the following points regarding nitrogen utilization by lactating cows. (a) Maintenance of ruminal ammonia nitrogen in excess of 5 mg/100 ml rumen fluid has no effect on microbial protein production. (b)Supplemental nonprotein nitrogen is not utilized in typical dairy and feedlot beef rations containing more than 12 to 13% crude protein(dry matter basis). (c)Nonprotein nitrogen is approximately equal to true protein as a source of nitrogen in typical dairy and feedlot rations containing not more than 12 to 13% crude protein. (d)A scheme based upon metabolizable protein (absorbable protein) for calculating requirements and comparing protein sources is superior to crude or digestible protein designations. Ultimate expression of the requirement may be in terms of crude protein for the sake of simplicity. (e)One kilogram of crude protein, regardless of nitrogen source, equals about .75kg metabolizable protein in typical dairy and feedlot beef rations containing not more than 12 to 13% crude protein. One kilogram of plant protein (true protein) fed in excess of an amount equivalent to 12 to 13% dietary protein equals about .3 kg metabolizable protein. (f)Protein supplementation of lactating cows might be related more to stage of lactation than to milk production. (g)Lactating cows having above average lactational ability may benefit from dietary protein as high as 16 to 17% (dry matter basis) during the first third of lactation. (h)Cows in the latter two-thirds of lactation appear to require 12.5% dietary protein or less. (i)Plant protein (true protein) should be the supplemental sources of nitrogen during the first third of lactation, with NPN providing most, if not all, the supplemental nitrogen during the last two-thirds of lactation.


Juvenile hysteria and primary shock: report of case. Hysterical aberrations may occur from circumstances other than fear and anxiety in a dental office. Irreversible shock from hysteria is well documented. It is important that causative factors of hysterical experiences be properly and promptly identified, and adequate measures be taken to allay these factors so that permanent adverse effects of the hysteria are aborted.


Surveying.

Surveying. Several systems for the survey and design for removable partial dentures are based on empiricism and misinformation. Presented here is a systematic approach to the basic requirements for precribing a prosthesis that will be received favorably by the patient.


Effect of humidity and temperature changes on orthodontic direct-bonding adhesive systems. Plastic brackets were bonded to 560 extracted human teeth with use of two orthodontic adhesive systems: (1) methyl methacrylate resin bonded to a sealant that was polymerized using ultraviolet light, and (2) self-polymerizing methyl methacrylate resin bonded directly to the etched enamel. Tensile and shearlike tests were performed. Prolonged exposure to heat, moisture, and severe temperature changes decreased the shearlike strength of both adhesives. Both systems were adequately strong to withstand routine orthodontic and estimated masticatory forces.


An outbreak of dysentery caused by Shigella dysenteriae type 1 on a coral island in the Bay of Bengal. An epidemic of severe dysentery occurred on St. Martin Island in the Bay of Bengal at a time when the island, with a population of 1,318, was isolated from the mainland because of the monsoon season. There were 434 cases and 28 deaths within a three-month period. Mortality (2%) was limited to the very young and the elderly. Shigella dysenteriae type 1 (Shiga bacillus) was isolated from 12 of 65 rectal swab specimens. All strains gave similar biochemical reactions and were resistant to tetracycline, chloramphenicol, streptomycin, and the sulfonamides but were sensitive to ampicillin, kanamycin, and gentamicin. No other organism was implicated in the outbreak. The disease disappeared from the island after institution of chlorination of the sources of drinking water and effective treatment of patients.


Effect of radiation-induced xerostomia on human oral microflora. A longitudinal study was performed to assess the effects of radiation-induced xerostomia on the human oral microflora. Pronounced microbial population shifts were found in each of five intraoral sites tested. Cariogenic microorganisms gained prominence at the expense of noncariogenic microorganisms in concert with the saliva shutdown. These changes occurred before the onset of clinical caries irrespective of whether or not a topical fluoride gel was used as a caries preventive.


Asymptomatic epidemic acquisition of group A Streptococcus: antibody response to extracellular and type-specific antigens. The antibody response of 40 institutionalized children involved in an epidemic associated with asymptomatic pharyngeal acquisition of a group A, M-11, T-11 typeable Streptococcus was studied. Titers of antibody to streptolysin O and to deoxyribonuclease B determined in sera collected from patients within two weeks of positive throat cultures were significantly higher than those in sera of controls (P smaller than 0.001). However, there was no rise in antibody titers in sera obtained from these patients after an interval of three weeks. Type-specific antibody to the group A Streptococcus (type M-11) was assayed in the sera of 24 patients. No detectable antibody activity was found either in the initial sera or in sera collected eight months after the epidemic. Thus, the asymptomatic nature of this epidemic could not be attributed to the presence of detectable type-specific antibody in this population at the time of the epidemic. These observations suggest that asymptomatic pharyngeal acquisition of group A Streptococcus may occur in epidemic fashion in certain populations and may not be associated with evidence of an antibody response to the streptococcal organism.


Susceptibility of coccidioides immitis, Candida albicans, and Cryptococcus neoformans to amphotericin B, flucytosine, and clotrimazole. Toxicity and failure of treatment with amphotericin B are stimuli for researchers to evaluate alternative antifungal antimicrobics. Also, data from susceptibility tests of Coccidioides immitis are sparse. With use of a defined, synthetic culture medium, C. immitis (25 strains). Candida albicans (21 strains), and Cryptococcus neoformans (21 strains) were tested against flucytosine, clotrimazole, and amphotericin B. Molecule for molecule, the sequency of activity was: clotrimazole greater than amphotericin B greater than flucytosine (totally inactive) C. immitis; and clotrimazole greater than amphotericin B greater than flucytosine with C. albicans and C. neoformans. With four strains of C. immitis, the minimal inhibitory concentration (of amphotericin B) was the same when inocula of arthrospores were tested as when corresponding spherules/endospores were tested simultaneously and identically. The clinical outcome of coccidioidomycosis in 17 patients treated with amphotericin B correlated best with minimal inhibitory concentration after incubation of cultures for 48 hr; a favorable response was associated with minimal inhibitory concentrations of less than or equal 1.0 mug/ml. Because clinical isolates of fungi appear to vary in susceptibility, in vitro tests may have clinical utility.


Transmissible drug resistance of Escherichia coli and Salmonella from humans, animals, and their rural environments. Isolates of Escherichia coli and Salmonella species obtained from humans, animals, and their rural environments were tested for their ability to transfer multiple drug resistance to competent recipients. In all, 125 (52.9%) of 242 isolates of E.coli transferred all or part of their resistance to E.coli CSH-2, and 103 (44.4%) of 232 isolates of E.coli transferred all or part of their resistance to Salmonella choleraesuis var. kuzendorf 214. The data suggest that the multiple drug resistance of E.coli transferred all or part of their resistance to Salmonella choleraesuis var kuzendorf 214. The data suggest that the multiple drug resistance of E. coli in the gut of humans who work with livestock may be due to the transfer of resistance determinants from animal strains to the resident flora of the human gut. Such transfer could occur during transient passage through the digestive tract. Multiple drug-resistant Salmonella were isolated from all sources. In all, 48 (80%) of 60 isolates of Salmonella transferred all or part of their resistance to E. coli CSH-2, and isolates of Salmonella transferred all or part of their resistance to E.coli CSH-2, and 42 (82.4%) of 51 isolates of Salmonella transferred all or part of their resistance to S. choleraesuis var, kuzendorf 214. Fifty-three (46.9%) of 113 antibiotic-sensitive isolates of E.coli possessed resistance transfer factor, and six (23.1%) of 26 isolates with resistance transfer factor mobilized resistance determinants in isolates unable to transfer drug resistance during conjugation experiments.


Colicin types of Shigella sonnei in relation to antibiotic resistance. Colicin typing of 436 strains of Shigella sonnei isolated in Dallas during a 10-year period was performed to determine whether resistance to ampicillin was associated with a single strain or was widespread among all S. sonnei types. One hundred ninety-three strains collected during a period of eight  and one-half years when ampicillin resistance was 1.6% were available for retrospective review. During the last one and one-half years of study, 20.6% of the 243 strains that represented almost all S. sonnei isolated in Dallas were resistant to ampicillin. More than 50% of the strains collected annually were colicin type 9,22% were untypable, and seven other types were less frequently encountered. Resistance to ampicillin increased slightly from 1.5% to 9.5% among the type 9 strains, and one type 8 strain (2.9%) was resistaiple resistance to seven antibiotics tested was found mainly in untypable strains. Thus it appears that the abrupt increase in ampicillin resistance in mid-1972 was due principally to resistance in a single biotype, and that resistance is not widespread among all types of S. sonnei.


Regression of cutaneous neoplasms following delayed-type hypersensitivity challenge reactions to microbial antigens or lymphokines. Induction of delayed-type hypersensitivity challenge reactions to microbial antigens at sites of neoplasms involving the skin resulted in regression of mycosis fungoides, reticulum cell sarcoma, superficial basal cell carcinoma and adenocarcinoma of the breast. Similar reactions induced by lymphokine preparations also resulted in regression of lesions of mycosis fungoides and superficial basal cell carcinoma. The role of the large monomuclear cells in the inflammatory infiltrate of the delayed hypersensitivity reaction in eliciting the tumor regression is discussed. It is proposed that these large mononuclear cells represent the effectors in a primitive surveillance mechanism for neoplastic cells.


VECTAN II: A computer program for the spatial analysis of the vectorcardiogram. This paper presents the operation of a digital computer program, VECTAN II, for the spatial analysis of the vectorcardiogram (VCG). The program incorporates a unique waveform recognition algorithm based on the spatial vector length which has been shown to perform better than previous algorithms. The waveform analysis employed by the program considers the vectorcardiogram as a three dimensional entity rather than as scalar or planar representations. VECTAN II is designed chiefly to measure and quantify the VCG response of normal subjects to a controlled stress by analyzing one VCG complex every five seconds throughout a long experiment. The program has been used to analyzeom the NASA Johnson Space Center Cardiovascular Laboratory, from the pre- and postflight medical examinations of the Apollo 15, 16 and 17 crewmen, and from onboard Skylab experiments.


The production of a temperature-sensitive persistent measles virus infection. A persistent infection of measles virus was established in HEp2 cells. All cells contained virus antigen when tested by specific immunofluorescence and approx. 50% were positive by haemadsorption. Infectious virus released into the supernatant medium was usually equivalent to no more than 0-001 p.f.u./cell, but between 10 and 40% of the infected cells produced plaques when plated on Vero cells. Passage of persistently infected cultures in the presence of measles antibody had no effect on the proportion of antigen-positive cells. Virus obtained from the supernatant medium of persistently infected cultures was temperature sensitive at 39-5 degrees C when tested on Vero cells whereas the original non-persistent virus produced infections on Vero cells at 39-8 degrees C. On passage of the persistently infected culture at 39-5 degrees C most of the surface antigens disappeared within 24 h whereas the intracellular virus antigens had not totally disappeared until the 5th passage.


Beta blockade in lithium tremor.

Beta blockade in lithium tremor. Practolol, propranolol, and placebo have been tested on an objective test of lithium induced tremor. Both beta-blocking agents produced significantly more tremor than the placebo. It is argued that lithium induced tremor is closer to essential than to physiological tremor.


Apomorphine and psychopathology.

Apomorphine and psychopathology. Forty men, mainly alcoholics, were administered either the dopamine receptor agonist, apomorphine HCl (1 mg), or distilled water subcutaneously three times a day for 14 days in a double blind study. None of the subjects developed an endogenous depression or schizophrenic symptoms. Scores on the Hamilton Rating Scale, Zung Self Rating Scale, and Brief Psychiatric Rating Scale showed improvement with both apomorphine and placebo. There were no significant differences between the two treatments on these rating scales. A significant incidence of spontaneous penile erections occurred after apomorphine treatment compared with placebo. Both treatments eliminated subjective craving for alcohol. Acute administration of apomorphine had no effect on psychomotor retardation or depressed mood in two patients with endogenous depression.


Threshold for pain from anterolateral quadrant stimulation as a predictor of success of percutaneous cordotomy for relief of pain. A sensitive quantitative index for predicting optimal electrode position in percutaneous anterolateral cordotomy was determined by electrical stimulation through the lesioning electrode. If the threshold for pain elicited by the stimulation electrode was less than 300 muA, a 5-sec radiofrequency lesion of 50 mA would produce complete contralateral analgesia with no weakness. When the pain threshold exceeded 300 muA, incomplete or no analgesia would result with the standard single lesion. The results further suggested that the fibers in the anterolateral quadrant that transmit pain are discretely rather than diffusely localized.


Meningomyelocele and progressive hydromyelia. Progressive paresis in myelodysplasia. Five cases of myelodysplasia with progressive paraparesis are presented. Three of the five patients developed spasticity, but dissociated sensory loss and loss of sphincter control was not a prominent feature. All were found to have compensated hydrocephalus and extensive communicating hydromyelia. The use of myelography and ventriculography in the diagnosis of hydromyelia is discussed. Ventricular drainage led to clinical improvement in two cases and radiological improvement in one. The relationship of compensated hydrocephalus, meningomyelocele, and progressive hydromyelia postnatally may support the hydrodynamic hypothesis of myelodysplasia.


Effect of ascorbic acid on the human electroencephalogram. Controversy exists over whether ascorbic acid (vitamin C) in doses in excess of known physiological requirements has demonstrable biological effects. The present study reports that such megadoses of vitamin C do significantly effect an objective electrophysiological measure, i.e., electroencephalogram (EEG) driving responses to photic stimulation. Four thousand milligrams of vitamin C administered 27 and 3 hours prior to assessment produced an effect on the EEGs of 18 healthy adolescent human males that was significantly different from a placebo. Fifty milligrams of ascorbic acid administered in this manner did not produce an effect on the EEG significantly different from placebo administration.


The histopathology of lepromatous leprosy in the nose. On the basis of clinical, histological and bacteriological assessments, 31 patients in Central India were selected and classified as having active but early lepromatous leprosy and 4 patients as having early borderline leprosy. From the nose of each patient an average of 4 biopsies were taken from particular sites of the septum and turbinates either by punch biopsy or dissection with a scalpel. The nasal tissues from all the lepromatous patients contained many acid-fast bacilli; no bacilli or abnormalities were seen in nasal tissues from the borderline patients. The histopathology of these highly bacilliferous tissues is described. Bacilli were universally seen in macrophages, but they were also seen in blood monocytes and polymorphs, fibroblasts, squamous and pseudo-columnar epithelium, keratin, peri- and endo-neurial cells of tiny nerve bundles, erectile tissue, vascular plain muscle, perivascular histiocytes and frequently and abundantly in the cytoplasm of endothelial lining cells of lymphatics and of small blood vessels and free within the lumina of these vessels. Five basic mechanisms of escape of bacilli from the submucosa on to the surface, and thus into the external environment, are described. Secondary infection, in the presence of an expansile lepromatous infiltrate, together with simple trauma to the surface epithelium, are the main factors in the discharge of bacilli. These histopathological observations are consistent with the findings from other recent studies on the nose in leprosy regarding (1) the large numbers of morphologically intact and viable Myco. leprae excreted in the nasal mucus of lepromatous patients; (2) the clinical changes observed in the nose of such patients, and (3) the similar nasal involvement and excretion of Myco. leprae from the nose of mice inoculated with leprosy bacilli of human origin. Of particular interest was the frequency and intensity of bacilli within the endothelial lining cells of small blood and lymph vessels and the presence of bacilli free within the lumina of these vessels or within monocytes and polymorphs. The possible dynamic significance of these observations in the pathogenesis of leprosy is discussed. The significance of all these observations in relation to (1) the spread of leprosy; (2) local factors in the nose which might favour the growth of Myco. leprae, and (3) the nose as a portal of entry, are discussed.


Viral hepatitis with intracytoplasmic cholestasis and cholesterol crystals in a drug addict. Electron-microscopical exammination of a liver biopsy from a young female drug addict with clinically relapsing serum hepatitis revealed virus-like particles free in the liver cell cytoplasm. In addition, there was intracellular cholestasis, and numerous clefts were found in the cytoplasm of hepatocytes, presumably caused by the presence of cholesterol crystals which were dissolved during the processing of the tissue. Such clefts have earlier been reported in liver cells in patients with cholestasis and hypercholesterolemia, but not in patients with viral hepatitis and a normal serum cholesterol concentration.


Yeast colonization in hospitalized and nonhospitalized children. Data have been accumulated to determine the prevalence of yeast colonization of the skin and digestive tract of hospitalized and nonhospitalized infants and children. There was no difference in the prevalence between hospitalized patients at the time of admission and nonhospitalized children. However, there was a positive correlation of the duration of hospitalization and the prevalence of yeast colonization. There were no correlations of hospitalization with site of colonization, age of the patient, or type of yeast isolated.


Early versus late introduction of continuous negative pressure in the management of the idiopathic respiratory distress syndrome. To evaluate the effectiveness of the early application of continuous negative pressure about the thorax, 23 infants with the idiopathic respiratory distress syndrome who had adequate alveolar ventilation were assigned to early or late application of CNP. There were 11 infants in the early CNP and 12 in the late CNP group. There were no differences between the two groups with respect to birth weight, gestational age, age at admission to the study, initial FIO2 requirements, or initial PaO2, PaCo2, and AaPO2. None of the ENCP patients required mechanical ventilation, whereas four of the LCNP group did so. The FIO2 requirements were significantly less for the ECNP infants when compared to the LCNP ones. The mean duration of FIO2 requirements greater than 0.6 was 28.3 hours for those receiving ECNP and 60.7 hours for those in LCNP (P LESS THAN 0.05). This study suggests that the course of RDS may be modified by the early application of CNP.


Traumatizing effect of breech delivery on infants with spina bifida. About one quarter of babies with myelomeningocele are born by breech delivery. Dissection of the locomotor system tissues of 49 spina bifida babies born by normal delivery and 15 spina bifida babies born by breech delivery revealed that the latter type of labor causes frequent and often severe trauma to the already handicapped muscles and nerves of the lower extremities. In cases where the birth damage is extensive, the baby's chances of survival may be reduced. The possibility should be considered that this type of labor, which traumatizes the baby's muscles and nerves, may influence the postnatal locomotor function and further contribute to the child's disability.


Glucocorticoid prophylaxis for respiratory distress syndrome: a review of potential toxicity. Much evidence suggests that glucocorticoids may increase the rate of lung development during late fetal life, and thereby decrease the risk of respiratory distress syndrome if the fetus is born prematurely. Results of human and animal studies are basically in agreement. The risks of this form of therapy are less well examined, especially in human studies. This review briefly outlines studies that relate to toxicity so that the "risk/benefit ratio" of this form of therapy may be assessed.


Osseous autografts. I. Clinical responses to bone blend or hip marrow grafts. A clinical evaluation was undertaken to compare regeneration of osseous defects following implantation of either osseous coagulum-bone blend from intraoral sources or autogenous iliac marrow-cancellous bone. Thirty-two transplants were performed in 15 male patients. The intraosseous defects in which marrow was placed (initial average depth = 7.18 mm) filled 60.7% (average fill = 4.36 mm). Defects in which osseous coagulum-bone blend was placed (initial average depth 4.0 mm) filled 73% (average fill = 2.93 mm). The difference in results between the two materials was not statistically significant. Therefore, similar levels of osseous regeneration apparently took place regardless of graft material used.


The clinical and histological response of periodontal pockets to root planing and oral hygiene. The study evaluated, clinically and microscopically, the soft tissue response of suprabony periodontal pockets treated by root planing and oral hygiene or by oral hygiene measures alone. The participants were 22 adults beginning treatment for inflammatory periodontal disease. For each patient, three clinically similar labial or lingual pockets were scored for gingival inflammation, pocket depth, and the extent of plaque and calculus. One pocket in each patient served as a control, and a gingival biopsy was obtained to determine the pretreatment level of inflammation. The second pocket was treated by root planing and by the patient's oral hygiene measures. The third pocket was treated only by the patient's daily oral hygiene measures. Fifty-six to 63 days later, treated areas were reevaluated clinically, and biopsies were secured. Each type of therapy reduced the mean pocket depth and the incidence and severity of gingivitis; however, root planing accompanied by oral hygiene measures resulted in a statistically greater improvement than did oral hygiene measures alone.


Cryopreservation of Trypanosoma cruzi bloodstream forms. Recovery rates of T. cruzi bloodstream forms subjected to several methods of cryopreservation in liquid nitrogen and at -73 C are reported.  Inoculations of animals with cryopreserved and nonpreserved trypomastigotes revealed that prolonged storage at -196 C apparently did not change the biologic characteristics of different T. cruzi strains.  The reproducibility and consistency of results suggest that "cryobanks" or "reference centers" may be established.


Ovulation induction and antifertility effects of an LH-RH analogue (AY 25,205) in cyclic rats. Pro-oestrous rats, treated with fluphenazine dihydrochloride to block ovulation, were used to compare the ovulation-inducing activity of synthetic LH-RH with one of its analogues, [D-Ala-6, des-Gly-NH2-10]-LH-RH ethylamide (AY-25,205). The lowest dose of LH-RH which produced a significant response was 125 ng compared with 2.9 ng AY-25,205. Statistical analysis of the data showed that AY-25,205 was approximately thirty-six times more potent than LH-RH. Treatment with AY-25,205 also induced partial ovulation in metoestrous rats (80 or 160 ng/rat) and apparently normal ovulation in dioestrous rats (10 to 160 ng/rat). The compound (160 ng) failed to induce further ovulation in oestrous rats which ovulated during the previous night. Advancement of premature ovulation in dioestrous rats with AY-25,205 prevented mating behaviour and pregnancy during the treatment cycle. The antifertility effect of the compound disappeared during the following cycle.


Cellular and subcellular compartmentalization of steroid metabolism in the rat testis. The following aspects are discussed in relation to the cellular and subcellular localization of steroid metabolism in the rat testis: (1) the localization of enzyme activities for steroid production in interstitial tissue and seminiferous tubules in intact cells and in isolated mitochondrial fractions: (2) steroid production by isolated cellular and subcellular fractions and by the intact testis in vivo; and (3) the effect of cofactors on steroid production by rat testis preparations in vitro. It is concluded that it is difficult to decide whether steroid production and the enzymes involved in steroid metabolism are restricted to specific compartments. Steroid production by the testis can, however, be quantitatively explained by that occurring in the isolated mitochondria of the interstitial tissue.


Antimalarial phenanthrene amino alcohols. 3. Halogen-containing 9-phenanthrenemethanols. A series of new 9-phenanthrene amino alcohols has been prepared in which each compound bears from one to five halogen or halogen-containing moieties. A number of these compounds are extremely active against Plasmodium berghei in the mouse. Some structural requirements for optimal efficacy are considered.


Synthesis and antibacterial evaluation of 1,2,3,4-tetrahydro-4-oxo-1,8-naphthyridine-3-carboxylic acid esters, carbonitriles, and carboxamides. A series of 1,2,3,4-tetrahydro-4-oxo-1,8-naphthyridine-3-carboxylic acid esters, carbonitriles, and carboxamides (2a-k) was synthesized and initially evaluated (dose range 50-400 mg/kg) in mice infected with Escherichia coli. Only two derivatives, the ethyl and butyl esters of 1-ethyl-1,2-dihydro-4-hydroxy-7-methyl-1,8-naphthyridine-3-carboxylic acid, protected the animals against E. coli and several other gram-negative bacterial pathogenic infections. A pro-drug type of mechanism appears to be operable since neither agent showed in vitro activity.


Inhibitors of protein synthesis. 4. Studies on the structure-activity relationship of gougerotin and some of its analogs. Seventeen structural analogs of gougerotin have been compared with the parent compound as inhibitors of the growh of E. coli B and as inhibitors of N-acetylphenylalanylpuromycin formation. The analogs comprise compounds with (1) an intact sarcosyl-D-seryl moiety and modifications at various positions of the carbohydrate moiety, (2) compounds which lack the peptidyl residue, and (3) compounds in which the sarcosyl-D-seryl moiety has been replaced by other aminoacyl or peptidyl moieties and in which various sites in the carbohydrate moiety have been modified in addition. These comparisons revealed that (1) the 3'-hydroxyl group makes a strong contribution to inhibitory activity, (2) the introduction of a double bond between the 2' and 3' positions of the carbohydrate moiety causes a marked decrease in activity; however, if in addition the sarcosyl-D-seryl moiety is replaced by the epsilon-N-methyl-beta-L-arginyl side chain, full activity is restored, (3) there is a marked degree of stereospecificity around the alpha carbon of the seryl moiety (replacement of the sarcosyl-D-seryl residue with the sarcosyl-L-seryl residue results in greatly reduced activity), and (4) replacement of the carboxamide group at position 5' with a hydroxymethyl function resulsts in reduced activity which is further decreased by alterations in the sarcosyl-D-seryl moiety. These alterations include (a) replacement of the D-seryl residue of the sarcosyl-D-seryl moiety by the D-alanyl or the D-phenylalanyl residues and (b) replacement of the whole sarcosyl-D-seryl side chain by the D-seryl, D-alanyl, D-phenylalanyl, p-methoxy-D-phenylalanyl, or p-methoxy-L-phenylalanyl moieties; the replacement of the sarcosyl-D-seryl side chain with the various aminoacyl residues did not lead to acceptor (puromycin-like) activity with N-Ac-Phe-tRNA as the donor.


1(((5-Nitrofuranyl)methylene)amino)-4 and/or-5-substituted 2-imidazolidinones. A series of 1-[[(5-nitrofuranyl)methylene]amino]-4- and/or -5-substituted 2-imidazolidinones was prepared utilizing three different reaction sequences. The structure of 4, the product derived from 4-methyl-2-imidazolidinone (2a), was verified by synthesis using an alternate, unequivocal route. The levo isomer l-4 was prepared by a series of reactions starting with L(+)-2-amino-1-propanol (l-10). All of the nitrofurans were examined for potential use as chemotherapeutic agents for urinary tract infections. Based on the high level of activity in the urine and the in vitro antibacterial activity (MIC) 4, l-4, and 16 are considered to be the most active as urinary tract agents.


Preparation and biological activity of some aminoacyl and peptidyl derivatives of 2'-amino-2'-deoxyuridine. Several aminoacyl and peptidyl derivatives of 2'-amino-2'-deoxyuridine have been synthesized. 2'-Amino-2'-deoxyuridine (I) and 2'-N-glycyl-2'-amino-2'-deoxyuridine (IIIa) were condensed with N-Cbz blocked p-nitrophenyl esters of the appropriate amino acids to afford the protected aminoacyl and peptidyl nucleosides. Removal of the protecting groups gave the title compounds in good yields. The biological effects of these derivatives are discussed.


A simulation laboratory for medical education. Simulation as an instructional technique is being utilized for the learning of clinical skills in the training of health professionals. The Center for Educational Development at the University of Illinois College of Medicine has established a Learning Resource and Development Center to assist medical faculty in the development and evaluation of instructional simulations. These are not designed to replace clinical experiences but to provide additional opportunities for students to strengthen skills in patient examination. Prior to the introduction of these learning tools in 1972, a study was undertaken to validate their instructional effectiveness. After an additional two years of operation and experimentation, a revalidation study was undertaken during the fall of 1974. Improvements are continually being made in the various simulation formats as a result of feedback from students and faculty.


Interaction of ethidium bromide with the transport system for monovalent cations in yeast. Ethidium was found to be taken up by yeast cells in a process that, at certain concentrations has the main following characteristics: a) a substrate is required; b) it presents cooperative kinetics, with n, according to the Hill equation approximately 3; c) ethidium can be concentrated more than 100-fold; d) the uptake is inhibited by Ca2+; e) the uptake of the dye is inhibited by monovalent cations with a selectivity pattern similar to that observed in their transport by yeast; f) ethidium inhibits the uptake of K+, and, at concentrations up to about 250 muM produces a competitive inhibition on the uptake of Rb+; and g) ethidium produces the same effects as K+ on respiration and the extrusion of H+. It is concluded that ethidium is taken up by yeast cells in a selective way by the same transport system normally employed for monovalent cation uptake.


The origin of mitochondria.

The origin of mitochondria. The endosymbiont and episome theories about the origin of mitochondria are reviewed.  Biochemical and genetic data, relevant to these theories are discussed.  An alternative theory is also proposed; this theory is that nuclear and mitochondrial DNAs developed from compartmentalized duplicate prokaryote DNAs.


Foreign-body tumorigenesis: in vitro isolation and expansion of preneoplastic clonal cell populations. Foreign-body reactions were induced in coisogenic CBA/H and CBA/H-T6 mice by sc implantation of 15 times 22 times 0.2-mm unplasticized vinyl chloride vinyl acetate copolymer films. At 6 months' post implantation, implants and unopened tissue capsules were transferred to recipient animals of the T6-different partner strain. After another 3 months, part of the film/capsule complex was transferred to (C57BL/10ScSn times CBA/H-T6)F1 mice for tumor development. Capsule-derived and film-attached cells of the other part were separately cultured. Cultures consisting initially of euploid cells were often gradually replaced by different cells with specific aneuploid karyotypes which were identical with, or closely related to, those of the corresponding tumors. The cultured cells implanted in hybrid recipients at different passage numbers frequently gave rise to homologous tumors. Hence, it was possible to prepare in vitro cells with prefixed specific tumor determinants at different stages of preneoplastic maturation.


Clinical applications of free flap transfer in the burn patient. Free microvascular flap transfer is indicated in situations in which flap coverage is required and no local flap is available. The technique of transfer of a groin flap based on the superficial circumflex iliac vessels was described. The procedure has been applied to two patients with acute electrical burns with extensive soft-tissue loss, three patients with scar contractures, and one with osteoradionecrosis. Satisfactory results were achieved in all patients. The procedure is contraindicated if recipient or donor vessels are abnormal or inadequate, and in the face of fulminating infection, general debility, and obesity. With scrupulous attention to indications and contraindications, the procedure is safe, reliable, and a valuable addition to the surgeon's armamentarium.


Clinical evaluation of the carbon dioxide laser for burn wound excisions: a comparison of the laser, scalpel, and electrocautery. The use of a 100 watt carbon dioxide laser for surgical excisions of burn eschar was evaluated in 13 patients with symmetrical burns. In each patient, laser excision was compared with either scalpel or electrocautery excision of a comparable area. Graft "take," blood loss, and operative speed were measured. Graft "take" following laser excisions was comparable to that obtained when either scalpel or electrocautery was used. Regression analysis indicates that laser excisions involved 29% of the blood loss encountered with scalpel excisions and that laser speed was 1.49 times scalpel speed. Although laser blood loss was 60% of electrocautery blood loss, laser speed was significantly slower: 73% of electrocautery speed. These differences, however, were of small clinical consequence. It is felt that the surgical arm of this laser is at present quite awkward and is in need of technological refinement if the full potential of the laser is to be realized.


Regulation of early mRNA synthesis after bacteriophage T4 infection of Escherichia coli. Regulation of T4-specific mRNA synthesis was studied during leucine starvation of a leucine-requiring stringent Escherichia coli B strain. This was done by imposing starvation prior to T4 infection and then letting RNA synthesis proceed for different time periods. Rifampin or streptolydigin was added to stop further RNA synthesis, and protein synthesis was restored by addition of leucine. Samples were withdrawn at different times, and the enzyme-forming capacities found that, during conditions which elicit the stringent response in uninfected bacteria, immediate early mRNA is not stringently regulated. This conclusion contradicts the earlier conclusion of others, obtained by measuring incorporation of radioactive uracil; this is explained by  the observation of Edlin and Neuhard (1967), confirmed and extended by us to the T4-infected cell, that the incorporation of uracil into RNA of a stringent strain is virtually blocked by amino acid starvation, whereas that of adenine continues at 30 to 50% of the rate seen in the presence of the required amino acid.


In vitro construction of different oligomeric forms of lambdadv DNA and studies of their transforming activities. Plasmid lambdadv1, which is in a dimeric form, was converted to a linear monomer duplex by the action of EcoRI restriction endonuclease that incises at a unique site in this plasmid genome. The resulting products were then joined by Escherichia coli DNA ligase to produce molecules with various oligomeric forms, and from these monomeric, dimeric, or trimeric circular molecules were purified. By transformation of cells with these DNAs, clones were obtained that carried lambdadv1 in a monomeric or dimeric form. The former type of clones have not been generated in vivo, except for one in a different host strain, and carriers of timeric or tetrameric lambdadv1's have not been obtained so far. It was observed that a considerable fraction of these oligomeric circular DNAs were converted to lower oligomers (e.g., from trimer to dimer) during transformation. The characteristics of the monomeric lambdadv1 carriers obtained were compared with those of dimeric lambdadv1 carriers. The stabilities of the plasmids of the two forms were the same. However, the monomeric plasmid carriers were less tolerant to lambdavir phage infection and perpetuated about 30% less plasmid genomes in monomer units. Furthermore, dimeric plasmid carriers appeared spontaneously and accumulated in cultures of the monomeric lambdadv1 carriers.


Genetic studies of coliphage P1. I. Mapping by use of prophage deletions. One hundred and ten amber mutants of coliphage P1 were isolated and localized into groups with respect to the existing genetic map by use of nonpermissive Escherichia coli K-12 strains lysogenic for P1 with deletions. These lysogens contain one of three types of deletion prophages: P1cry and its derivatives, P1dlacs, and P1dpros. Fourteen such lysogens were tested for their ability to rescue the amber mutants which were then assigned to one of nine deletion segments of the P1 genome defined by the termini of the various prophage deletions. The relationship of the nine deletion segments with the published P1 map is described, two new segments having been added. The deletions of the 14 prophages overlapped sufficiently to indicate that the P1 genetic prophage map should be represented in circular form, which is consistent with the fact that P1 is normally a circular plasmid in the prophage state. The distribution of mutants into deletion segments is nonrandom for at least one segment. In addition, the deletion termini of the 14 defective prophages coincided in five out of nine regions separating the nine deletion segments. Various possible explanations are discussed for the nonrandom recurrence of these deletion termini, including the evidence of hot spots of recombination.


Rifampin inhibition of bacteriophage phiX174 parental replicative-form DNA synthesis in an Escherichia coli dnaC mutant. The Escherichia coli dnaC protein is not absolutely required in vivo for bacteriophage phiX174 parental replicative-form synthesis (Kranias and Dumas, 1974). However, when rifampin is present at a concentration that inhibits DNA-dependent RNA polymerase, phiX174 parental replicative-form synthesis is dependent on the dnaC protein activity. We conclude that E. coli DNA-dependent RNA polymerase can substitute for the dnaC protein in phiX174 parental replicative-form DNA synthesis, presumably in its initiation. The implications of this result with respect to the in vitro synthesis of the complementary strand of phiX174 DNA are discussed.


Escherichia coli capsule bacteriophages. VII. Bacteriophage 29-host capsular polysaccharide interactions. Different interactions between particles of Escherichia coli capsule bacteriophage 29 and its receptor, the E. coli serotype 29 capsular polysaccharide have been studied. The inactivation of phage 29 (8 x 10(3) PFU/ml) by isolated host capsular glycan was found to be physiologically insignificant (50% inactivation dose equals 100 mug after 1 h at 37 C). No adsorption (less than 2 x 10(4) PFU/mug) of the viruses to K29 polysaccharide-coated erythroyctes (at 0 or 37 C) was observed either. The phage particles were, however, found to catalyze the hydrolysis of beta-D-glucosido-(1leads to 3)-D-glucuronic acid bonds (arrow) in the receptor polymer, leading, ultimately, to the formation of a mixture of K29 hexasaccharide (one repeating unit), dodecasaccharide, and octadecasaccharide: (see article). Testing derivatives of K29 polysaccharide, as well as 82 heterologous bacterial (mainly Enteriobactericeae) capsular glycans, the viral glycanase was found to be highly specific; in accordance with the host range of phage 29, only one enzymatic cross-reaction (with the Klebsiella K31 polysaccharide) was observed. These and previous results, as well as the electron optical findings of M. E. Bayer and H. Thurow (submitted for publication), are discussed in terms of a unifying mechanism of phage 29-host capsule interaction. We propose that the viruses penetrate the capsules by means of their spike-associated glycanase activity, which leads them along capsular polysaccharide strands to membrane-cell wall adhesions where ejection of the viral genomes occurs.


Identification of bacteriophage T4-specific precursor tRNA by using a host mutant defective in the methylation of tRNA. A mutant of Escherichia coli K-12 that is defective in the synthesis of 5-methyluridine (ribothymidine) in tRNA was used to identify precursors to phage T4-specific tRNA. The precursor molecules, isolated by gel filtration, were more than twice the size of tRNA. This method is suitable for isolation of rather large amounts of such precursor molecules.


Ureteroneocystostomy versus ureteropyelostomy: a comparison in the same renal allograft series. Of the 132 transplants in our series 44 patients underwent reconstruction by ureteropyelostomy (group 1) and 88 underwent reconstruction by ureteroneocystostomy (group 2). The rates of major urologic complications were 11 percent in group 1 and 4 per cent in group 2. These results support the widely held opinion that ureteroneocystostomy is the superior method for urinary reconstruction in renal allograft cases. However, the alternative of ureteropyelostomy should be part of every transplant surgeons' armamentarium, especially for cadaver transplants when ureteral blood supply may be compromised.


Limulus lysate test for gram-negative bacterial meningitis. Bedside application. The limulus lysate on cerebrospinal fluid was evaluated in 335 infants and children as a method for the rapid diagnosis of Gram-negative bacterial meningitis. Positive limulus tests were obtained within one hour in 33 of 34 cases of Hemophilus influenzae meningitis; four additional patients with Gram-negative meningitis also showed positive limulus lysate tests. Conversely, 13 patients with Gram-positive bacterial meningitis all yielded negative limulus assays. All 48 cases of aseptic meningitis and 236 children with no meningitis showed negative limulus assays. Antibiotic therapy prior to hospitalization did not vitiate the validity of the test. A bedside adaptation of the limulus test, performed by house officers and medical students, showed approximately 98% agreement with the laboratory assay.


The Johns Hopkins rechargeable pacemaker. Historical aspect. Several groups have attempted to develop a clinically usable rechargeable cardiac pacemaker. Because of the technical problems, of the 11 reported efforts, only one has met with success, the unit developed at the Johns Hopkins University. A brief review of rechargeable pacemakers is given, along with a discussion of technical problems that required solution before the rechargeable pacemaker could be used clinically. The rechargeable pacemaker, because of its size, should minimize patient problems of discomfort, cosmesis, and skin erosion. The anticipated life of the rechargeable power cell should eliminate the need for pulse generator replacement during the patients's lifetime.


Exercise-induced asthma. Pharmacological assessment of single drugs and drug combinations. The relative effectiveness of ephedrine, theophylline, and hydroxyzine hydrochloride and their combinations was studied in a two-part investigation of 16 youths who has asthma and exercise-induced asthma. The beneficial and adverse effects of the drugs in the control of asthma were studied in the first part of the investigation. Both theophylline and ephedrine were judged superior to placebo when given alone. When combined they were somewhat more effective than either agent alone, although adverse effects precluded their use in almost half of the subjects. The addition of hydroxyzine diminished the side effects sufficiently to make the combination acceptable to four subjects who refused to take ephedrine/theophylline. In the second part of the study, the relief of exercise-induced asthma was evaluated. Ephedrine had no effect, hydroxyzine had a weak effect on hastening recovery, but theophylline modified the postexercise response significantly. The three drugs together produced an additive effect superior to that of theophylline alone.


[Transmission of drug-resistance through conjugation in Yersinia enterocolitica and Yersinia pseudotuberculosis. (II). With special reference to streptomycin resistant strains isolated from men and domestic animals (author's transl)]. Seven strains of Y. enterocolitica, of which 2 strains being resistant to SM, TC and SA, 2 to SM and SA 3 to SM, and 3 strains of Y. pseudotuberculosis resistant to SM have been isolated from men and domestic animals. Tests were carried out with these 10 isolates for transfer of the drug resistance by bacterial conjugation, and evidence was obtained for transfer of resistance from 1 SM-resistant Y. enterocolitica and 2 Y. pseudotuberculosis strains to the sensitive E. coli K-12. Moreover, secondary transfer of this resistance to sensitive strains of the donor species Y. enterocolitica and Y pseudotuberculosis was also demonstrated. The findings indicate the spontaneous occurrence of organisms carrying the resistance transfer factor, R(Sm), among the populations of these two species in the natural environment.