Naturally occurring sites within the Shigella dysenteriae tryptophan operon severely limit tryptophan biosynthesis.

We investigated the structural, functional, and regulatory properties of the Shigella dysenteriae tryptophan (trp.) operon  in transduction hybrids in which the cysB-trp-region of Escherichia coli is replaced by the corresponding region from S. dysenteriae. Tryptophan biosynthesis was largely blocked in the hybrids, although the order of the structural genes was identical with that of E. coli. Nutritional tests and enzyme assays revealed that the hybrids produced a defective anthranilate synthetase (ASase). Deletion mapping identified two distinct sites in trpE, each of which was partially responsible for the instability and low activity of ASase. We also discovered a pleiotropic site trpP (S) that maps outside the structural gene region and is closely linked to the S. dysenteriae trp operator. trpP (S) reduced the rate of trp messenger ribonucleic acid synthesis, and consequently trp enzyme levels, 10-fold relative to wild-type E. coli. In recombinants in which the structural genes of E coli were under the control of the S. dysenteriae promoter, enzyme levels were also reduced 10-fold. In some fast-growing revertants of the original hybrids, the rates of trp messenger ribonucleic acid synthesis and levels of tryptophan synthetase were restored to values characteristic of wild-type E.coli. Thus, the Trp auxotrophy associated with the S dysenteriae trp operon derives from the combination of a defective ASase and decreased expression of the entire operon imposed by trpP (S).


Tryptophan operon regulation in interspecific hybrids of enteric bacteria.

We examined tryptophan regulation in merodiploid hybrids in which a plasmid carrying the trp operon of Escherichia was introduced into Trp mutants of other enteric genera, or in which a plasmid carrying the trpR+ (repressor) gene of E. coli was transfered into fully constitutive trpR mutants of other genera. In these hybrids the trp operon of one species is controlled by the repressor of a different species. Similar investigations were possible in transduction hybrids in which either the trp operon or the trpR+ locus of Shigella dysenteriae was introduced into E. coli. Our measurements of trp enzymes levels in repressed and nonrepressed cells indicate that Trp regulation is normal, with only minor quantitative variations, in hybrids between E coli and Shigella dysenteriae, Salmonella typhimurium, Klebsiella aerogenes, Serratia marcescens, and Proteus mirabilis. Our results support the idea that a repressor-operator mechanism for regulating trp messenger ribonucleic acid production evolved in a common ancestor of the enteric bacteria, and that this repressor-operator recognition has been conversed during the evolutionary divergence of the Enterobacteriaceae.


Kinetic studies of Bacillus polymyxa nitrogenase.

Nitrogenase from the facultative anaerobe Bacillus polymxa was separated into its component proteins, which were recombined in the ratio that produced optimal specific activity (125 to 175 nmol of C2H2 reduced/min per mg of total protein). The apparent Michaelis constants (Km)for the magnesium adenosine triphosphate complex, reducible substrates azide, acetylene, and N2 and the nonphysiological electron donor hydrosulfite (S2O42-) were determined to be 0.7, 0.7, 0.2, 0.06, and 0.03 MM, respectively. These apparent Km values are in reasonable agreement with those reported for the nitrogenases of Azotobacter vinelandii and Klebsiella pneumoniae. Either a total lack of cooperativity between binding sites or a single binding site for reducible substrates is indicated by analysis of Hill plots. Hill plot slopes of approximately 1.7 suggest that multiple binding sites exist for both ATP and S2O42-.


Fatty acid transport by the lipophilic bacterium Nocardia asteroides.

Hexadecanoate was translocated in Nocardia asteroides by a constitutive transport system(s), which transported short, medium, and long-chain fatty acids. Inhibition of hexadenocanoate transport by homologues suggested that at least two systems are present: one specific for short-chain fatty acids and the other specific for medium- and long-chain fatty acids. Saturation kinetics typical of a carrier-mediated transport system (Kt = 870 muM)were observed, and concentration of fatty acids against a gradient was achieved. Inhibitor studies indicated that free sulfhydryl groups, a functional respiratory chain, and energy are required for translocation. Efflux of [14C]hexadecanoate in the presence of excess unlabeled hexadecanoate or 2,4-dinitrophenol and the cytoplasmic localization of acyl-coenzyme A synthetase (acid:coenzyme A ligase [adenosine monophosphate]; EC 6.2.1.3) (Calmes and Deal, 1973) are consistent with the hypothesis that fatty acids are transported and released intracellularly as free fatty acids.


cheA, cheB, and cheC genes of Escherichia coli and their role in chemotaxis.

Motile but generally nonchemotatic (che) mutants of Escherichia coli were isolated by a simple screening method. A total of 172 independent mutants were examined, and four genes were defined on the basis of mapping and complemenvestigated by determining their null phenotypes with nonsense or bacteriophage Mu-induced mutations. The cheA and cheB products were essential in producing changes of swimming direction and flagellar rotation. The checC product appeared to be an essential component of the flagellum; however, specific mutational alterations of this component allowed flagellar assembly but prevented directional changes in swimming. Since some cheB mutants changed directions incessantly, this gene product may also serve to control the direction of flagellar rotation in response to chemoreceptor signals. Thus most or all of the common elements in the signalling process were involved in the generation and regulation of changes in the direction of flagellar rotation.


Germ tube formation from zonal rotor fractions of Candida albicans.

Homogenous cell populations of increasing cell volume may have been isolated from exponential and stationary culture of Candida albicans by centrifugation on a sucrose gradient. Observations of the yeast-mycelial transition using these populations showed the following. (i) No fraction from early logarithmic phase cells was unable to undergo morphological transition. (ii) The time of initiation of germ tube production was correlated with cell size in stationary-phase cultures. (iii) The rate of appearance of germ tubes was nearly identical in all fractions measured. (iv) Addition of N-acetyl-D-glucosamine to homogeneous cell populations decreased the time of initial appearance of germ tubes but did not affect the rate of appearance after initiation.


Thymidine uptake and utilization in Escherichia coli: a new gene controlling nucleoside transport.

A commonly used strain of Escherichia coli K-12 was shown to be deficient in the transport of a number of nucleosides, including thymidine. Thymidine incorporation was unaffected. Strain AB2497 exhibited a strikingly lower thymidine pulse-label incorporation at low (less than 1 mug/ml) thymidine concentrations than do many other strains. The deficiency appeared to be due to mutation in a single gene. This gene, which we designated nup (for nucleoside uptake), is located at 10 to 13 min on the E. coli linkage map. In nup+ strains, the transport of a given nucleoside was relatively insensitive to large excesses of other nucleosides but was competitively inhibited by the same nucleoside. Mutants deficient inthymidine kinase are deficient in thymidine uptake but normal in deoxyadenosine uptake. A two-step model for nucleoside transport is presented in which the first step, utilizing the nup gene product, is a nonspecific translocation of nucleoside to the interior of the cell. In the second step, the individual nucleosides are modified by cellular enzymes (e.g., nucleosides kinases) facilitate accumulation.


Clo DF13 plasmid deoxyribonucleic acid-directed in vitro synthesis of biologically active cloacin DF13 and clo DF13 immunity protein.

Clo DF13 plasmid deoxyribonucleic acid (DNA) was used as a template to direct transcription and translation in a DNA-dependent cell-free system prepared from Escherichia coli. Analysis of the invitro products on sodium dodecylsulfate-polyacrylamide gels revealed that Clo DF13 DNA directs the synthesis of at least 10 polypeptides ranging in molecular weight from approximately 7,000 to 70,000. Two of these polypeptides could be identified, with respect to their physiochemical and biological characteristics, as the products of the Clo DF13 genes coding for cloacin DF13 and Clo DF13 immunity protein. These results confirm previous findings, obtained which Clo DF13-harbouring minicells of E. coli, that the structural tenes for the latter proteins residue on the Clo DF13 genome.


Initial characterization of Aspergillus nidulans mutants blocked in the nuclear replication cycle.

Several hundred temperature-sensitive mutants of Aspergillus nidulans were screened for ability of their conidia to produce germ tubes at the nonpermissive temperature while still remaining with the original single conidial nucleus.


Determination of the execution points of mutations in the nuclear replication cycle of Aspergillus nidulans.

Cultures of nuclear replication cycle mutants of Aspergillus nidulans were transferred to the nonpermissive temperature, and the fraction of nuclei still able to reach mitosis was determined. For the determinations, benomyl [methyl-1(butylcarbomoyl)benzimidazolecarbamate] was added to trap nuclei in mitosis, and these were detected by staining with aceto-orcein. The assumptions and controls required to relate the experimentally determined fractions to the points where a mutation blocks the nuclear cycle are discussed. Nine genetically distinct mutants were tested. Two of these were blocked early in the cycle, two in the middle, and five close to, or during, mitosis.


Periplasmic protein related to the sn-glycerol-3-phosphate transport system of Escherichia coli.

Two-dimensional gel electrophoresis of shock fluids of Escherichia coli K-12 revealed the presence of a periplasmic protein related to sn-glycerol-3-phosphate transport (GLPT) that is under the regulation of glpR, the regulatory gene of the glp regulon. Mutants selected for their resistance to phosphonomycin and found to be defective in sn-glycerol-3-phosphate transport either did not produce GLPT or produced it in reduced amounts. Other mutations exhibited no apparent effect of GLPT. Transductions of glpT+ nalA phage P1 into these mutants and selection for growth on sn-glycerol-3-phosphate revealed a 50% cotransduction frequency to nalA. Reversion of mutants taht did not produce GLPT to growth on sn-glycerol-3-phosphate resulted in strains that produce GLPT. This suggests a close relationship of GLPT to the glpT gene and to sn-glycerol-3-phosphate transport. Attempts to demonstrate binding activity of GLPT in crude shock fluid towards sn-glycerol-3-phosphate have failed so far. However, all shock fluids, independent of their GLPT content, exhibited an enzymatic activity that hydrolyzes under the conditions of the binding assay, 30 to 60% of the sn-glycerol-3-phosphate to glycerol and inorganic orthophosphate.


Centrifugal separation of irradiated cultures of Escherichia coli cells into viable and nonviable populations.

Incubation of ultraviolet-irradiated Escherichia coli B/r cultures with 0.7% Triton X-100 resulted in a large decrease in turbidity. Under phase-contrast optics, most of the irradiated detergent-treated cells were smaller than normal and of low phase density; only a small percentage were normal or larger than normal and of normal phase density. Irradiated cells not treated with detergent showed fewer pronounced morphological changes. Irradiated cells treated with detergent lost large amounts of proteins and ribonucleic acid, but not of deoxyribonucleic acid. Such cultures could be separated by centrifugation into populations of (i) slowly sedimenting cells consisting of small, phase-light cells of low viability and (ii) large cells of normal phase density and high viability (100%). A similar separation was effected in gamma-irradiated cultures.


Pyrimidine dimer excision in surviving and nonsurviving cells of ultraviolet-irradiated cultures of Escherichia coli.

We compared dimer excision in viable and nonviable cells fractions separated from Escherichia coli B/r cultures exposed to ultraviolet (UV) irradiation. For cells grown on minimal medium with glycerol as a carbon source, both fractions from the irradiated (20 J/m2, 5% survival) culture excised 60 to 70% of the thymine dimers from prelabeled DNA within 120 min. This percentage was, within experimental error, the same as that obtained from unseparated cultures. When isolated viable and nonviable populations were given a second UV exposure (20 J/m2) both types of cells were again able to excise dimers. The UV survival curve for the isolated viable population indicates that these cells are no more sensitive to radiation than exponentially growing cells not previously exposed to UV. The extent of dimer excision after UV irradiation was also the same in viable and nonviable cells separated from cultures grown on a glucose minimal medium in which both populations excised about 85% of the dimers within 120 min. These results show that the extent of removal of pyrimidine dimer from deoxyribonucleic acid is not precisely correlated with survival of repair-competent bacterial cells after exposure to UV light.


In vivo methylation by Escherichia coli K-12 mec+ deoxyribonucleic acid-cytosine methylase protects against in vitro cleavage by the RII restriction endonuclease (R. Eco RII).

We have analyzed the susceptibility of the deoxyribonucleic acid (DNA) of phage fd replicative form (RF) and of Escherichia coli to in vitro cleavage by purified RII restriction endonuclease (R. Eco RII). The results are summarized as follows: (i) fd, mec- RFI, isolated from infected E. coli K-12 mec- bacteria (a mutant strain lacking DNA-cytosine methylase activity), is cleaved into at least two fragments, whereas fd. mec+ RFI, isolated from the parental mec+ strain, is not cleaved. (ii) E. coli mec- DNA is extensively degraded, whereas mec+ DNA-cytosine methylase acts as an RII modification enzyme.


Association-dissociation behavior and subunit structure of heat-released nitrate reductase from Escherichia coli.

Nitrate reductase, released from the membrane fraction of Escherichia coli by a neutral heat treatment, was purified to homogeneity by gel filtration chromatography. The purified enzyme behaved as an associating-dissociating system, exhibiting concentration-dependent sedimentation constants which ranged from 24 S at high concentrations in the ultracentrifuge down to 10 S at low concentrations in sucrose gradients. The molecular weight determined at high concentrations by sedimentation equilibrium was 880,000 +/- 30,000. Large and small enzyme species were detected on polyacrylamide disc gels run with diluted samples of enzyme. The ratio of the two species was concentration-dependent and the dissociation was reversible. The purified enzyme appeared to be homogeneous and monodisperse in the ultracentrifuge, on sucrose gradients, during gel filtration on Bio-Gel and on polyacrylamide gels, but it had a heterogeneous subunit composition as determined by sodium dodecyl sulfate gel electrophoresis. Enzyme species with different subunit compositions were partially resolved by gel filtration. The fractions with the highest specific activity contained subunits of 150,000 and 55,000 daltons in a ratio of approximately 1:1. Other fractions contained reduced amounts of the 55,000-dalton subunit and correspondingly increased amounts of 51,000-, 45,000-, and 10,000-dalton subunits, suggesting that the heterogeneity was the result of proteolytic degradation of the 55,000-dalton subunit. The enzyme contained approximately 12 non-heme irons, 12 acid-labile sulfides, 24 cysteine residues, and 1 molybdenum per 200,000 daltons.


Mode of action of bottromycin A2. Release of aminoacyl- or peptidyl-tRNA from ribosomes.

Bottromycin A2 inhibited MS2 phage RNA-dependent protein synthesis as well as polyuridylic acid-(poly(U))- or polyadenylic acid (poly(A))-dependent polypeptide synthesis. When the ribosomal complex with N-acetyl-[14C]phenylalanyl-tRNA (N-acetyl-[14C]Phe-tRNA) at the A site was subjected to bottromycin A2, the release of N-acetyl-[14C]Phe-tRNA was observed while no release of N-acetyl-[14C]Phe-tRNA from the donor site was observed, indicating that the action of bottromycin A2 is specific to the A site of ribosomes. Due to bottromycin's capacity to release [14C]Phe-tRNA or N-acetyl-[14C]Phe-tRNA from the ribosomal acceptor site (A site), bottromycin A2 inhibited the nonenzymatic binding of N-acetyl-[14C]Phe-tRNA and elongation factor T (EF-T)-dependent binding if the concentration of EF-Tu-GTP-[14C]Phe-tRNA ternary complex was low. Our data are consistent with the possibility that the inhibition of overall polypeptide synthesis by bottromycin A2 is at least partly due to bottromycin A2's activity to release aminoacyl- or oligopeptidyl-tRNA from ribosomes. Among 10 antibiotics tested, bottromycin A2 and lincomycin released aminoacyl-tRNA from ribosomes.


Transfer ribonucleic acid biosynthesis. Substrate specificity of ribonuclease P.

Bacteriophage T4 synthesizes proline and serine tRNA species which are derived from a common precursor RNA. The processing of this precursor RNA involves the replacement of a U-A-A terminus in serine tRNA by C-C-A prior to precursor cleavage. In the present work we have examined in detail the cleavage of T4 proline-serine precursor RNA by the previously identified ribonuclease P. Ribonuclease P accurately cleaves precursor RNA terminating in either C-C-A or U-A-A to generate the 5' termini characteristic of both mature tRNA species. These cleavages do not depend solely on the nucleotide sequence of the precursor RNA since isolated oligonucleotides spanning the cleavage sites are not substrates for the enzyme. Two types of experiments show that RNase P kinetically favors precursor RNA ending C-C-A over that ending U-A-A. Isolated preparations of precursor RNA containing the C-C-A sequence were cleaved more rapidly by RNase P than precursor RNA ending U-A-A. In addition, the serine tRNA generated by limited cleavage of a mixed population of precursor RNA ending C-C-A or U-A-A was enriched 3-fold in the C-A-A sequence relative to the starting material. Bacteriophage T4 proline-serine precursor RNA, in contrast to other tRNA precursors, accumulates in measurable amounts in wild type cells. This accumulation would appear to be a consequence of the requirement for the generation of the C-C-A sequence prior to RNase P cleavage. The enzymic specificity of RNase P in vitro therefore reflects the in vivo pathway for serine tRNA biosynthesis, where the C-C-A sequence is synthesized while the serine tRNA sequence is still a part of the large precursor RNA.


An antimutator deoxyribonucleic acid polymerase. I. Purification and properties of the enzyme.

The DNA polymerase induced by an antimutator T4 phage has been purified to apparent homogeneity and has been compared to the wild type polymerase. The mutant enzyme resembles the wild type in thermal stability, pH optimum, salt activation, divalent metal ion requirement, inhibition by a sulfhydryl reagent, and apparent affinity for DNA. However, the mutant enzyme differs from the wild type in its 8-fold higher 3'-exonuclease activity and in its decreased apparent affinity for deoxyribonucleoside triphosphates. Inhibition studies indicate that the exonuclease of the mutant enzyme is more vulnerable to physical and chemical modification than its wild type counterpart.


An antimutator deoxyribonucleic acid polymerase. II. In vitro and in vivo studies of its temperature sensitivity.

Upon infection of Escherichia coli with the temperature-sensitive bacteriophage T4 mutant, L141, at 43 degrees, DNA polymerase activity is induced, yet there is a marked decrease in DNA synthesis. Temperature shift experiments of infected cultures show dramatic changes in the rate of DNA synthesis, while the polymerase activity in extracts remains constant. Unlike most temperature-sensitive systems in which the lesion can be traced to an unstable enzyme, L141 provides a unique example in which the catalytic properties of the enzyme and the change in the nature of the template caused by the increased temperature combine to reduce the rate of DNA synthesis.


Factors affecting the acyl selectivities of acyltransferases in Escherichia coli.

In Escherichia coli the synthesis of phosphatidic acid from glycerophosphate involves first the intermediate formation of 1-acyl glycerophosphate. This reaction is catalyzed by the membrane-bound acyl-CoA(acyl-ACP):glycerophosphate acyltransferase. The 1-acylglycerophosphate is then converted to phosphatidic acid by acyl-CoA(acyl-ACP):1-acylglycerophosphate acyltransferase (Okuyama, H., and Wakil, S.J. (1973) J. Biol. Chem. 248, 5197-5205). In vitro both acyltransferases utilize various saturated and unsaturated acyl-CoAs at comparable rates, resulting in the incorporation of both saturated and unsaturated fatty acids into position 1 as well as position 2 of the glycerophosphate moiety. Thus, the specificities of acyltransferase systems as compared with regard to the maximal velocities for various acyl-AoAs, do not explain the positional distribution of the individual fatty acid in phospholipid molecules. The selectivities of the acyltransferases for acyl-CoAs are variable depending upon the acceptor concentration. In the presence of both palmitoyl-CoA and oleoyl-CoA and at low concentrations of the acceptors comparable to those found in vivo, the acylation at position 1 of glycerophosphate by acyl-CoA:glycerophosphate acyltransferase showed higher preference for palmitate, whereas the acylation at position 2 by acyl-CoA:1-acylglycerophosphate acyltransferase showed higher selectivity for oleate. In the presence of saturating amounts of the acceptors, the acylation at position 1 or position 2 was less selective for the acyl-CoAs. The ratios of saturated acyl-CoA to unsaturated acyl-CoA also affect the ratios of the fatty acids incorporated in vitro into positions 1 and 2 of phosphatidic acid; relatively more palmitate was incorporated when the proportion of palmitoyl-CoA among the acyl donors was higher and vice versa. Thus, highly selective positioning of various acyl-CoAs observed at lower concentrations of the acceptors in phosphatidic acid synthesis in vitro helps to explain the selective distribution of saturated and unsaturated fatty acids at positions 1 and 2 of glycerophospholipids in the membranes. Another factor, the availability of acyl donors, affects the proportions of different molecular species of phosphatidic acid, which at least partly explains the variability of molecular species of phospholipids found in vivo.


Utilization of exogenous GDP-mannose for the synthesis of mannose-containing lipids and glycoproteins by oviduct cells.

Suspensions of oviduct cells were prepared by subjecting oviduct tissue to sequential incubations with EDTA, alpha-chymotrypsin, and crude collagenase, followed by a final incubation with EDTA. Cells isolated in this way incorporate mannose from exogenous GDP-mannose into mannosyl-lipid, oligosaccharide-lipid, and glycoprotein(s). Based on several criteria, the mannosyl-lipid is identical with mannosyl-phosphoryldolichol. Similarly, the oligosaccharide-lipid appears to be identical with the oligosaccharide-lipid synthesized in vitro (Lucas, J. J., Waechter, C. J., and Lennarz, W. J. (1975) J. Biol. Chem. 250, 1992-2002). In contrast, the glycoproteins are much lower in molecular weight than those labeled in cell-free preparations. Using intact oviduct cell suspensions it was found that: (a) exogenous GDP-mannose, not its breakdown products, serves as the direct mannosyl donor; (b) experiments using mixtures of known proportions of broken and intact cells, as well as studies with metabolic inhibitors, indicate that greater than 50% of the observed incorporation of mannose from GDP-mannose was catalyzed by enzymes associated with intact cells, rather than broken cells or membrane fragments; (c) incorporation of mannose from GDP-mannose into the mannosyl acceptors does not require energy and proceeds without significant uptake of GDP-mannose into trichloroacetic acid-soluble components of the cells; (d) under conditions where labeled guanosine incorporation into nucleic acids is readily detected, no incorporation of the guanosine moiety of [3H]GDP-mannose is observed. These results indicate that the enzymes catalyzing synthesis of lipid-linked intermediates involved in glycoprotein synthesis are not only associated with intracellular membranes, but with the plasma membrane as well.


Cr(III) complexes as active site probes of yeast inorganic pyrophosphatase.

The inert coordination complex of Cr(III) with PP1 (CrPP1) is not a substrate for yeast inorganic pyrophosphatase and does not inhibit the Mg2+-dependent activity of this enzyme. These results, when compared with the effects of CrPP1 on other enzymes and with the effects of other divalent metal ions on this enzyme, suggest that yeast inorganic pyrophosphatase binds substraather than the phosphate groups. Mg2+ partially protects against a slow inactivation of the enzyme by Cr(H2O)6(3)+; this exchange inert ion may provide a means of labeling the metal ion binding site of this enzyme.


Function and structure in phage Qbeta RNA replicase. Association of EF-Tu-Ts with the other enzyme subunits.

Qbeta replicase is a complex of four nonidentical subunits readily dissociable into two subcomplexes: 30 S ribosomal protein S1 and the phage-coded polypeptide (Subunits I + II) and protein synthesis elongation factors EF-Tu and EF-Ts (Subunits III + IV). The affinity of the two subcomplexes for one another increases with increasing ionic strength. The enzyme is capable of initiation of RNA synthesis with synthetic templates only when in the low ionic strength conformation. Elongation of initiated polynucleotide chains is not affectedby ionic strength. Addition of Qbeta RNA to the enzyme also alters its quaternary structure: the EF-Tu-Ts cannot be covalently attached to the other enzyme subunits with bifunctional cross-linking reagents in the presence of RNA. This conformational change is not influenced by ionic strength. The addition of Qbeta RNA to the enzyme, does not result in the release of EF-Tu-Ts from the other enzyme subunits: whereas free EF-Tu-Ts binds GDP independently of salt concentration, this binding by Qbeta replicase is sensitive to high ionic strength and remains so in the presence of Qbeta RNA. Furthermore, RNA does not allow the release of EF-Ts from EF-Tu by GTP as measured by sensitivity of EF-Ts activity to N-ethylmaleimide.


Structural and functional interdependence of the protomers of Escherichia coli K 12 tryptophanase during binding of pyridoxal 5'-phosphate.

It has been shown previously that the binding of pyridoxal 5'-phosphate to Escherichia coli K 12 tryptophanase brings about an important conformational change of the protein. The way in which this structural change is transmitted from holoprotomers to apoprotomers is investigated here, using hybrid molecules (between apoprotomers and irreversibly saturated holoprotomers). It is shown that the binding of two or three coenzyme molecules per tetramer stabilizes the whole molecule against thermal inactivation and cold-induced dissociation. The change in conformation induced on an apoprotomer by the proximity of three holoprotomers is described, using three structural probes: the kinetics of binding of the cofactor and of 5'-phosphopyridoxyl-tryptophan (an analog of an intermediate in the catalytic reaction), and the reactivity of the essential cysteines. The kinetic anticooperatively in the binding of pyridoxal 5'-phosphate is confirmed, some of its parameters are determined, and its mechanism is interpreted in relation to the coupling between the protomers.


The dissociated tryptophanase subunit is inactive.

The dissociation into dimers of apotryptophanase has been studied from two points of view: the nature of the interactions which govern the dimer-tetramer equilibrium and the effect of dissociation on the functional properties of the enzyme. It is shown that the order in which different anions are able to shift the dimer-tetramer equilibrium is that of the Hofmeister series, thus showing that the main contribution to the interaction between two dimers is of hydrophobic nature. It is also shown that, when dimeric apotryptophanase is incubated in the presence of its cofactor and substrate, the kinetics of appearance of active molecules is of second order in enzyme and is independent of the pyridoxal-P concentration; its rate constant has been determined (5-10(4) M-1 S-1). These results indicate that the reassociation of dimers into tetramers is the rate-limiting step in the appearance of enzymatic activity, and that the tryptophanase dimer is not functional.


Transition state analogs for thiamin pyrophosphate-dependent enzymes.

Many of the transition states that are formed from thiamin pyrophosphate in enzymic reactions are expected to have structures in which the thiazolium ring of thiamin pyrophosphate has lost most of its positive charge. We have synthesized thiamin thiazolone pyrophosphate from the unphosphorylated compound. The sulphur-containing ring of thiamin thiazolone pyrophosphate is uncharged, and thus the compound resembles these transition states. In agreement with the prediction from the transition state theory of reaction rates, thiamin thiazolone pyrophosphate binds to Escherichia coli pyruvate dehydrogenase complex (EC 1.2.7.1) much more strongly than thiamin pyrophosphate itself. An upper limit for the value of the dissociation constant, calculated from the extent of inactivation of the enzyme by a low concentration of thiamin thiazolone pyrophosphate, is 5 X 10(-10) M at 3 degrees in 0.5 mM MgCl2/10 mM potassium phosphate, pH 6.6. The dissociation constant for thiamin pyrophosphate under similar conditions is about 10(-5) M. The kinetics of inactivation of pyruvate dehydrogenase complex by thiamin thiazolone pyrophosphate are first order with respect to both enzyme and thiamin thiazolone pyrophosphate; the value of the second order rate constant is 5.7 X 10(5) M-1 min-1 at 3 degrees in 0.5 mM MgCl2/10 mM potassium phosphate, pH 6.6. An analysis of the decrease in the rates of inactivation caused by thiamin pyrophosphate indicates that thiamin thiazolone pyrophosphate binds at the coenzyme sites. We have also synthesized thiamin thiothiazolone pyrophosphate and obtained very similar results with this compound.


Antibiotic-impregnated acrylic bone cement.

In vitro and in vivo experiments were performed to study the antibacterial and mechanical properties of Simplex and Palacos acrylic bone cements utilized for the production of acrylic cement-antibiotic composites. Acrylic cement without antibiotic had no bacteriostatic effect on Staphylococcus aureus, Escherichia coli, and Pseudomonas. Oxacillin, cefazolin, and gentamicin are stable in acrylic cement and are released in a microbiologically active form. The three antibiotics diffused from the Palacos in larger amounts daily and for a significantly longer time than from the Simplex. Mechanical tests demonstrated that admixture of antibiotics and acrylic cement had no influence on the compressive and diametral tension strengths of the cement. Bacteriostatic concentrations of oxacillin in wound hematomas were measured for fourteen days after implantation of an oxacillin-Simplex composite in dogs. High bactericidal concentrations of the antibiotic were measured in the surrounding bone for twenty-one days after implantation.


The 1974 Gordon Richards Memorial Lecture: trends in the organization of cancer services.

The development of cancer programs and organizations in Canada is traced from their beginnings until the present time, and tribute is paid to our pioneers, of whom Dr. Richards was an outstanding example in this field. Although provinical cancer control measures vary, there is a marked similarity among some, so that they can be categorized into two or three patterns. The desirable requirements for the establishment and progressive development of cancer centres is described together with their relationship to increasing integration of teaching and research activities. An attempt is made to predict the future pattern of our "cancer clinic" system in which there will be increasing reliance on an interdisciplinary approach.


Malignant lymphoma of the testis.

Three cases of patients suffering from malignant lymphoma of the testicle are reported. The clinical aspects and characteristic pathological features are discussed. The evolution of lymphoma of the testicle differs from lymphomas in other sites and appears to be more agressive. It is recommended that a more extensive radiation therapy be applied because of frequent extra-nodal spread which is often excluded when using conventional fields. Chemotherapy provides long periods of remission in malignant lymphoma. This therapeutic weapon should be combined with radiation therapy for a better control of the disease.


Topographical differences in the distribution of surface coat components and intramembrane particles. A cytochemical and freeze-fracture study in culture forms of Trypanosoma cruzi.

A regional specialization of the cell surface of T. cruzi culture forms was found at the cytostome as a localized thick surface coat rich in carbohydrate-containing components. The prominent surface coat was located over a region of the plasma membrane where intramembranous particles were exceedingly low in number. In turn, the particle-poor region was related to specialized submembrane fibrils not present under other regions of the plasma membrane. The cystostome region provides a striking example of a stable regional differentiation of the plasma membrane, involving the outer surface, the membrane interior, and the underlying cytoplasm. In addition, independence of Con A receptors, colloidal iron binding sites, and ruthenium red-stainable surface components from membrane particles was demonstrated at the flagellar membrane.


Identification of glucagon-producing cells (A cells) in dog gastric mucosa.

An immunocytochemical technique using specific antiglucagon serum reveals the presence of glucagon-containing cells situated exclusively in the oxyntic glandular mucosa of the dog stomach. Electron microscope examination of the mucosa demonstrated endocrine cells containing secretory granules with a round dense core surrounded by a clear halo, indistinguishable from secretory granules of pancreatic A cells. Like the alpha granules of pancreatic A cells, the granules of these gastric endocrine cells exhibited a peripheral distribution of silver grains after Grimelius silver staining. Moreover, the granules of these cells were found to be specifically labeled with reaction product, using the peroxidase immunocytochemical technique at the ultrastructural level. Accordingly, these cells were named gastric A cells. These data suggest that the gastric oxyntic mucosa contains cells indistinguishable cytologically, cytochemically, and immunocytochemically from pancreatic A cells. It is believed that gastric A cells are responsible for the secretion of the gastric glucagon.


Visualization of stimulated nerve endings by preferential calcium accumulation of mitochondria.

Calcium was detected by X-ray microanalysis in the mitochondria of electrically stimulated nerve endings. The phenomenon described here offers a simple means for identifying the stimulated nerve endings in the electron microscope and appears to be a promising new method for following spontaneous and drug-stimulated translocation of calcium in relation to the regulation of neurotransmitter release.


Lymphoma with large intracavitary metastasis to the heart. Report of a case.

A case if reported of a 2 1/2-year-old boy with an extensive filtration of the retroperitoneum, mediastinum, and almost every organ of the body with a very primitive type of tumor identified as a poorly-differentiated mixed histiocyticlymphocytic lymphoma. The right atrium was completely filled with a large intracavitary metastasis which caused obstruction of the venous return to the heart with subsequent death.


[Atheromatous stenosis and occlusion of the brachiocephalic arterial trunk. Apropos of 24 operated cases].

The author reports an unpublished series of 24 operated cases and considers the surgical problems raised by atheromatous stenosis and obstruction of the innominate artery. At present, excellent results may be obtained thanks to: 1) better precision of the indications, excluding patients with neurological complications undergoing aggravation or permanent neurological signs; 2) at present, operations by the cervical route should be performed when the state of the heart and lungs prevents direct surgery by sternotomy, which remains, however, the method of choice.


[Wounds of the flexor tendons of the fingers at the level of the hand. Results of primary sutures].

The authors review in 100 patients, 182 wounds of flexor tendons of the fingers at the level of the hand emphasize the definite preponderance in young workers, their frequency which is all the greater as one moves away from the thumb, and approches the area of the fibrous pulleys, their common misdiagnosis, especially at the level of the thumb, their prognosis which is all the worse as one approaches the fifth finger and the areas of the pulleys. The poor results simply confirm their reputation of seriousness from an economic and social point of view. The technical problems raised by stiffening of the proximal interphalangeal joints and, above all, the formation of adhesions, are far from solved, it seems that an improvement in the results might be obtained if these wounds were repaired by specialised surgeons in a unit dealing exclusively with surgery of the hand which, under present conditions, is not always the case.


Limitations of the FPD and ECD in atmospheric analysis: a review.

This paper reviews the use of electron capture and flame photometric detectors in ambient air measurements, with particular emphasis on the factors which may affect the sensitivity, precision, and accuracy of the analyses. The interference effects of oxygen, water, and other common constituents in air samples are discussed. In addition to those anomalies that are due to the nature of the analytical samples, certain limitations are imposed by the inherent response characteristics of the detectors. These notable difficulties and limitations are defined along with their potential consequences in atmospheric applications. The basic intent of the discussion is to warn environmental scientists, who use the ECD and FPD, that significant errors can occur in their results unless they adhere to the analytical requirements of the analysis. Therefore, it is necessary to validate the ECD and FPD measurement techniques for their appropriateness in each new analytical application.


Gonadotropin output and response to LRH administration in congenital virilizing adrenal hyperplasia.

Three premenarchial, 2 primary amenorrheic, and 5 post-menarchial patients with congenital virilizing adrenal hyperplasia (CAH) were examined, utilizing frequent blood sampling techniques and the administration of synthetic luteinizing hormone-releasing hormone (LHRH) to determine whether a normal pattern of gonadotropin output occurs in CAH. Pulsatile gonadotropin output was not observed in premenarchial patients, but was seen in all who had had spontaneous menses. Bone age did not correlate with baseline or episodic gonadotropin output, or with response to LRH, but did reflect the past or present therapeutic control. The normal developmental progression of gonadotropin output was only documented in those patients who had been maintained at doses of suppressive glucocorticoids appropriate for body surface.


Demonstration of antibodies to testicular basement membrane by immunofluorescence in a patient with multiple primary endocrine deficiencies.

Circulating antibodies directed against testicular basement membrane and germinal cells are demonstrated in a patient with multiple primary endocrine gland deficiencies involving the thyroid, adrenals, testes, and pernicious anemia. Testicular biopsy revealed focal destruction of seminiferous tubules and interstitial fibrosis. The tubular basement membranes showed sharply positive immunofluorescence when stained with the patient's FITC conjugated gamma globulins.


The functional changes of the pituitary gonadotrophs during the menstrual cycle.

Submaximal doses of LRF, administered over a period of several hours, either by repeated pulses (10 mug at 2 h intervals X 5) or by constant infusion (0.2 mug/min X 4 h), have permitted the assessment of changes in the releasable gonadotropin during the menstrual cycle. Quantitations in the acute releasable and ultimately releasable gonadotropins were made which represent, respectively, the sensitivity and reserve of the gonadotrophs. The functional expression of these two components of gonadotropin release exhibited profound changes during the menstrual cycle and were in synchrony with the cyclicity of ovarian steroid levels; during the early follicular phase, both sensitivity and reserve were at a minimum, but with increasing levels of E2, a preferential increase in reserve over sensitivity (P less than 0.005) was found. Although both sensitivity and reserve increased dramatically near the midcycle, the relative change in these two components was reversed from the late follicular phase to the midcycle surge. The presence of this phenomenon may be causally related to the development of a self-priming effect of LRF at this time, as evidenced by an augmentation of gonadotropin release to the 2nd pulse of LRF. Thus, a build-up in pituitary store consequent to the greater increase in reserve than in sensitivity, together with the appearance of the self-priming effect of LRF induced by progressively rising levels of E2, may constitute the essential dynamics required for the development of the midcycle gonadotropin surge. Pituitary sensitivity and reserve continued to be high during the early luteal phase but reduced progressively thereafter. In all studied, FSH responses were less obvious but showed remarkable parallelism to the pattern of LH responses. We have concluded that the functional capacity of the gonadotrophs exhibits a remarkable cyclic change and that the adenohypophysis represents a critical feedback site in the development of pre-ovulatory gonadotropin surge.


Medium-dependent inhibition of Peptostreptococcus anaerobius by sodium polyanetholsulfonate in blood culture media.

Of 13 species of anaerobic cocci, Peptostreptococcus anaerobius was the only species tested that was sensitive to 0.1% sodium polyanetholsulfonate (SPS). However, the sensitivity of P. anaerobius to SPS varied according to the media in which the cultures were grown. In supplemented peptone (B-D) and brain heart infusion media, most strain of P. anaerobius were not inhibited by SPS. Gelatin and proteose peptone were the medium components which were protective. The minimal inhibitory concentration of SPS for P. anaerobius was approximately 60-fold higher in media. However, the concentration of SPS required to neutralize the bactericidal properties of human serum was only four fold higher in media containing geltain. In a commerical medium containing SPS (0.03%) and gelatin (1.2%), SPS-sensitive strains of P. anaerobius were not inhibited by SPS, and the bactericdal action of human blood on Escherichia coli C and Serratia marcescens SM 29 was eliminated.


Analysis of cost and accuracy of alternative strategies for Enterobacteriaceae identification.

Analysis of the cost of time and material required for the diagnosis of Enterobacteriacea isolates indicated that a conventional 17-tube (20-test) setup costs $7.98 per isolated identified. Using the API 20E, a similar identification cost $3.02. A conventional 7-tube (10-test) setup cost $3.60, whereas the comparable cost by 30% while increasing the number of isolate identified correctly by 3%. Other strategres using the API 20E or a deoxyribounclease test were also evaluated for cost and accuracy.


Clinical isolates of anaerobic gram-negative rods with a formate-fumarate energy metabolism: Bacteroides corrodens, Vibrio succionogenes, and unidentified strains.

Strains of anaerobic, gram-negative bacteria, isolated from human clinical specimens and from studies of human normal flora, that have energy metabolism similar to Vibrio succinogenes are described. Included are four human isolates of V. succinogenes, five similar strains of motile straight rods, three strains of Bacteroides corrodens, and two unidentified strains. All strains studied grew poorly in usual anaerobic broth media but produced good turbidity in overnight broth cultures in media containing fromate and fumarate, indicating that all have an energy metabolism similar to V. succinogenes: they gain energy by the transfer of electrons from formate or hydrogen to fumarate.


Fluorescent antibody test for the serological diagnosis of gonorrhea.

An indirect fluorescent antibody technique has been developed for the serological diagnosis of gonorrhea. The selected strain(s) of Neisseria gonorrhoeae possesses a heat-labile surface antigen (L-antigen). Sera are diluted 1:10, and an aliquot is hear inactivated at 59 C for 30 min. The treated and untreated aliquots are then examined for human immunoglobulin G anti-L-antigen. In a prelimiary study of 495 sera, 95% of those from women with a bacteriologically confirmed diagnosis of gorrhea and 87% of those from male patients were reactive in this test, whereas only 1.4% fo the sera from presumably normal individuals were reactive.


Simple method for immunofluorescent identification of mycoplasma colonies.

General purpose rubber stoppers proved to be a simple, rapid, and satisfactory method of transferring mycoplasma colonies from agar to microscope slides for immunofluorescence.


Duodenal isolation of Salmonella typhi by string capsule in acute typhoid fever.

Three of seven volunteers with acute typhoid fever had Salmonella typhi isolated from the duodenum using a string capsule device. The string capsule device provides a simple method for culturing S. typhi from the duodenal contents. Its possible use in typhoid carriers is discussed.


Use of CO2 for more rapid growth of the Nocardia species.

Growth of the Nocardia species is facilitated by use of 10% CO(2) for initial isolation from clinical material or from laboratory stock cultures.


Survival of isolated human islets of Langerhans maintained in tissue culture.

Transplantation of human pancreatic islets to diabetic patients may require that donor islets be kept viable in vitro for extended time periods before transfer to the recipient. We have maintained isolated pancreatic islets obtained from the human cadaveric pancreas in tissue culture for 1-3 wk, after which we studied the structure and function of the islets. Electron micrographs of the cultured islets showed a satisfactory preservation of both beta-cells and alpha 2-cells. After culture for 1 wk, the islet oxygen uptake proceeded at a constant rate at a low glucose concentration (3.3 mM) and was significantly enhanced by raising the glucose concentration to 16.7 mM. Likewise, after culture for 1 wk, the islets responded with an increased insulin release when exposed to 16.7 mM glucose with or without added theophylline (10 mM). Islets cultured for 1-3 wk were able to incorporate [3H]leucine into proinsulin, as judged by gel filtration of acid-alcohol extracts. Glucagon release from the cultured islets was reduced significantly by 16.7 mM glucose alone, but stimulated by glucose (16.7 mM) plus theophylline (10 MM). It is concluded that viable pancreatic islets can be isolated from the pancreas of adult human donors and maintained in tissue culture for at least 1 wk without loss of the specific functions of the alpha 2- and beta-cells. It remains to be established whether such islets will survive and remain functionally competent after transplantation to human recipients.


Differential diagnosis of psychiatric patients with the Minnesota Percepto-Diagnostic Test.

This study investigated whether normals, personality disorders, psychotics and organics could be differentiated by the Minnesota Percepto-Diagnostic Test. There were significant differences between all the groups except the psychotics and personality disorders. It appears that, in general, perceptual disturbance lies along a pathological continuum. Similar performances of psychotics and personality disorders were discussed.


Characterization of the inappropriate gonadotropin secretion in polycystic ovary syndrome.

To evaluate gonadotropin release in polycystic ovary syndrome (PCO), one or more of the following hypothalamic-pituitary function tests were performed on 24 patients with the syndrome. These tests included (a) the pulsatile pattern and day-to-day fluctuation of gonadotropin release; (b) effects of exogenous estrogen and antiestrogen (clomiphene) administration on gonadotropin release; and (c) pituitary responsiveness to maximal (150 mug) and submaximal (10 mug) luteinizing hormone-releasing factor (LRF) injections. In 10 of the 14 patients sampled frequently (15 min) for 6 h, luteinizing hormone (LH) levels were elevated above the concentration seen in normal cycling women (except the LH surge). These high LH concentrations appeared to be maintained by and temporally related to the presence of exaggerated pulsatile LH release, either in the form of enhanced amplitude or increased frequency. In all subjects, levels of follicle-stimulating hormone (FSH) were low or low normal, and a pulsatile pattern was not discernible. In four patients, daily sampling revealed marked day-to-day fluctuation of LH but not FSH. That the elevated LH levels were not related to a defect in the negative-feedback effect of estrogen was suggested by the appropriate fall of LH in four patients given an acute intravenous infusion of 17beta-estradiol. This infusion had no effect on FSH levels. In addition, clomiphene elicited rises of both LH and FSH that were comparable to the ones observed in normal women given the same treatment. The clomiphene study also suggested that the positive-feed-back mechanism of estrogen on LH release was intact when the preovulatory rises of 17beta-estradiol induced appropriate LH surges. The elevated LH levels appeared to be related to a heightened pituitary responsiveness to the LRF. This was found in the 11 and 2 patients given maximal (150 mug) and submaximal (10 mug) doses of LRF, respectively. The augmented pituitary sensitivity for LH release correlated with the basal levels of both estrone (P less than 0.025) and 17beta-estradiol (P less than 0.02). The net increase in FSH was significantly greater (P less than 0.001) in the PCO patients than the normal women with maximal doses of LRF. With the smaller dose study none of the injections had a discernible effect on FSH concentrations in either subject. The disparity between LH and FSH secretion could be explained by the preferential inhibitory action of estrogen on FSH release, coupled with a relative insensitivity of FSH release. These data indicate that in these PCO patients the abnormalities of the hypothalamic-pituitary regulation of gonadotropin secretion was not an inherent defect but represented a functional derangement consequent to inappropriate estrogen feedback, which led to a vicious cycle of chronic anovulation and inappropriate gonadotropin secretion.


Judgments of educators and child-care personnel about appropriate treatment for mentally retarded or normal, overactive or withdrawn, boys.

Special educators and child-care personnel ranked the appropriateness of various psychological treatments for a boy described either as mentally retarded or of normal intelligence and whose behavior was described either as withdrawn or overactive. The predicted IQ level by behavior interaction was found only for drug treatment, which was ranked as relatively more appropriate for the retarded overactive child (p less than .05). Drug treatment and behavior modification were perceived as more appropriate for the overactive child (p less than .01, .05). Play therapy, family therapy, and no treatment were deemed more appropriate for the withdrawn child (p less than .05). Educators favored behavior modification and school consultation more than did child-care personnel (p less than .05).


Personality factors and drug effects in a controlled study of cyclazocine.

This paper investigated the relationship between measurable personality factors and level of effects shown to cyclazocine and placebo in a controlled study. An attempt also was made through case analysis to examine the association between dynamic aspects of personality and adverse drug effect. Hysteria scores on the MMPI were found to be related significantly to self-reported effects under both the drug and placebo conditions. Clinical observations were examined retrospectively for three cases with adverse reactions and cited to support a dynamic theory that associated drug reactivity to personality factors.


A pilot study of the short-term psychotropic effects of GPA 2640.

Thirty-two anxious nonpsychotic inpatients participated in a four-day, double-blind repeat crossover study of a new drug, GPA 2640, and placebo. Each treatment period lasted for one day, and the study lasted for four days. Twenty patients completed the study, and 28 completed two or three days. The doses were individually adjusted and mostly ranged between 1100 and 1300 mg daily. Several valid observer-rating and self-rating scales were used. There was a slight trend for GPA 2640 to yield somewhat lower anxiety scores on drug days than on placebo days, but none of the differences between rating and self-rating scores reached a significant level. A few patients reported a marked improvement in mood after two to four days, but the significance of this finding cannot be evaluated from this design. Two patients developed temporary paranoid delusions which may have been drug related. Two patients had raised SGOT levels after the study. There were no significant differences in vital signs between drug days and placebo days. The results signify that the drug has no immediate antianxiety effects (such as have been demonstrated with benzodiazepines in two previous studies utilizing the same design). This does not preclude the possibility of delayed anxiolytic effects or of other delayed psychotropic effects such as can be observed with antipsychotic drugs or antidepressants.


Clinical and bacteriological evaluation of amikacin in severe gram-negative infections.

Amikacin was administered to 51 patients with serious Gram-negative infections. A favorable clinical outcome was observed in 32 patients (62 per cent). No major untoward effects were noted. The severity of the underlying disease and the site of the infection were important for the outcome. Urinary tract infections responded in 21 (78 per cent) out of 27 patients, but bronchopulmonary infections responded in only 3 out of 10 patients (30 per cent). The degree of antibacterial activity of the serum of the patients receiving amikacin was found to be related to the outcome. An activity greater than or equal to 1/8 was associated with 78 per cent of favorable responses and was found more often in patients receiving 1500 mg daily.


Rate of climbing fiber degeneration in rabbit cerebellum following parafloccular stalk and medullopontine lesions.

To resolve inconsistencies in experimental studies which use reduced silver methods to detect cerebellar climbing fiber sources within the brain stem, an evaluation of the temporal course of degeneration was undertaken. The paraflocculus of the rabbit is uniquely situated within the temporal bone and connects with the corpus cerebelli via a stalk passing through a bony foramen. A unilateral electrolytic lesion deafferented the paraflocculus without disturbing blood supply or causing damage (18 animals). Comparatively, another group of unilateral lesions was placed at the dorsolateral pontomedullary junction, adding significantly to the cortical area deafferented (12 animals). Animals of each group were killed at successively longer intervals commencing at 18 hours, and a modified Fink-Heimer impregnation process was applied to the cerebellar cortex and opposite-sided controls. In parafloccular lesions, degeneration was detected at 24 hours in the climbing fibers of the molecular layer. At successively longer intervals, degeneration became increasingly evident in granule layer and white matter. Subsequent to dorsolateral pontomedullary lesions, climbing fiber degeneration was first observed in the molecular layer at 24 hours and was clearly evident there at 36 hours. By 72 hours degeneration had reached the white matter, meanwhile disappearing in the molecular layer where it was first seen. Using techniques of Nauta-Fink-Heimer to display secondary climbing fiber degeneration in cerebellar cortex, it was found that too long post-operative intervals could preclude its detection, since in both groups of animals it commenced earlier and disappeared sooner in the molecular layer.


The effectiveness of diphenhydramine HCI in pediatric sleep disorders.

Fifty children with a variety of sleep disorders were studied in a placebo-controlled, double-blind clinical trial of diphenydramine elixir given in a dosage of 1.0 mg/kg at bedtime. Sleep records measuring latency time, number of awakenings, and duration of sleep were compiled by the patient's parent. A global assessment as to the severity of sleep disturbance and the therapeutic effect of the medication and placebo was made on a weekly basis. Diphenhydramine was significantly better than placebo in reducing sleep latency time and the number of awakenings per night, while sleep duration was marginally increased. There were no essential differences between diphenhydramine and placebo in the other study parameters, i.e., restlessness, nightmares, and difficulty awakening. Diphenhydramine was judged to be more effective than placebo on the weekly global assessment. The results indicate that diphenhydramine is a safe, effective bedtime sleep aid for pediatric patients.


Receptive fields of barrels in the somatosensory neocortex of the rat.

Peripheral receptive fields of "barrels" in the somatosensory neocortex (SmI) of rats were determined with microelectrope recording and anatomical techniques. A method of flattening each hemisphere prior to embedding was devised so that the organization of layer IV could be seen through the full extent of SmI on a single section. All of the barrels in the rat are located within layer IV of Smi. sinus hairs on the lateral and rostral face and furry buccal pad project to a continuous sheet of barrels in the head area of the contralateral SmI. The pattern of projections in this area indicates that each barrel receives information from one sinus hair. Large whisking vibrissae project to the largest cell-dense barrels and smaller non-whisking sinus hairs project to smaller barrels that are relatively cell-sparse. Barrels were also found outside of the head area in regions that receive projections from the forepaw and hindfoot. Receptive fields of these barrels are located on glabrous and hairy surfaces of the appendages that do not include any sinus hairs. Other parts of the body project to cell-dense areas of SmI that do not appear to contain barrels. Barrel subgroups and other somatotopic areas are separated by narrow barrel-free zones. As a result of this pattern of contrasting cell densities, an outline of the rat's body is delineated within layer IV.


A comparison between the amino acid compositions of 2 chymosin-sensitive polypeptides and C-terminal segments of kappa-casein.

Two distinct types of chymosin-sensitive polypeptide were found in the soluble fraction obtained when a solution of crude kappa-casein in 0-4 M-NaCl was adjusted to pH 3. One corresponded to a segment of kappa-casein comprising residues 94-169. The other was very similar to the section Gln77-Val169 in the protein, but contained a phosphate group plus isoleucine and glycine in the region between the N-terminus and the chymosin-sensitive bond. If these polypeptides form as a result of a specific cleavage of kappa-casein, the data indicate that there is a variant of this protein with a number of amino acid replacements on the N-terminal side of the chymosin-sensitive bond. The shorter polypeptide and kappa-casein are both rapidly cleaved by chymosin at pH 6-7 which suggests that the contribution of residues 1-93 to the sensitivity of the Phe-Met bond in kappa-casein is a minor one.


Immunochemical, chromatographic, and milk-clotting activity measurements for quantification of milk-clotting enzymes in bovine rennets.

Monospecific rabbit antibodies were produced against bovine chymosin (rennin) and bovine pepsin A and used for quantitative measurements of these enzymes by rocket immunoelectrophoresis. Bovine pepsin B was identified and measured quantitatively in tandem crossed immunoelectrophoresis, using a polyspecific antibody preparation. Chromatographically purified chymosin B and bovine pepsins A and B were used as standards. The contents of chymosin and bovine pepsin A were also determined by 2 forms of DEAE chromatography, and by an assay utilizing the ratio of milk-clotting activity at pH 6-0 and 6-5. Results obtained by measurements on 3 bovine rennets (calf rennet, adult bovine rennet, and a mixture of such types) by the different methods of analysis were in good agreement. The advantages of the immunochemical methods are direct and simultaneous identification and quantitation of enzymes.


A long-term study of penfluridol in chronic schizophrenia.

Penfluridol, a member of the novel diphenylbutylpiperidine class of antipsychotic drugs, is the first long-acting oral neuroleptic. The population of the present study consisted of 24 chronic schizophrenic patients (14 males, ten females) whose treatment with penfluridol was initiated in our previous open/double-blind trial lasting 32 weeks 1; mean age was 42.2 years and mean duration of illness, 15.4 years. During one additional year in an uncontrolled clinical study, penfluridol in the form of 20-mg capsuent procedures included BPRS, CGI, NOSIE, vital signs, and laboratory measurements. During this long-term treatment with penfluridol, the scores of a cluster of BPRS items that included emotional withdrawal, conceptual disorganization, motor retardation, uncooperativeness, and blunted affect showed a progressively significant improvement. This indicated that the Bleulerian primary symptoms in chronic schizophrenics can be improved by the long-term administration of this long-acting neuropleptic with concomitant betterment of social adaptation and activity. The percentage of failure was very low (four patients) and was marked by instability of psychopathology with periods of excitation. The incidence of extrapyramidal reactions necessitating the administration of an antiparkinsonian drug during the length of trial was 35 per cent. No serious effects nor significant laboratory test changes were observed.


An evaluation of papaverine in tardive dyskinesia.

An evaluation of papaverine was carried out in nine hospitalized chronic patients with moderate to servere tardive dyskinesia. Changes in movements were assessed on the Simpson and AIMS scales by two trained rates blind to the study design. Results showed a modest 20-25 per cent improvement in oral dyskinesia, but only two of the nine patients showed clinically obvious improvements in their movement disorders. No drug-induced side effects were found. The mechanism of pharmacologic action of papaverine in movement disorders needs to be clarified, but dopamine receptor blockade may be primarily involved.


Cariogenic flora: establishment, localization, and transmission.

The foregoing is a review of current information on various aspects of the development of the cariogenic flora and it illustrates some of the complexities involved. The multifactorial nature of dental caries makes it necessary to look at this disease in a special way. Etiologic forces represented by a complex bacterial flora and a variable dietary influence are in competition with resistance factors of the host, which include the immune system. This creates a situation where it is difficult to segregate individual aspects of the disease for study. One approach is to design study models that emphasize the factor under investigation in relation to other contributing factors. For example the currently used animal caries test models are deliberately exaggerated in terms of the cariogenic challenge applied. Very young animals are continuously exposed to a high sucrose diet and are infected with a high dosage of cariogenic organisms. The clinical counterpart of this model should use subjects with high caries activity, because it is reasonable to assume that etiologic factors are exaggerated in human populations where the disease is rampant. However, if the purpose is to study resistance factors such as immunity, it may be more profitable to investigate their influence in either animal or human models where the cariogenic challenge is reduced. This, in effect, proportionally increases the influence of a comparatively subtle factor such as immunity. Davies has made a similar point in relation to the influence of hereditary factors in dental caries. He states the belief that regions of low caries prevalence would be the most promising for epidemiological investigation of resistance factors. Investigations into the role of immunity in dental caries have not been too productive in the past. However, it now is possible to focus on selected target organisms and to apply recently acquired information about the immune system operating in the oral cavity. Detailed knowledge of the origins and development of the cariogenic flora and the mechanisms by which it is maintained in individuals and in populations should permit a rational attack on the problem.


Streptococcus mutans serotypes: some aspects of their identification, distribution, antigenic shifts, and relationship to caries.

For an immunologic point of view, several facts are worth consideration. S mutans can be separated into at least seven serotypes. Five of the types are based on antigens that may be specific for S mutans. One type, e, is related to the Lancefield group E streptocci, and one type, f, may lack an antigen that shows serological specificity. Analyses of plaque samples from individuals with a high caries activity have, in most instances, shown the presence of c, d, and possibly the g types. This does not necessarily mean that they are per se more cariogenic than the other types, but if all the serotypes cannot be combatted simultaneously, the c, d, and g types are an obvious first choice. S mutans strains do have antigens other than those used for serological identification, and it is not known which antigens can evoke antibodies with the highest protective capacity in humans. The phenomenon of antigenic shifts may make it possible for the bacteria to elude antibodies. However, the number of possible changes may be restricted. If certain antigens are of importance for the cariogenicity of S mutans, a change in their structure might result in a less cariogenic flora.


Immunization experiments using the rodent caries model.

Taken together, the immunization experiments which have been performed in the rat caries model system appear to suggest a correlation between the presence of salivary antibody to S mutans and reductions in caries caused by these bacteria. However, the multifactorial nature of this disease does not permit at present the conclusion that the presence of this antibody is both necessary and sufficient to give rise to the demonstrated effects on pathogenesis. To clarify the role of salivary antibody, several refinements may be required in the current model. Immunization procedures that elicit only a local antibody response would both simplify interpretations of effects and would be more desirable for use as a vaccine. Such procedures might include intraductal installation of antigen in the parotid gland which has been demonstrated to result in this type of response. An additional refinement stems from the knowledge that the kinds of immunization procedures currently used stimulated both cellular immune and soluble antibody systems, potentially giving rise to a rather broad spectrum of immune responses. Therefore, it might be useful to study the effects on S mutans pathogenesis in rats in which certain of these responses have been repressed, for example, by thymectomy, antilymphocyte serum, and so on. Also, each of these approaches would be measurably enhanced by more sensitive techniques to monitor immunological events in the oral cavity. Refinements in the selection and use of relevant antigens of S mutans also are necessary to delineate the in vivo mechanism of immunological interference in the pathogenesis of cariogenic streptococci. Approaches involve the use of purified GTF antigens or cell surface antigens both in the investigation of these mechanisms in in vitro models using antibody specifically directed to these antigens and in rat immunization experiments using immunogenic preparations of these materials. In addition, alterations in the diet and challenge dosage of infecting cariogenic organisms might permit more sensitive detection of effects in vivo. Clearly, the evidence suggests immunological interference with S mutans pathogenesis in the rat model system. However, several gaps exist in our basic understanding of this interference and of the appropriate system in which to observe these effects. The potential significance of this phenomenon should spur efforts to fill these gaps to establish definitively the role of immunity as an ecological determinant in the oral cavity.


Antigens in Streptococcus mutans cross reactive with human heart muscle.

An antigen that cross reacts with mammalian heart tissue has been shown to be present in several strains of S mutans. Inoculation of S mutans into rabbits elicited heart-reactive antibody as part of the immunologic response. This heart-reactive antibody was demonstrated to be tissue-specific in that it only bound heart and smooth muscle tissue. Similar results have been previously reported using group A streptococci. Adsorption studies using sarcolemmal sheaths, group A streptoccal membranes, and fractions of cariogenic streptococci demonstrated the presence of a similar antigen. The aforementioned fractions could remove heart-reactive antibody from both immune rabbit serums and serums from patients with acute rheumatic fever. These findings dictate the necessity for testing any future caries vaccine containing fractions of S mutans for the presence of this cross-reactive antigen to avoid a possible autoimmunization.


A comparison of polyglycolic acid (Dexon), nylon and silk sutures in skin surgery.

Synthetic polyglycolic acid (Dexon) sutures were compared with silk or nylon in 50 patients requiring skin surgery. After some practice, polyglycolic acid is an easily handled as nylon or silk. Skin reactions occurred in 50% of the patients sutured with nylon, in 10% of those sutured with polyglycolic acid, and in none sutured with silk. Wound healing was satisfactory in all cases.


Use of skin flaps in dermatologic surgery of the face.

Different flap techniques for plastic repair of defects are described, namely, local flaps (advancement, transposition and rotation flaps) and regional flaps. Such use of skin of similar structure and pigmentation gives excellent cosmetic results. The indications for using local or regional flaps depends on size and location of the defect. Total extirpation of cutaneous malignancy particularly by microscopically monitored serial excision is a prerequisite to reconstruction.


Subcuticular skin closure--description of technique.

A technique of subcuticular wound closure is described in which both ends of a running suture are buried in the wound. Thus, there are no suture ends left on the skin. This method is only applicable to absorbable sutures.


Betamethasone valerate compared by the oral and inhaled routes in childhood asthma.

The value of betamethasone valerate by inhalation in the prophylactic therapy of severe childhood asthma has been established. To determine whether the efficacy of this drug is due to a local or a systemic action a double-blind crossover study of 28 days' treatment with oral betamethasone valerate and 28 days' treatment with inhaled steroid was carried out in 10 asthmatic children. Daily doses used were 1 mg orally and 800 mug by inhalation. Nine patients had fewer symptoms, higher peak expiratory flow rates, and a lower bronchodilator requirement on inhaled than on oral therapy. Exercise-induced bronchoconstriction was diminished on inhaled therapy. Five children requested early termination of the oral therapy period because of unacceptable symptoms. Nine parents stated a preference for the period of inhaled therapy. It is concluded that betamethasone valerate is highly effective by inhalation but that a comparable oral dose has no appreciable clinical effect.


Refractory period to aspirin in a patient with aspirin-induced asthma.

Oral aspirin challenge was used to detect unrecognized aspirin intolerance in a select group of 50 asthmatic patients who denied aspirin intolerance. A double-blind protocol was used to further study those patients who reacted to initial graded aspirin challenge. In one patient the use of a double-blind protocol led to the serendipitous discovery of a 72-hr refractory period to the adverse effects of aspirin, after initial ingestion of aspirin resulted in respiratory and systemic symptoms.


General Washington and the patriot soldiers. They won a war with little food. A Bicentennial study.

The troops in the first action of the American Revolution in Massachusetts were well provisioned through the efforts of Joseph Trumbull of Connecticut, whom the Second Continental Congress appointed Commissary General, at General Washingtion's request. The ration, as ordered, would have provided-by today's standards-more calories, twice as much protein, and adequate minerals and vitamins, except possible vitamin A and ascorbic acid. Unfortunately, all items in the ration were seldom available to the soldiers. Each man was issued his ration and was responsible for cooking it. Trumbull held his post for two years, with little support from the Congress and no departmental organization. When the Army moved to New York, difficulties in provisioning the troops on the march began-and continued until 1781. Following the retreat to Morristown, New Jersey, in 1776, flour and beef, but little else, were available during the winter and into the following summer. Washington pleaded with the Congress for help with food supplies, but to no avail. Finally, Trumbull resigned. Thereafter, followed a series of four reorganizations of the commissariat by the Congress-each disastrously conceived-which brought no relief to the ill-fed soldiers, whose physical condition progressively worsened. Finally, a fifth reorganization in 1781 put into operation a contract plan of procuring food; no longer was it necessary for Washington to order impressment of food from farmers or for the troops to live off the land. The Continental Army "starved, not because the country could not furnish the food, but becasue the people were unwilling to endure taxation and Congress did not understand the importance of administrative centralization; ...the greater part of the hardships" were avoidable. Yes, General Washington and the patriot soldiers won the war with little food.


Primary lymphosarcoma of the breast: review of the literature on occurrence in elderly patients.

A case of primary lymphocytic lymphosarcoma of the breast in a 74-year-old woman is presented, together with a review of the literature on 23 cases of primary breast lymphosarcoma in patients aged 60 or older. In this age group, simple local excision or mastectomy is probably sufficient.


Complex surveillance of Streptococcus pyogenes. III. Immunological characteristics of streptococcal resistance in blood and new aspects of type nonspecific protection.

The influence of nonspecific immunity factors in indifferent human blood on the resistance of M non-typable field streptococci and collection M strains in the absence of anti-M antibodies was studied. The experimental results indicate that primary resistance is conditioned on balanced equipment of the strain with M protein and the nonspecific cofactor receptor [R]. Disbalanced strains were strongly phagocytized, with participation by nonspecific opsonins (complement C3 and nonspecific cofactor), irrespective of their M protein content. M + R--disbalance (selected collection strains) and M--R + disbalance (field strains on artificial media) were observed. The M and R resistance factor equipment of the streptococci probably represents plus and minus variants in the diverse process of the asymptomatic dissemination and pathogenic activity of streptococci in the human population. The given results are manifestly related to the general manner of existence of streptococci, to questions of conditional pathogenicity and to the concept of nonspecific defence against streptococci.


Comparison of methods for the isolation of genital mycoplasmas from men.

Two hundred nine men were studied to determine the optimal method of obtaining cultures for genital mycoplasma. Ureaplasma urealyticum (T-mycoplasmas) was isolated from 95 (45.5%) of the participants. Urethral cultures obtained by means of urethrogenital calcium alginate swabs identified 82 (86%) of the 95 colonized men. Urethral cultures taken with cotton-tipped applicators (76%) urine cultures (27%), and cultures of the coronal sulcus (24%) detected fewer colonized men. All men who were colonized with U. urealyticum were identified by one of the two urethral cultures. Mycoplasma hominis was recovered from 73 (34.9%) of the 209 men. Urethral cultures identified most of the circumcised men who were colonized with M. hominis (11 of 14; 79%). In contrast, cultures from the coronal sulcus detected most of the colonized uncircumcised men (49 of 59; 83%). More than 90% of the men who were colonized with M. hominis were identified by either urethral culture or culture of the coronal sulcus. A similar study was conducted among 143 normal college students yielded comparable results.


Nonspecific resistance to infection induced in mice by a water-soluble adjuvant derived from Mycobacterium smegmatis.

The effect of a nontoxic, water-soluble adjuvant (Neo-WSA) from delipidated cells of Mycobacterium smegmatis on the susceptibility of mice to infection with four challenge organisms was studied. An intravenous dose of 1 mg of Neo-WSA per mouse 24 hr before challenge enhanced resistance to infection with a fungus (Candida albicans), a gram-negative bacterium (Klebsiella pneumoniae), and a gram-positive bacterium (Streptococcus pneumoniae). Protection by Neo-WSA was not significant when the mice were challenged with a malarial parasite. Plasmodium berghei. When 1 mg of Neo-WSA was given intravenously to mice 10 min before challenge with C. albicans, protection was significant, but when the same dose was given two or six days prior to challenge, mice were not protected. The concentration of iron in serum had not changed significantly 1 or 24 hr after the intravenous injection of 1 mg of Neo-WSA. Thus Neo-WSA is capable of inducing nonspecific resistance to certain experimental infections in mice. The protection afforded by administration of Neo-WSA 10 min before challenge, the lack of protection afforded by administration of Neo-WSA six days before challenge, and the lack of significant change in the serum iron concentration clearly separate this compound from bacterial endotoxins, which are classical inducers of nonspecific resistance to infection.


Delayed hypersensitivity to fungal antigens in mice. II. Characterization of the active component in immunogenic RNA extracts.

In a mouse model, cell-mediated immunity to Coccidioides immitis, as assayed by the delayed hypersensitivity skin test, was transferred with whole immunogenic RNA extract and its greater than 33S and 6S-13S sedimentation fractions. Both fractions were cleaved by RNase, but the products retained their transfer activity. The greater than 33S fraction of immunogenic RNA extract was inactivated by pronase, whereas the 6S-13S fraction was resistant to the proteolytic enzyme; however, after RNase treatment the latter fraction was sensitive to pronase. This finding suggests a protective role for RNA. Dialysis of immunogenic RNA extract yielded a dialysate with a ratio of absorbance at 260 nm to that at 280 nm (A260:A280) of 1.02. Similarly, the dialysis product of RNase-treated RNA is active and has an A260:A280 ratio of 1.34. The data indicate that at least part of the active moiety of immunogenic RNA extracts is an RNA-associated, pronase-labile peptide or nucleopeptide. Furthermore, it is possible that the dialyzable transfer factor may be the same peptide or nucleopeptide cleaved from immunogenic RNA during preparation of the transfer factor.


Resistance of Shigella dysenteriae type 1 to ampicillin and other antimicrobial agents: strains isolated during a dysentery outbreak in a hospital in Mexico City.

In June 1972, an epidemic of dysentery began in a hospital ward lodging 22 children with tuberculosis. Fifteen of them developed the disease and five children died. The age of the children ranged from five months to four years. A rectal swab culture taken from all hospitalized children three weeks after the initiation of the outbreak revealed Shigella dysenteriae type 1 in five of the patients (28%). The strains isolated were susceptible to cephalothin, gentamicin, kanamycin, colistin, trimethoprim, and nalidixic acid, but were resistant to ampicillin (greater than 5,000 mug/ml), chloramphenicol (300 mug/ml), streptomycin (400 mug/ml), tetracycline (100 mug/ml), and sulfadiazine (1,000 mug per disk). Transfer experiments to Escherichia coli K-12 indicated that these strains were infected with two different plasmids; one was responsible for resistance to chloramphenicol, tetracycline, streptomycin, and sulfonamides, and the other caused resistance to ampicillin. The epidemiological and clinical importance of these findings is emphasized.


[Bonding strength between porcelain and gold alloys. (2) The effects of iron, indium and tin (author's transl)].

Ten kinds of alloys were prepared for testing by addition of 0-0.75 wt% iron, indium and tin (total amount 0.75 wt%) to base alloy, 88 Au-6 Pt-5 Pd-1 Ag. Opaque porcelain (Ceramco Inc.) was fused to these alloys according to the manufacturer's directions. The bonding strength was determined by the shear bond test that had been described in the preceding paper and the effects of the trace elements were examined.


[Flow of molten metal in denture base in horizontal centrifugal casting procedure. (Part 1) Flow, inflow volume and casting time of molten metal passing through single aprue into disk type mold (author's transl)].

A pyrex glass plate was fitted at the bottom of casting ring, and disk type wax pattern (thickness. 0.43 mm) was put on the plate. Five types of sprueing were applied. Pure tin was casted using holizontal centrifugal casting machine. Flow of molten metal was filmed by the motor drive camera with the method of stroboscope. The results were summarized as follows. 1) When the sprue was attached at the center of the disk type mold vertically, moten metal flowed like a concentric circle at the early stage of casting. It was affected gradually by the direction of gravity and revolution, and it filled the mold from the lower part to the upper part. 2) When the sprue gate was attached to the side edge of the mold, and the sprue gate was placed to the forward and backward direction against the revolution direction, molten metal filled from lower part to the upper part. 3) When the sprue gate was placed against upper edge, molten metal flow was affected by the direction of gravity and revolution. When the sprue gate was placed against lower edge, molten metal filled quietry from the lower part to the upper part. 4) Inflow volume per unit time (inflow rate) was small at the early stage of casting. Inflow rate increased and became constant at the next stage. At the latter stage it became small again. 5) Inflow rate increased with the increase of area of sprue. 6) The time which was necessary to fill the volume of 1 cm (about 80% of the mold volume) became short with the increase of area of sprue. It was also influenced by the type of sprueing.


[Mechanical studies on the dental bridges. (XI) Studies on the apportionment loads to be affected by the supporting conditions of two abutment teeth (author's transl)].

Abutment teeth have been treated as the elastic behavior at the supported conditions and apportionment loads are depended upon the elastic properties of the pontics and abutment teeth. So, this paper shows the apportionment loads of dental bridges and the dependence of the elastic constants of abutment teeth. Abutment teeth are rotated and sinked with digestive power on the pontics. So, the elastic constants of abutment teeth are given with k and K, which k, K are the sinked and rotated coefficients, which are expressed the elastic properties of these abutment teeth. Apportionment loads to be connected with the flexural rigidity of the pontics are calculated for two numerical examples (for the values of 3.6 x 10(3) kg-mm2 and 3.6 x 10(8) kg-mm2 of the concentrated and distributed loads).


[Slip casting of stainless steel powder (author's transl)].

Slip casting of stainless steel powder (AISI type 316 L) was investigated as means of forming medical and dental porous restorations. This research was undertaken to evaluate the effects of the particle size and aging of casting slip and firing conditions. Bulk density was used as a measure of the degree of sintering. Water contents of casting bodies decreased with the particle size and its casting rates, bulk densities and bending strengths increased. Aging of slip decreased casting rates, water contents and bending strengths of the casts. The bulk densities of the sintered stainless steel increased with sintering time and temperature. The porosities of the materials decreased with the particle size and the elevating temperature. The bending strengths of the materials increased sharply with the decreasing particle size. The optical micrographs did not always show the uniform elimination of pores in the sintered. Aging of slip increased a little the bulk densities of the materials and decreased the porosity and the bending strength.


Immunity to the group B streptococci: interaction of serum and macrophages with types Ia, Ib, and Ic.

The opsonization and phagocytosis of group B streptococci of types Ia, Ib, and Ic were studied in vitro by measuring the uptake of radioactivity by coverslip cultures of rabbit alevolar macrophages during incubation with radiolabeled, nonviable bacteria which had been exposed to rabbit serum. The uptake of counts per minute was quantitative, reproducible, and reversibly inhibited by cold, indicating that it was largely a measurement of phagocytic ingestion rather than of attachment of bacteria-immunoglobulin complexes to macrophage membranes. Moreover, suspended macrophages killed approximately 90% of viable streptococci in the presence of specific antiserum. The opsonic activity of immune serum was heat stable, and phagocytosis of streptococci was insignificant after incubation with normal serum and antiserum to some heterologous group B streptococci. By absorption studies, it was possible to identify the effect of antibodies to specific bacterial antigens. Phagocytosis of streptococci containing the corresponding antigens was maximal after opsonization with homologous or heterologous sera containing antibody to IaCHO, IbCHO, or Ibc protein. Phagocytosis of all three serotypes was intermediate when opsonization could be attributed to anti-IabcCHO. The opsonization of a specific group B streptococcus is complex and may involve two or more antigen-antibody systems.


Susceptibility of mice to group B coxsackie virus is influenced by the diabetic gene.

A positive correlation was found between genetic predisposition to diabetes in the mouse and susceptibility to group B Coxsackie virus in this host. Male mice of the inbred strain C57BL/Ks and the following genetic variants were used; mice homozygous for the autosomal recessive gene for diabetes (db/db), the phenotypically normal heterozygous (db/+), and the normal mice which lacked the diabetic gene (+/+). The mortality response of the +/+ mice to intraperitoneal inoculation with Coxsackie virus B4 differed from the response of the two genetic variants (db/db and db/+) derived from this strain. The db/+ variant was more susceptible to Coxsackie virus B4 than the parental background strain (+/+). The db/db variant was more susceptible than either of the other genotypes. Pathological findings of the pancreas of the three genotypes during the acute stage of infection closely paralleled the genotypically dependent susceptibility of the host.


Influence of embryonic estrogens on adult feather coloration in the duck Anas platyrhynchos.

Embryos of the Rouen duck were treated on day 11 to 18 with a single injection of estradiol benzoate in the air chamber. After hatching, skin grafts were exchanged between male ducklings that had been treated with estrogen in ovo and control males. The results showed that the skin of male embryos can be permanently feminized to the same extent as untreated female embryos simply by exposing embryonic male skin to female sex hormones during embryogenesis.


Selective reinnervation in skin rotation grafts in Rana pipiens.

Skin rotation grafts were performed in Rana pipiens tadpoles producing misdirected reflexes in adult frogs. The pattern of innervation fields of dorsal and ventral rami of segmental nerves 3, 4, 5 and 6 were investigated by recording from the severed rami with suction electrodes. Innervation patterns of regenerated fields were influenced by graft borders and adjacent fields. Misdirected reflexes were found for ventral skin on the dorsal side only when innervated by ventral rami in a manner never seen in normal or sham operated animals. This suggested that selective reinnervation has occurred.


Radioimmunoassay technology in mass drug screening: an evaluation of an absorbent paper disk transport system.

These studies have demonstrated the feasibility of using urine-saturated paper disks in place of urine in the RIA system for drug abuse detection. Results with the disks are consistent with those using urine. A satisfactory procedure has been devised which provides reproducibility of results with no loss of sensitivity or specificity. Further, the procedure is essentially the same as the current procedure requiring urine except that a paper disk punched from a filter paper strip impregnated with urine is used. Complete flexibility is retained to switch from urine to disk. No new or additional equipment is required. It is envisioned that the urine would remain at the collection site and dried filter paper strips containing urine under test be shipped to toxicology laboratories. Sould the disk assay be positive, the urine specimen identified with that disk could then be shipped to the laboratory for confirmation by gas-liquid chromatography or other acceptable methods. The time and expense incurred in shipping large volumes of urine would thus be eliminated.


R factor-mediated resistance to ultraviolet light in strains of Escherichia coli deficient in known repair functions.

The expression of resistance to u.v. irradiation mediated by R factor R46 has been studied in strains deficient in excision repair and recombination repair. The R factor protected wild-type bacteria and also wild-type cells in which repair had been inhibited by the substitution of bromouracil for chromosomal thymine. It increased the survival of strains defective in the endonucleolytic (uvr), repolymerizing (pol) and joining (lig) stages of the excision repair process. Recombination deficient bacteria mutant at the recB or recC loci were protected by R46, but the R factor had little effect on the survival of a recA strain or a recA recB double mutant. R46 increased the survival of cells that had been treated with chloramphenicol before u.v. irradiation, but did not protect cultures treated with chloramphenciol after irradiation. It is concluded that R46 confers resistance to the lethal effects of u.v. irradiation by a mechanism that is independent of excision repair. Resistance appears to be mediated by an inducible gene product, which is possibly a nuclease and dependent on a functional host recA gene for expression.


Derivation and properties of F-prime factors in Escherichia coli carrying nitrogen fixation genes from Klebsiella pneumoniae.

A His+ Nif+ Escherichia coli K12, Hfr strain (UNF43) was constructed by an intergeneric mating between a Klebsiella pneumoniae donor strain (HF3) and a his-HFR E. coli strain (SBI824) which transfers his as an early marker. An F-prime nif plasmid, FN39, carrying genes which correspond to the E. coli chromosomal region, metG gnd his shiA, but excluding purF and aroD, was isolated from UNF43. Translocation of carbenicillin resistance genes from a P-type R-factor, R68, to FN39 increased the stability of his and nif on the derivative F-prime, FN68. Sedimentation analysis of both F-primes in sucrose gradients revealed our covalently closed circular(CCC) DNA species of molecular weights 279 +/- 9, 136 +/- 3, 90 +/- 1 and 44 +/- 1 megadaltons. It is suggested that the two smallest CCC-DNA species are component replicons of the composite F-primes of molecular weight 136 +/- 3 megadaltons, and that the molecules of 279 +/- 9 megadaltons are CCC-dimers. FN68 was transferable in intergeneric matings to Klebsiella aerogenes, K. pneumoniae and Salmonella typhimurium but not to Proteus mirabilis; only carbenicillin resistance and sex factor activity were transferred to Erwinia herbicola. nif genes on FN68 were expressed in a Nif- mutant of K. pneumoniae and also in S. typhimurium, which in conventional tests is naturally non-nitrogen-fixing; expression of the his determinant of FN68 became temperature-sensitive in S. typhimurium.


Genetic analysis of deletions of R100-1 that are both transfer-deficient and tetracycline-sensitive.

The extent of the deletions of five Tets Tra- mutants of R100-1 was determined by complementation experiments with wild-type and tra mutants of Flac. The presence or absence of the origin of transfer on the mutants was also investigated. Using the results, a tentative map of this region of the R factor was drawn: it was essentially similar to the analogous region of the E. coli K12 F factor, except that tet was located between traJ and traA. Some of the deletions had removed the promoter for the transfer operon. This allowed detection of the transcription of traC and distal genes from a weak, traJ-independent promoter. This is probably the Is2 promoter, since R100-1 carries and Is2 insertion sequence located immediately to the left of traC in the correct orientation. Since neither the transfer operon promoter nor the Is2 promoter seemed  to be required for transcription of traI, it was concluded that, unlike the F factor, this was located in a separate operon.


Intra-species tranduction with Proteus mirabilis high frequency transducing phages.

The properties of three additional Proteus mirabilis hosts for the high frequency transducing (HET) phages 5006MHFTkappa and 5006MHFTak are described. The phages transduce resistance to kanamycin and to ampicillin plus kanamycin, respectively, and were produced by ultraviolet induction of derivatives of P. mirabilis strain PM5006. Strain PM804 could not be shown to adsorb the phages although it yielded a few transductants. All transductants, even those produced at low multiplicities of phage input were lysogenic and segregated markers at high frequency.  Ultraviolet induced phage lysates of these transductants transduced PM804 at higher frequencies and PM5006 at lower frequencies than the original phages. Strain PM804 or its derivatives did not adsorb phage from these lysates. Tranmission experiments through PM5006 of phage in the transductant lysates confirmed that PM804 had a host-controlled modification system which modified HFT phage from PM5006. That PM804 also possessed a restriction system was inferred from the greater numbers of transductants obtained with page which bore a compatible modification pattern. Strain N and a restrictionless mutant of it named NHI, adsorbed the HET phages avidly and failed to modify their host range. Transduction frequencies of the phage markers were about 10(-4)/p.f.u. adsorbed to strain NHI and only about tenfold lower to strain N which did not plate the phages. Tranductants also had the features of heterogenotes and those obtained at low multiplicites of infection were non-lysogenic. The latter transductants adsorbed homolgous phage while NHI transductants also plated the HFT lysates. Strain NHI, lysogenized by the parent phage 5006M, did not adsorb the HFT phages. These findings suggest that the HFT phages were most likely defective for lysogenic conversion to homologous phage non-adsorption. The postulated restriction enzymes of PM804 and strain N could not be shown to be thermolabile.