Physicochemical and Biological Properties of Sonically Treated Vi Antigen

Electrophoretically purified Vi antigen from Citrobacter freundii 5396/38 was depolymerized by sonic treatment. The treatment caused an 80% reduction in specific viscosity and a reduction in molecular weight from 1.6 x 10(6) to 3.9 x 10(4). The O-acetyl and N-acetyl contents of the antigen and its infrared spectrum remained unchanged. The sonically treated antigen was only 1% as effective as the original antigen in eliciting protection in mice against challenge with Salmonella typhi. Sonically treated antigen also elicited lower antibody titers after single injections in mice and rabbits. No loss in ability to precipitate antibody or to sensitize red blood cells for hemagglutination was observed.     

Magnetic resonance and kinetic studies of pyruvate, phosphate dikinase. Interaction of oxalate with the phosphorylated form of the enzyme.

Pyruvate, orthophosphate dikinase (EC 2.7.9.1) carries out its catalytic function in three successive partial reactions, the final step being the reaction of pyruvate with a stable phosphoenzyme intermediate to give phosphoenolpyruvate and free enzyme (Evans, H.J., and Wood, H. G. (1968), Proc. Natl. Acad. Sci. U.S.A. 61, 1448). Interactions of oxalate, a structural analog of enolpyruvate, with the phosphorylated form of the enzyme have been investigated by kinetic inhibition measurements and by magnetic resonance studies of manganous ion complexes with the enzyme. Oxalate inhibits the reaction catalyzed by pyruvate, phosphate dikinase, and the inhibition is linearly competitive with respect to pyruvate. The inhibitor constant for oxalate of 25 mu-M is fourfold lower than the Michaelis constant for pyruvate. The enhancement in the longitudinal relaxation rate of water protons (PRR) which occurs upon binding of Mn(II) to the enzyme has been used to monitor binding of oxalate to Mn(II)-enzyme complexes. PRR titrations indicate that the dissociation constant of oxalate from the Mn(II) complex of the free form of the enzyme is an order of magnitude weaker than the kinetically determined Ki. On the other hand, titrations of solutions which contain the phosphorylated form of the enzyme reveal a much stronger binding of oxalate. Moreover, the strength of oxalate binding to the phosphorylated enzyme is a function both of the species and of the concentration of monovalent cations in the solution. In the presence of Tl+, which has the most favorable activator constant for the final partial reaction, the dissociation constant for oxalate from its complex with the phosphorylated enzyme is less than 1 mu-M. Electron paramagnetic resonance (EPR) spectra for the enzyme-bound Mn(II) are sensitive to structural perturbations which occur upon binding of substrates or of oxalate to the enzyme. The EPR spectrum for the Mn(II)-phosphoenzyme-oxalate species is distinguished from spectra for other complexes of the enzyme by unusually narrow line widths and consequent resolution of fine structure from electronic quadrupole splitting. The narrow lines in the EPR spectrum are indicative of a rigid, pseudocrystalline environment for the bound Mn(II). The magnitude and frequency dependence of the PRR for the Mn(II)-phosphoenzyme-oxalate complex indicate that if any water molecules are bound to the Mn(II), their exchange with the bulk water is severely retarded. The kinetic and magnetic resonance studies support the hypothesis that oxalate mimics the reactive intermediate, enolpyruvate, in a complex with the phosphorylated enzyme which may resemble the structure of the transition state of the final partial reaction.     

The pathogenicity of an entomopoxvirus fromOthnonius batesi [Col.: Scarabaeidae] and its possible use as a control agent

In a natural population ofOthnonius batesi Oll. at Glen Innes, N.S.W., 4.0% of larvae in 1972 and 2.2% in 1973 were exhibiting symptoms of virus infection, whilst 0.8% of pupae and adults from the same population were infected in 1972. These figures, and field observations of infected larvae, suggested that the pathogenicity of the virus was low. In laboratory experiments withO. batesi the infection had little effect on mortality, and no significant effect on duration of the first instar, food intake, or larval growth. The vast accumulation of virus in the fat body probably results in mortality prior to, or during, the pupal period. Temperature had a marked effect on both % infection (optimum 30°C) and production of viral spheroids (optimum 25°C). Very little viral development occurred below 20°C. In a host range study onlyO. batesi, Rhopaea verreauxi Blanch. andR. morbillosa Blkb. were infectedper os. The possible use of the virus as a control agent is discussed.     

BACE-1 inhibitors part 2: identification of hydroxy ethylamines (HEAs) with reduced peptidic character

This paper describes the discovery of non-peptidic, potent, and selective hydroxy ethylamine (HEA) inhibitors of BACE-1 by replacement of the prime side of a lead di-amide 2. Inhibitors with nanosmolar potency and high selectivity were identified. Depending on the nature of the P(1)(') and P(2)(') substituents, two different binding modes were observed in X-ray co-crystal structures.     

Evaluating the Use of Blood Cultures in the Management of Children Hospitalized for Community-Acquired Pneumonia

BackgroundBlood cultures are often recommended for the evaluation of community-acquired pneumonia (CAP). However, institutions vary in their use of blood cultures, and blood cultures have unclear utility in CAP management in hospitalized children.ObjectiveTo identify clinical factors associated with obtaining blood cultures in children hospitalized with CAP, and to estimate the association between blood culture obtainment and hospital length of stay (LOS).MethodsWe performed a multicenter retrospective cohort study of children admitted with a diagnosis of CAP to any of four pediatric hospitals in the United States from January 1, 2011-December 31, 2012. Demographics, medical history, diagnostic testing, and clinical outcomes were abstracted via manual chart review. Multivariable logistic regression evaluated patient and clinical factors for associations with obtaining blood cultures. Propensity score-matched Kaplan-Meier analysis compared patients with and without blood cultures for hospital LOS.ResultsSix hundred fourteen charts met inclusion criteria; 390 children had blood cultures obtained. Of children with blood cultures, six (1.5%) were positive for a pathogen and nine (2.3%) grew a contaminant. Factors associated with blood culture obtainment included presenting with symptoms of systemic inflammatory response syndrome (OR 1.78, 95% CI 1.10–2.89), receiving intravenous hydration (OR 3.94, 95% CI 3.22–4.83), receiving antibiotics before admission (OR 1.49, 95% CI 1.17–1.89), hospital admission from the ED (OR 1.65, 95% CI 1.05–2.60), and having health insurance (OR 0.42, 95% CI 0.30–0.60). In propensity score-matched analysis, patients with blood cultures had median 0.8 days longer LOS (2.0 vs 1.2 days, P < .0001) without increased odds of readmission (OR 0.94, 95% CI 0.45–1.97) or death (P = .25).ConclusionsObtaining blood cultures in children hospitalized with CAP rarely identifies a causative pathogen and is associated with increased LOS. Our results highlight the need to refine the role of obtaining blood cultures in children hospitalized with CAP.     

Analysis of Euglena gracilis chloroplast deoxyribonucleic acid with a restriction endonuclease, EcoRI.

Cleavage of chloroplast deoxyribonucleic acid (DNA) of Euglena gracilis Z with restriction endonuclease RI from Escherichia coli (EcoRI) yielded 23 bands upon electrophoresis in gels of agarose. Four of the bands contained twice the stoichiometric amount of DNA. One of these bands contained two similarly sized fragments. The sum of the molecular weight of the 24 different fragments equaled the molecular weight of the circular molecule. The restriction fragments had different buoyant densities, with four having distinctly heavy densities in CsCl. Restriction fragments with a high buoyant density were preferentially lost when broken chloroplast DNA was purified by equilibrium density gradient centrifugation. Hybridization of chloroplast ribosomal ribonucleic acid to intact chloroplast DNA determined that there are two cistrons for 16S and 23S ribosomal ribonucleic acid. These two cistrons are located on six restriction fragments, all of which have buoyant densities greater than the intact molecule of chloroplast DNA.     

Eavesdropping in crabs: an agency for lady detection

Although conspicuous courtship displays are an effective way of attracting the attention of receptive females, they could provide valuable information to rival males on the location of these females. In fiddler crabs, males that see a receptive female wave their single, greatly enlarged claw in a highly conspicuous courtship display. We test whether other males use this courtship display to alert them to the presence of receptive females that they cannot directly see. We show that male fiddler crabs (Uca mjoebergi) eavesdrop on the courtship displays of nearby males to detect mate-searching females. This allows males to begin waving before a female becomes visible. Furthermore, males appear to adjust their waving according to the information available: eavesdropping males wave 12 times faster than non-courting males but only 1.7 times slower than males in full visual contact with the female.     

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.     

Proline transport and osmotic stress response in Escherichia coli K-12.

Proline is accumulated in Escherichia coli via two active transport systems, proline porter I (PPI) and PPII. In our experiments, PPI was insensitive to catabolite repression and was reduced in activity twofold when bacteria were subjected to amino acid-limited growth. PPII, which has a lower affinity for proline than PPI, was induced by tryptophan-limited growth. PPII activity was elevated in bacteria that were subjected to osmotic stress during growth or the transport measurement. Neither PPI nor uptake of serine or glutamine was affected by osmotic stress. Mutation proU205, which was similar in genetic map location and phenotype to other proU mutations isolated in E. coli and Salmonella typhimurium, influenced the sensitivity of the bacteria to the toxic proline analogs azetidine-2-carboxylate and 3,4-dehydroproline, the proline requirements of auxotrophs, and the osmoprotective effect of proline. This mutation did not influence proline uptake via PPI or PPII. A very low uptake activity (6% of the PPII activity) observed in osmotically stressed bacteria lacking PPI and PPII was not observed when the proU205 lesion was introduced.     

Increased insulin-stimulated Akt pSer473 and cytosolic SHP2 protein abundance in human skeletal muscle following acute exercise and short-term training.

The purpose of the present study was to determine in human skeletal muscle whether a single exercise bout and 7 days of consecutive endurance (cycling) training 1) increased insulin-stimulated Akt pSer(473) and 2) altered the abundance of the protein tyrosine phosphatases (PTPases), PTP1B and SHP2. In healthy, untrained men (n = 8; 24 +/- 1 yr), glucose infusion rate during a hyperinsulinemic euglycemic clamp, when compared with untrained values, was not improved 24 h following a single 60-min bout of endurance cycling but was significantly increased ( approximately 30%; P < 0.05) 24 h following completion of 7 days of exercise training. Insulin-stimulated Akt pSer(473) was approximately 50% higher (P < 0.05) 24 h following the acute bout of exercise, with this effect remaining after 7 days of training (P < 0.05). Insulin-stimulated insulin receptor and insulin receptor substrate-1 tyrosine phosphorylation were not altered 24 h after acute exercise and short-term training. Insulin did not acutely regulate the localization of the PTPases, PTP1B or SHP2, although cytosolic protein abundance of SHP2 was increased (P < 0.05; main effect) 24 h following acute exercise and short-term training. In conclusion, insulin-sensitive Akt pSer(473) and cytosolic SHP2 protein abundance are higher after acute exercise and short-term training, and this effect appears largely due to the residual effects of the last bout of prior exercise. The significance of exercise-induced alterations in cytosolic SHP2 and insulin-stimulated Akt pSer(473) on the improvement in insulin sensitivity requires further elucidation.