Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the M_r-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.     

The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+−K+ pump in erythrocytes

Cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the $\text{Na}{}^{\mathrm{+}}\text{?K}{}^{\mathrm{+}}$ pump. This difference in sensitivity to membrane lipid order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.     

Microsomal cytochromes of Candida tropicalis grown on alkanes

A comparison of methods used in isolating microsomes and in measuring microsomal cytochrome P-450 demonstrated that separation following protoplast lysis gave the best results. By this latter technique a high amount of cytochrome P-450 (0.2–0.3 nmol/mg) was recovered but cytochrome P-420, considered as the denatured form, was absent.The alkanes specifically induce cytochromes P-450 and b₅ localized on the microsomes. The denaturation in vivo of cytochrome P-450 into cytochrome P-420 even occurs during storage at 1 °C. This degradation is increased during preparation of subcellular fractions if no preventive measures are taken.     

Exploring Beneficial/Virulence Properties of Two Dairy-Related Strains of Streptococcus infantarius subsp. infantarius

The genus Streptococcus includes various species, remarkably different in their behavior, applications, virulence, and safety. Taxonomically Streptococcus infantarius subsp. infantarius belonging to the Streptococcus bovis group, which includes several pathogen species, however, has been found as predominant species in some African dairy products that are widely consumed and considered to be safe. Streptococcus infantarius subsp. infantarius’ safety may be questioned due to the association of this species with clinical cases. In this study, isolates from dairy origin were selected based on their bacteriocinogenic potential and differentiated by their RAPD-PCR profiles. Two strains were identified by 16S rRNA sequencing as St. infantarius subsp. infantarius and investigated regarding their potential beneficial properties and factors related to virulence and safety. A series of in vitro tests included properties related to survival in the gastrointestinal tract and beneficial intestinal activities. Production of bacteriocin/s, detection of related genes, and partial characterization of expressed antimicrobial protein were evaluated. Genes related to folate biosynthesis were detected in both studied strains. Evaluation of physiological tests related to strains virulence, adhesion, and resistance to antibiotics and detections of virulence and biogenic amines production-related genes were also investigated. Taking in consideration all the aspects of the specific nature of St. infantarius subsp. infantarius K1–4 and K5–1 (beneficial properties and virulence characteristics), both strains cannot be considered safe for human or other animals application, even though they have been isolated from dairy products. This study is highlighting the importance of evaluation for presence of potential virulence factors in newly characterized strains in order to be confident in their safety.

     

mTORC1 activity is supported by spatial association with focal adhesions

The mammalian target of rapamycin complex 1 (mTORC1) integrates mitogenic and stress signals to control growth and metabolism. Activation of mTORC1 by amino acids and growth factors involves recruitment of the complex to the lysosomal membrane and is further supported by lysosome distribution to the cell periphery. Here, we show that translocation of lysosomes toward the cell periphery brings mTORC1 into proximity with focal adhesions (FAs). We demonstrate that FAs constitute discrete plasma membrane hubs mediating growth factor signaling and amino acid input into the cell. FAs, as well as the translocation of lysosome-bound mTORC1 to their vicinity, contribute to both peripheral and intracellular mTORC1 activity. Conversely, lysosomal distribution to the cell periphery is dispensable for the activation of mTORC1 constitutively targeted to FAs. This study advances our understanding of spatial mTORC1 regulation by demonstrating that the localization of mTORC1 to FAs is both necessary and sufficient for its activation by growth-promoting stimuli.     

Shadoo/PrP (Sprn0/0/Prnp0/0) double knockout mice

Shadoo (Sho) is a brain glycoprotein with similarities to the unstructured region of PrP^C. Frameshift alleles of the Sho gene, Sprn, are reported in variant Creutzfeldt-Jakob disease (vCJD) patients while Sprn mRNA knockdown in PrP-null (Prnp^0/0) embryos produces lethality, advancing Sho as the hypothetical PrP-like “pi” protein. Also, Sho levels are reduced as misfolded PrP accumulates during prion infections. To penetrate these issues we created Sprn null alleles (Daude et al., Proc. Natl. Acad. Sci USA 2012; 109(23): 9035–40). Results from the challenge of Sprn null and TgSprn transgenic mice with rodent-adapted prions coalesce to define downregulation of Sho as a “tracer” for the formation of misfolded PrP. However, classical BSE and rodent-adapted BSE isolates may behave differently, as they do for other facets of the pathogenic process, and this intriguing variation warrants closer scrutiny. With regards to physiological function, double knockout mice (Sprn^0/0/Prnp^0/0) mice survived to over 600 d of age. This suggests that Sho is not pi, or, given the accumulating data for many activities for PrP^C, that the pi hypothesis invoking a discrete signaling pathway to maintain neuronal viability is no longer tenable.     

Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms'' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions.     

Molecular cloning,expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.     

Extending a Model of Shift‐Work Tolerance

The present study contributes to theory and practice through the development of a model of shift‐work tolerance with the potential to indicate interventions that reduce nurses' intention toward turnover and increase job satisfaction in hospital‐based settings. Survey data from 1257 nurses were used to conduct structural equation modeling that examine the direct and indirect effects of supervisor and colleague support, team identity, team climate, and control over working environment on time‐based work/life conflict, psychological well‐being, physical symptoms, job satisfaction, and turnover intention. The analysis of the proposed model revealed a good fit The chi‐square difference test was non‐significant (χ²(26)=338.56), the fit indices were high (CFI=.923, NFI=.918, and NNFI=.868), the distribution of residuals was symmetric and approached zero, the average standardized residual was low (AASR=.04), and the standardized RMR was. 072. In terms of the predictor variable, the final model explained 48% of the variance in turnover intention. The data revealed considerable evidence of both direct effects on adjustment and complex indirect links between levels of adjustment and work‐related social support, team identity, team climate, and control. Nurses with high supervisor and coworker support experienced more positive team climates, identified more strongly with their team, and increased their perceptions of control over their work environment. This in turn lowered their appraisals of their time‐based work/life conflict, which consequently increased their psychological well‐being and job satisfaction and reduced their physical health symptoms and turnover intention. The type of shift schedule worked by the nurses influenced levels of turnover intention, control over work environment, time‐based work/life conflict, and physical symptoms.