Biochemical changes during sporulation of Bacillus stearothermophilus.

The process of sporulation was studied in Bacillus stearothermophilus. A medium is described that supports good growth and sporulation of the organism. In this medium, which contains glucose, salts, and amino acids, acetate starts to accumulate before any of the glucose is catabolized. Enzymes of the tricarboxylic acid cycle are present at all times during growth and sporulation and are found in dormant spores. As the glucose in the culture is consumed, acetate rapidly increases and the pH of the medium drops. The acetate rapidly disappears during sporulation and the pH rises. Dipicolinic acid appears during sporulation and several key-enzyme activities fluctuate in a characteristic pattern.     

Metabolism of gamma-glutamyl amino acids and peptides in mouse liver and kidney in vivo.

The metabolism in vivo of gamma-glutamyl amino acids and peptides was studied in the mouse after administration of loading doses of L-gamma-glutamyl-2-aminobutyrate and several other gamma-glutamyl compounds, including glutathione. A great and rapid accumulation of glutamate, glutamine, aspartate and pyrrolidone carboxylate was observed in the kidney. Similarly, after administration of a tracer dose of L-gamma-[14C]glutamyl-L-2-aminobutyrate a rapid incorporation of label into kidney glutamate, glutamine and aspartate was found. These results suggest that both the hydrolytic and gamma-glutamyl transfer reactions catalyzed by gamma-glutamyl transpeptidase are active in the renal handling of gamma-glutamyl compounds. Indirect evidence was obtained that L-gamma-glutamyl-2-aminobutyrate is partially taken up by the kidney cell in an intact form. In contrast to the kidney, administration of several gamma-glutamyl derivatives did not cause an increase in liver glutamate, glutamine and pyrrolidone carboxylate. After administration of L-gamma-glutamyl-2-aminobutyrate only a slight increase in liver aspartate and pyrrolidone carboxylate was observed. Experiments with L-gamma-[14C]glutamyl-L-2-aminobutyrate suggest that this derivative is largely first degraded to its component amino acids (probably in the kidney) before entering into the metabolism of the liver cell. gamma-Glutamyl transpeptidase may function in the metabolism and transport of glutathione and other gamma-glutamyl compounds in a manner analogous to the function of dipeptidases and disaccharidases in the metabolism and transport of dipeptides and disaccharides respectively.     

Inactivation of glucose 6-phosphate dehydrogenase during germination and outgrowth of Bacillus cereus T endospores.

The specific activity and total activity of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) under conditions of complete cell breakage fall 10-20-fold during a 3h period of spore germination and outgrowth. The spores must germinate (lose refractility), but do not have to undergo outgrowth, for the loss of activity to occur. Glucose 6-phosphate dehydrogenase activity from cells as any stage of development is completely stable in extracts at 4 degrees C or 30 degrees C. All of the enzyme activity is found in a soluble (50000g supernatant) fraction and remains completely soluble throughout development. Soluble protein and total cellular protein remain constant for about 2h. Proteinases could not be detected or protein turnover demonstrated during the morphogenetic process. Phenylmethanesuophony fluoride and o-phenanthroline, inhibitors of proteolytic enzymes, do not prevent glucose 6-phosphate dehydrogenase inactivation when added to whole cells. Mixing experiments show no inhibitor of glucose 6-phosphate dehydrogenase to be present in late-stage cells. The enzyme is not excreted into the culture medium. Chloramphenicol and rifampicine immediately stop protein synthesis and development but not the inactivation of glucose 6-phosphate dehydrogenase. NaN3, 2,4-dinitrophenol or anaerobiosis immediately stop development and prevent the loss of enzyme activity. A requirement for metabolic energy is therefore probable. Extracts of spores pre-labelled with L[14C]leucine were made at various stages of morphogenesis and subjected to polyacrylamide-gel electrophoresis. Glucose 6-phosphate dehydrogenase, which was identified by a specific stain, did not lose 14C label, and therefore may not be degraded during the inactivation process.     

Determination of pyrrolidone carboxylate and gamma-glutamyl amino acids by gas chromatography.

Gas chromatographic methods for the quantitation of pyrrolidone carboxylate and γ-glutamyl amino acids are described. These intermediates of the γ-glutamyl cycle were separated by ion exchange chromatography and converted to their N-acyl-ester derivatives in a reaction with a mixture of 2,2,3,3,3-pentafluoro-1-propanol and pentafluoropropionic anhydride. The derivatives have excellent electron capture properties thus making possible their determination even in small amounts of material of biological origin. The method was applied for the determination of concentrations of pyrrolidone carboxylate in human urine and cerebrospinal fluid, and in the brain, liver, and kidney of the mouse. It was also used to demonstrate the formation in mouse tissues of several γ-glutamyl derivatives of amino acids after administration of the corresponding free amino acid.     

Analytical validation of quantitative immunohistochemical assays of tumor infiltrating lymphocyte biomarkers

Tumor infiltrating lymphocytes (TIL), especially T-cells, have both prognostic and therapeutic applications. The presence of CD8+ effector T-cells and the ratio of CD8+ cells to FOXP3+ regulatory T-cells have been used as biomarkers of disease prognosis to predict response to various immunotherapies. Blocking the interaction between inhibitory receptors on T-cells and their ligands with therapeutic antibodies including atezolizumab, nivolumab, pembrolizumab and tremelimumab increases the immune response against cancer cells and has shown significant improvement in clinical benefits and survival in several different tumor types. The improved clinical outcome is presumed to be associated with a higher tumor infiltration; therefore, it is thought that more accurate methods for measuring the amount of TIL could assist prognosis and predict treatment response. We have developed and validated quantitative immunohistochemistry (IHC) assays for CD3, CD8 and FOXP3 for immunophenotyping T-lymphocytes in tumor tissue. Various types of formalin fixed, paraffin embedded (FFPE) tumor tissues were immunolabeled with anti-CD3, anti-CD8 and anti-FOXP3 antibodies using an IHC autostainer. The tumor area of stained tissues, including the invasive margin of the tumor, was scored by a pathologist (visual scoring) and by computer-based quantitative image analysis. Two image analysis scores were obtained for the staining of each biomarker: the percent positive cells in the tumor area and positive cells/mm² tumor area. Comparison of visual vs. image analysis scoring methods using regression analysis showed high correlation and indicated that quantitative image analysis can be used to score the number of positive cells in IHC stained slides. To demonstrate that the IHC assays produce consistent results in normal daily testing, we evaluated the specificity, sensitivity and reproducibility of the IHC assays using both visual and image analysis scoring methods. We found that CD3, CD8 and FOXP3 IHC assays met the fit-for-purpose analytical acceptance validation criteria and that they can be used to support clinical studies.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

The cardiac electrogenic sodium/bicarbonate cotransporter (NBCe1) is activated by aldosterone through the G protein-coupled receptor 30 (GPR 30)

The sodium/bicarbonate cotransporter (NBC) transports extracellular Na⁺ and HCO₃^? into the cytoplasm upon intracellular acidosis, restoring the acidic pH_i to near neutral values. Two different NBC isoforms have been described in the heart, the electroneutral NBCn1 (1Na⁺:1HCO₃^?) and the electrogenic NBCe1 (1Na⁺:2HCO₃^?). Certain non-genomic effects of aldosterone (Ald) were due to an orphan G protein-couple receptor 30 (GPR30). We have recently demonstrated that Ald activates GPR30 in adult rat ventricular myocytes, which transactivates the epidermal growth factor receptor (EGFR) and in turn triggers a reactive oxygen species (ROS)- and PI3K/AKT-dependent pathway, leading to the stimulation of NBC. The aim of this study was to investigate the NBC isoform involved in the Ald/GPR30-induced NBC activation. Using specific NBCe1 inhibitory antibodies (a-L3) we demonstrated that Ald does not affect NBCn1 activity. Ald was able to increase NBCe1 activity recorded in isolation. Using immunofluorescence and confocal microscopy analysis we showed in this work that both NBCe1 and GPR30 are localized in t-tubules. In conclusion, we have demonstrated that NBCe1 is the NBC isoform activated by Ald in the heart.     

Overrepresentation of interactions between homologous proteins in interactomes

It is well proved that the probability that a protein interacts with itself is higher than that it interacts with another protein. It has been recently shown that the probability of interaction is also higher for proteins with significant sequence similarity. In this paper we show that proteins sharing identical PFAM domains interact more often than expected by chance in Saccharomyces cerevisiae and Escherichia coli. We also analyze the variety of domain interfaces used by homologous proteins to interact and show that the overrepresentation of interactions between homological proteins is not caused by small number of pairs of identical "sticky domains" shared between interacting proteins.     

Locally enhanced sampling molecular dynamics study of the dioxygen transport in human cytoglobin

Cytoglobin (Cyg)--a new member of the vertebrate heme globin family--is expressed in many tissues of the human body but its physiological role is still unclear. It may deliver oxygen under hypoxia, serve as a scavenger of reactive species or be involved in collagen synthesis. This protein is usually six-coordinated and binds oxygen by a displacement of the distal HisE7 imidazole. In this paper, the results of 60 ns molecular dynamics (MD) simulations of dioxygen diffusion inside Cyg matrix are discussed. In addition to a classical MD trajectory, an approximate Locally Enhanced Sampling (LES) method has been employed. Classical diffusion paths were carefully analyzed, five cavities in dynamical structures were determined and at least four distinct ligand exit paths were identified. The most probable exit/entry path is connected with a large tunnel present in Cyg. Several residues that are perhaps critical for kinetics of small gaseous diffusion were discovered. A comparison of gaseous ligand transport in Cyg and in the most studied heme protein myoglobin is presented. Implications of efficient oxygen transport found in Cyg to its possible physiological role are discussed.