Complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase from rat mammary gland

The complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase (thioesterase II) from rat mammary gland is presented. Most of the sequence was derived by analysis of peptide fragments produced by cleavage at methionyl, glutamyl, lysyl, arginyl, and tryptophanyl residues. A small section of the sequence was deduced from a previously analyzed cDNA clone. The protein consists of 260 residues and has a blocked amino-terminal methionine and calculated Mr of 29,212. The carboxy-terminal sequence, verified by Edman degradation of the carboxy-terminal cyanogen bromide fragment and carboxypeptidase Y digestion of the intact thioesterase II, terminates with a serine residue and lacks three additional residues predicted by the cDNA sequence. The native enzyme contains three cysteine residues but no disulfide bridges. The active site serine residue is located at position 101. The rat mammary gland thioesterase II exhibits approximately 40% homology with a thioesterase from mallard uropygial gland, the sequence of which was recently determined by cDNA analysis [Poulose, A.J., Rogers, L., Cheesbrough, T. M., & Kolattukudy, P. E. (1985) J. Biol. Chem. 260, 15953-15958]. Thus the two enzymes may share similar structural features and a common evolutionary origin. The location of the active site in these thioesterases differs from that of other serine active site esterases; indeed, the enzymes do not exhibit any significant homology with other serine esterases, suggesting that they may constitute a separate new family of serine active site enzymes.     

Human hemoglobin Portland II (zeta 2 beta 2). Isolation and characterization of Portland hemoglobin components and their constituent globin chains

Two types of embryonic hemoglobins (Hb) containing zeta chains have been identified in the blood of several neonates of Chinese origin with homozygous alpha-thalassemia. In addition to Hb Portland I (zeta 2 gamma 2) which was previously reported, another embryonic hemoglobin has been detected and found to contain zeta chains and beta chains. It is being designated Hb Portland II and has the formula (zeta 2 beta 2). It has a mobility slightly slower than that of Hb A on starch gel electrophoresis at pH 8.6 and has been found in the hemolysates of blood of some but not all hydropic infants. Another component with a mobility faster than that of Hb A2 on starch gel has been isolated from the blood of some hydropic neonates. This latter component is postulated to be zeta 2 delta 2. The occurrence of Hb Portland I and Hb Portland II in these hydropic neonates is consistent with the hypothesis that, in the absence of normal alpha chain production, zeta chains are continued to be produced at later states of development than normal and form tetramers with each of the beta-like globin chains. Because Hb Portland II has not been found in blood from all hydropic neonates, we postulate that the presence of this hemoglobin in these fetuses may be correlated with the gestational age of the fetus at the time of birth.     

Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase

The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP- GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B- D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP- GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.      

Time-course of cardiac myocyte injury due to oxidative stress

Time course of changes in cell morphology, cation content, lipid peroxidation and high energy phosphates was examined in isolated rat cardiac myocytes exposed to oxygen radicals for 0 to 20 min. Xanthine (2 mM) and xanthine oxidase (10 U/L) mixture was used as a source of oxygen radicals. A significant decrease in the number of rod-shape cells with a concomitant increase in the number of hypercontracted cells was observed within 5 min of exposure to xanthine-xanthine oxidase (x-xo). At 10,15 and 20 min of exposure to x-xo, there was a time-dependant increase in the number of round cells. Lipid peroxide content, as indicated by the thiobarbituric acid reactive material, was significantly and progressively increased between 10 to 20 min of perfusion with x-xo. In myocytes exposed to x-xo, Ca²⁺ and Na⁺ were increased by 15% and 45% at 15 min and by 55% and 100% at 20 min respectively. Levels of adenosine tri- and di- phosphates were significantly depressed and that of adenosine mono- phosphate were higher at 20 min. These data support the hypothesis that reactive oxygen intermediates can directly influence myocyte structure and function, but these changes seem to occur more slowly in isolated myocytes than in whole hearts.     

Coronavirus and Paramyxovirus Shedding by Bats in a Cave and Buildings in Ethiopia

EcoHealth - Bats are important hosts of zoonotic viruses with pandemic potential, including filoviruses, MERS-Coronavirus (CoV), SARS-CoV -1, and likely SARS-CoV-2. Viral infection and transmission...     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Fungal fludioxonil sensitivity is diminished by a constitutively active form of the group III histidine kinase

The fungicide fludioxonil is used to control plant-pathogenic fungi by causing improper activation of the Hog1-type MAPK. However, the appearance of fludioxonil resistant mutants, mostly caused by mutations in the group III histidine kinases, poses a serious problem. Moreover, such mutations cause also hyperosmotic sensitivity and the underlying mechanism has been elusive for a long time. Using Saccharomyces cerevisiae as an experimental host, we show that those phenotypes are conferred by a constitutively active form of the group III histidine kinase. Our results explain the different reasons for fludioxonil resistance conferred by its deletion and missense mutation.     

Endotoxin enhances microvascular thrombosis in mouse cremaster venules via a TLR4-dependent, neutrophil-independent mechanism

Endotoxemia promotes adhesive interactions between platelets and microvascular endothelium in vivo. We sought to determine whether endotoxin (lipopolysaccharide, LPS) modified platelet thrombus formation in mouse cremaster venules and whether Toll-like receptor 4 (TLR4) and neutrophils were involved in the response. Intravital videomicroscopy was performed in the cremaster microcirculation of pentobarbital-anesthetized mice; venular platelet thrombi were induced with a light/dye endothelial injury model. C57BL/6 mice treated with Escherichia coli endotoxin had enhanced rates of venular platelet thrombus formation: the time to microvessel occlusion was reduced by approximately 50% (P < 0.005) compared with saline-treated animals. Enhanced microvascular thrombosis was evident as early as 2 h after LPS administration. LPS had no effect on thrombosis in either of two mouse strains with altered TLR4 signaling (C57BL/10ScNJ or C3H/HeJ), whereas it enhanced thrombosis in the control strains (C57BL/10J and C3H/HeN). LPS also enhanced platelet adhesion to endothelium in the absence of light/dye injury. Platelet adhesion, but not enhanced thrombosis, was inhibited by depletion of circulating neutrophils. LPS failed to enhance platelet aggregation ex vivo and did not influence platelet P-selectin expression, a marker of platelet activation. These findings support the notion that endotoxemia promotes platelet thrombus formation independent of neutrophils and without enhancement of platelet aggregation, via a TLR4-dependent mechanism.     

Substitutions of 2S and 7U chromosomes of Aegilops kotschyi in wheat enhance grain iron and zinc concentration

Biofortification through genetic manipulation is the best approach for improving micronutrient content of the staple food crops to alleviate hidden hunger, namely, the deficiency of Fe and Zn affecting more than two billion people worldwide. An interspecific hybridization was made between T. aestivum line Chinese Spring (CS) and Aegilops kotschyi accession 3790 selected for high grain iron and zinc concentration. The CS × Ae. kotschyi F₁ hybrid with low chromosome pairing was highly male and female sterile. This was backcrossed with wheat cultivars to get seed set. The selfed BC₁F₁ and BC₂F₁ plants with high grain iron and zinc concentration were selected in subsequent generations. The selected derivatives showed 60–136% enhanced grain iron and zinc concentration and 50–120% increased iron and zinc content per seed as compared to the recipient wheat cultivars. Thirteen cytologically stable, fertile and agronomically superior plants with high grain iron and zinc concentrations were selected for molecular characterization. The application of anchored wheat SSR markers, transferable to Ae. kotschyi, to the high grain iron and zinc containing derivatives indicated introgression of group 2 and group 7 chromosomes of Ae. kotschyi. GISH and FISH analysis of some derivatives confirmed the substitution of chromosomes 2S and 7U for their homoeologues of the A genome, suggesting that some of the genes controlling high grain micronutrient content in the Ae. kotschyi accession are on these chromosomes.