Restriction fragment length polymorphisms of horse class II MHC genes observed using various human alpha-and beta-chain cDNA probes

Genomic DNA isolated from 20 horses was digested with up to six restriction endonucleases and subjected to southern blot hybridization analysis using various human class II alpha- and beta-chain cDNA probes. A high degree of restriction fragment length polymorphism (RFLP) was found for the DQ alpha, DP beta, DQ beta and DR beta probes, about 20 polymorphic bands being detected for each. DR alpha showed 2-4 polymorphic bands, whereas no evidence for DP alpha-like genes was found. A number of correlations of RFLPs with individual alloantisera were apparent.     

Values,Errors, and Precautions

In the environmental health literature, errors in interpreting studies or data are not infrequent. Many are of the Type II variety. Common solecisms of this type are: treating the criterion of p < 0.05 as a sacrament; demanding complete confounder control; arguing for the existence of phantom confounders; arguing that the effect size is trivial; building nonveridical models; arguing for no effect from inadequate sample size; demanding causal proof; arguing that causality is reversed; conducting a ballot of published studies. These are examined in this paper.     

Ischemia-Induced Translocation of Ca2+/Calmodulin-Dependent Protein Kinase II: Potential Role in Neuronal Damage

The activities of Ca2+/calmodulin (CaM)-dependent, Ca2+/phospholipid-dependent, and cyclic AMP-dependent protein kinases (CaM-KII, PKC, and PKA, respectively) were determined in rat brains after global ischemia. Both CaM-KII and PKC activities were significantly depressed in both hippocampal and cerebral cortical regions of ischemic animals, whereas no change was detected in PKA activity. The loss of CaM-KII activity was more dramatic and more sustained than the loss of PKC activity and correlated with the duration of ischemia. These decreases in enzyme activity were found in both supernatant and pellet fractions from crude homogenates. When the supernatant and pellet were analyzed for the amount of CaM-KII 50-kDa protein, a significant decrease was detected in supernatant fractions that paralleled a gain in the amount of CaM-KII in the pellet. Thus, the loss of CaM-KII activity in the supernatant can be explained by translocation of the enzyme to the pellet. Whether inactivation of CaM-KII occurs during or after the enzyme translocates from the supernatant to the pellet is unknown. Our results indicate that loss in CaM-KII activity parallels neuronal damage associated with ischemia; down-regulation of CaM-KII activity coincided with translocation of the enzyme to the particulate fraction, and it is proposed that this may be, in fact, a mechanism for controlling excessive CaM-KII phosphorylation.     

Synthesis of ABO histo-blood group type I and II antigens

The ABO histo-blood group system is one of the most clinically important antigen families. As part of our overall goal to prepare the entire set of the A, B and H type I-VI antigens for a range of biochemical investigations, we report herein the synthesis of the type I and II antigens with a 7-octen-1-yl aglycone. This linker was chosen to facilitate not only the future conjugation of the antigens to a protein or solid support but also the synthesis of the H type I and II octyl glycosides for enzyme kinetic studies.     

Altered clearance of human α2-macroglobulin complexes following reaction with cis-dichlorodiamineplatinum(II)

Clearance studies were performed in mice using α₂-macroglobulin (α₂M), α₂M-trypsin comlex and α₂M-CH₃NH₂ complex. All three species were incubated with cis-dichlorodiamine platinum(II) (cis-DDPt) at concentrations between 9.0 μM and 1.67 mM for 4 h and then dialyzed. The clearance rate of native α₂M was unchanged following incubation with cis-DDPt. α₂M-trypsin and α₂M-CH₃NH₂ cleared rapidly from the ciruculation; however, reaction with cis-DDPt significantly decreased the plasma elimination rate of both complexes. Non-denaturing gel electrophoresis and α₂M activity assays demonstrated relative stability following incubations with cis-DDPt which markedly altered clearance. Evidence for cis-DDPt crosslinking of α₂M subunits was obtained: however, whether this crosslinking is involved in altered clearance remains undetermined. Iodoacetamide treatment of α₂M did not duplicate the effect of cis-DDPton α₂M clearance, nor did it inhibit the effect of cis-DDPt on α₂M clearance. Plasma elimination of α₂M complex was also unaltered by pretreatment of mice with intravenous free cis-DDPt.     

Parameters that specify the timing of cytokinesis.

One model for the timing of cytokinesis is based on findings that p34(cdc2) can phosphorylate myosin regulatory light chain (LC20) on inhibitory sites (serines 1 and 2) in vitro (Satterwhite, L.L., M.H. Lohka, K.L. Wilson, T.Y. Scherson, L.J. Cisek, J.L. Corden, and T.D. Pollard. 1992. J. Cell Biol. 118:595-605), and this inhibition is proposed to delay cytokinesis until p34(cdc2) activity falls at anaphase. We have characterized previously several kinase activities associated with the isolated cortical cytoskeleton of dividing sea urchin embryos (Walker, G.R., C.B. Shuster, and D.R. Burgess. 1997. J. Cell Sci. 110:1373-1386). Among these kinases and substrates is p34(cdc2) and LC20. In comparison with whole cell activity, cortical H1 kinase activity is delayed, with maximum levels in cortices prepared from late anaphase/telophase embryos. To determine whether cortical-associated p34(cdc2) influences cortical myosin II activity during cytokinesis, we labeled eggs in vivo with [(32)P]orthophosphate, prepared cortices, and mapped LC20 phosphorylation through the first cell division. We found no evidence of serine 1,2 phosphorylation at any time during mitosis on LC20 from cortically associated myosin. Instead, we observed a sharp rise in serine 19 phosphorylation during anaphase and telophase, consistent with an activating phosphorylation by myosin light chain kinase. However, serine 1,2 phosphorylation was detected on light chains from detergent-soluble myosin II. Furthermore, cells arrested in mitosis by microinjection of nondegradable cyclin B could be induced to form cleavage furrows if the spindle poles were physically placed in close proximity to the cortex. These results suggest that factors independent of myosin II inactivation, such as the delivery of the cleavage stimulus to the cortex, determine the timing of cytokinesis.     

Incorporation of [35S]methionine in higher plants reveals that stimulation of the D1 reaction centre II protein turnover accompanies tolerance to heavy metal stress

The effects of cadmium stress (CdCl₂) on photochemical activity and protein behaviour of photosystem II (PSII) were studied in vivo and in vitro . Treatments of pea ( Pisum sativum ) and broad bean ( Vicia faba ) plants with 0·05–5 m M cadmium (CdCl₂) modified PSII activity with a resulting increase in electron transfer followed by an inhibition and damage to the oxygen-evolving complex. Pulse-chase experiments with [³⁵S]methionine in vivo followed by the separation of the radiolabelled thylakoids into grana and stroma exposed regions indicated that the synthesis, degradation and assembly of the D1 protein were greatly affected by cadmium. Initially D1 synthesis increased, later slowing down when the stress became advanced; at the same time the D1 degradation was increased. Binding studies with radiolabelled [¹⁴C]herbicide revealed that the Q_B pocket activity was also altered. However, the primary consequence of cadmium stress was the disassembly of the stacked regions. The measurements indicated differential tolerance to cadmium stress between the two plant species, which was not caused by either differential metal uptake or binding to the PSII complex. This suggests that the resulting changes in D1 turnover are a consequence of an unknown primary effect of cadmium on the PSII apparatus. However, we show that the higher tolerance to heavy metal stress found in broad bean plants relative to pea is accompanied by stimulation of D1 turnover. These experiments supported by previous data suggest that modulation of D1 turnover under stress is a commonly occurring process.     

Antibiogram,prevalence of methicillin-resistant and multi-drug resistant Staphylococcus spp. in different clinical samples

Methicillin-resistant Staphylococcus spp. (MRSS) are causing numerous forms of illness in humans ranging from mild to fatal infections. We need to investigate the resistant pattern for different clinical isolates to control the resistance phenomena. This study was designed to provide the resistance pattern of isolated Staphylococcus spp. from various clinical samples in Khartoum State and to elucidate the frequencies of Multidrug-resistant (MDR), Extensively drug-resistant (XDR) and pan-drug resistant (PDR). Two hundred and ten bacterial isolates were from different sources (catheter tip, sputum, vaginal swab, urine, tracheal aspirate, blood, pus, nasal swab, stool, throat swab, pleural fluid, and ear swab). Isolates were identified based on their morphological characters and biochemical reaction. Antibiotics susceptibility screening was performed using twenty-three antibiotics from eighteen classes against all isolated Staphylococcus spp. following the Clinical and Laboratory Standards Institute (CLSI) guideline. The result revealed that out of 63 Gram-positive isolated bacteria, 52 (82.5%) were Staphylococcus spp. with a high incidence of S. aureus 37(71.2%). Out of all Staphylococcus spp., 38 (73.1%) were Methicillin-resistant (MR). The prevalence of MDR was higher in S. aureus (89.2%) than in S. epidermidis (75%). All Staphylococcus spp. displayed resistance to ampicillin and penicillin, while all S. aureus were sensitive to daptomycin and fosfomycin. One isolate was XDR possible PDR, while no PDR was reported in all isolated bacteria. This study provided evidence for the antimicrobial-resistant (AMR) burden in Sudan and highlighted the need for a practical and functional stewardship program to reduce the unreasonable costs of antibiotics.     

Quantitative secretome and glycome of primary human adipocytes during insulin resistance

Adipose tissue is both an energy storage depot and an endocrine organ. The impaired regulation of the secreted proteins of adipose tissue, known as adipocytokines, observed during obesity contributes to the onset of whole-body insulin resistance and the pathobiology of type 2 diabetes mellitus (T2DM). In addition, the global elevation of the intracellular glycosylation of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) via either genetic or pharmacological methods is sufficient to induce insulin resistance in both cultured cells and animal models. The elevation of global O-GlcNAc levels is associated with the altered expression of many adipocytokines. We have previously characterized the rodent adipocyte secretome during insulin sensitive and insulin resistant conditions. Here, we characterize and quantify the secretome and glycome of primary human adipocytes during insulin responsive and insulin resistant conditions generated by the classical method of hyperglycemia and hyperinsulinemia or by the pharmacological manipulation of O-GlcNAc levels. Using a proteomic approach, we identify 190 secreted proteins and report a total of 20 up-regulated and 6 down-regulated proteins that are detected in both insulin resistant conditions. Moreover, we apply glycomic techniques to examine (1) the sites of N-glycosylation on secreted proteins, (2) the structures of complex N- and O-glycans, and (3) the relative abundance of complex N- and O-glycans structures in insulin responsive and insulin resistant conditions. We identify 91 N-glycosylation sites derived from 51 secreted proteins, as well as 155 and 29 released N- and O-glycans respectively. We go on to quantify many of the N- and O-glycan structures between insulin responsive and insulin resistance conditions demonstrating no significant changes in complex glycosylation in the time frame for the induction of insulin resistance. Thus, our data support that the O-GlcNAc modification is involved in the regulation of adipocytokine secretion upon the induction of insulin resistance in human adipocytes.