A Substrate for Direct Measurement of L-iduronic Acid 2-sulfate sulfatase

Commercially available sodium heparinate has been sequentially treated with methanolic 0.06M hydrogen chloride and nitrous acid. The nondegraded material was separated by gel filtration from the nonsulfated and monosulfated disaccharides produced. The latter ones, obtained in 10% yield, have been used as a substrate for the direct measurement of the enzyme L-iduronic acid 2-sulfate sulfatase present in human plasma and fibroblast homogenates. Studies of the kinetics and pH optimum of the enzyme, by use of plasma of a patient with mucolipidosis II, indicated an apparent K_m of 2.5mM and a pH optimum of 4.6-4.8. The levels of activity in normal plasma and plasma of a patient with Hunter's disease were found to be 20.4 ± 1.22 units (μmol sulfate/24 h/g protein) and 3.25 ± 0.35 units, respectively. In homogenates of cultured skin fibroblasts, the levels were 137.6 ± 10.7 units for normal controls and 6.4 ± 5.1 for patients with Hunter's disease. The plasma of two obligated heterozygotes gave intermediate levels of activity, whereas the plasma of two possible heterozygotes gave either intermediate levels or entirely normal levels of activity.     

Shifts in ATP synthesis during preimplantation stages of mouse embryos.

The actual and potential activities of the cyochrome system were studied in cleavage-stage mouse embroys. Activities were determined by assaying embroys for total ATP and the rates of [32-P]ATP synthesis both before and after their incubation in medium supplemented either with an energy coupling site inhibitor (antimycin, amytal or cyanide) or with the FADH-linked substrate, succinate. The data indicate that there are three major shifts in the mode of ATP production during preimplantation stages: the first, between the two-cell and late four-cell stages; the second, between the eight-celland late morula stages; and the third, between the late morula and late blastocyst stages. These data are discussed in relation to studies on the energy metabolism of cleavage and blastocyst stage mouse embryos.     

Hexon peptides of type 2, 3, and 5 adenoviruses and their relationship to hexon structure.

Peptides of hexons from type 2 and 5 (subgroup III) and type 3 (subgroup I) adenoviruses were produced by treatment with cyanogen bromide and were separated by isoelectric focusing in polyacrylamide gels containing 8 M urea. Peptides with identical isoelectric points, but from different hexon types, were considered to have structural similarities. According to this criterion for chemical relatedness, about two-thirds of the type 2 and 5 hexon peptides may be considered similar. In contrast, the majority of the type 3 hexon peptides differed chemically from peptides of type 2 and 5 hexons. Virions and free hexons were iodinated with 125-I in the presence of lactoperoxidase and H-2-O-2. When 125-I-labeled virions were disrupted and the hexon was purified, the highly labeled cyanogen bromide peptides were labeled. When purified hexons from the excess cellular pool were iodinated, peptides common to types 2, 3, and 5 (peptides 12 and 14) were most extensively labeled. Thus, hexons assembled in virions and those free in solution were iodinated differently. The data suggest that immunologically the hexons in viral capsids react differently from unassembled hexons because the polypeptide chains assume slightly different folding configurations in the two hexon forms and therefore expose different regions of the protein to antibodies.     

Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovrius defective in viral DNA synthesis.

Infection of African green monkey kidney cells with type 5 adenovirus leads to the synthesis of two infected, cell-specific proteins with approximate molecular weights of 72,000 and 48,000, that bind specifically to single-stranded but not double-stranded DNA. The production of these two proteins was studied after infection with two DNA-negative adenovirus mutants belonging to different complementation groups (H5 ts36 and H5 ts 125). Both DNA binding proteins were detected in cells infected with either mutant at the permissive temperature (32 C) AND ALSO IN H5 ts36-infected cells at the nonpermissive temperature (39.5 C). In H5 ts125-infected cells at 39.5 C, however, less than 5% of the normal wild-type level of these DNA binding proteins was detectable. When H5 ts125-infected cells were labeled with radioactive leucine at 32 C and subsequently shifted to 39.5 C in the presence of unlabeled leucine (chase), the level of DNA binding proteins found in these infected cells was markedly reduced compared to cultures not shifted to 39.5 C. These data suggest that the DNA binding proteins themselves were temperature sensitive. This conclusion was confirmed by experiments in which the DNA binding proteins were eluted from DNA cellulose with buffers of increasing temperatures (thermal elution). The H5 ts 125 proteins were shown to elute at lower temperatures than either wild-type or H5 ts36 proteins. These results are taken to indicate that the H5 ts125 mutant codes for a DNA binding protein that is thermolabile for continued binding to single-stranded DNA.     

Histochemical evaluation of sodium aurothiomalate inhibition of mouse sperm enzymes

Histochemical procedures for the mouse sperm enzymes hyaluronidase, esterase and acrosin were used to test the inhibitory effects of the low molecular weight hyaluronidase inhibitor sodium aurothiomalate (Myocrisin): hyaluronidase and esterase, but not acrosin, were inhibited. These enzymes were also inhibited in testis homogenates when assayed spectrophotometrically. These results suggest that the antifertility effects of sodium aurothiomalate may be due to the inhibition of several sperm enzymes including both hyaluronidase and esterase. These histochemical assays may be useful for in-vivo detection of chemicals that affect male fertility.     

Statins in cardiometabolic disease: what makes pitavastatin different?

The term cardiometabolic disease encompasses a range of lifestyle-related conditions, including Metabolic syndrome (MetS) and type 2 diabetes (T2D), that are characterized by different combinations of cardiovascular (CV) risk factors, including dyslipidemia, abdominal obesity, hypertension, hyperglycemia/insulin resistance, and vascular inflammation. These risk factors individually and interdependently increase the risk of CV and cerebrovascular events, and represent one of the biggest health challenges worldwide today. CV diseases account for almost 50% of all deaths in Europe and around 30% of all deaths worldwide. Furthermore, the risk of CV death is increased twofold to fourfold in people with T2D. Whilst the clinical management of CV disease has improved in Western Europe, the pandemic of obesity and T2D reduces the impact of these gains. This, together with the growing, aging population, means the number of CV deaths is predicted to increase from 17.1 million worldwide in 2004 to 23.6 million in 2030. The recommended treatment for MetS is lifestyle change followed by treatment for the individual risk factors. Numerous studies have shown that lowering low-density lipoprotein-cholesterol (LDL-C) levels using statins can significantly reduce CV risk in people with and without T2D or MetS. However, the risk of major vascular events in those attaining the maximum levels of LDL-C-reduction is only reduced by around one-third, which leaves substantial residual risk. Recent studies suggest that low high-density lipoprotein-cholesterol (HDL-C) (<1 .0 mmol/l; 40 mg/dl) and high triglyceride levels (≥1.7 mmol/l; 150 mg/dl) are independent risk factors for CV disease and that the relationship between HDL-C and CV risk persists even when on-treatment LDL-C levels are low (<1.7 mmol/l; 70 mg/dl). European guidelines highlight the importance of reducing residual risk by targeting these risk factors in addition to LDL-C. This is particularly important in patients with T2D and MetS because obesity and high levels of glycated hemoglobin are directly related to low levels of HDL-C and high triglyceride. Although most statins have a similar low-density lipoprotein-lowering efficacy, differences in chemical structure and pharmacokinetic profile can lead to variations in pleiotropic effects (for example, high-density lipoprotein-elevating efficacy), adverse event profiles, and drug-drug interactions. The choice of statin should therefore depend on the needs of the individual patient. The following reviews will discuss the potential benefits of pitavastatin versus other statins in the treatment of patients with dyslipidemia and MetS or T2D, focusing on its effects on HDL-C quantity and quality, its potential impact on atherosclerosis and CV risk, and its metabolic characteristics that reduce the risk of drug interactions. Recent controversies surrounding the potentially diabetogenic effects of statins will also be discussed.     

Leptin modulates lymphocytes' adherence to hepatic stellate cells is associated with oxidative status alterations

We investigated leptin effects on lymphocyte interactions with hepatic-stellate-cells (HSCs). Leptin showed pro-fibrotic effects on HSCs with oxidative status imbalance.In co-cultures, leptin activates HSCs and consequently adhered HCV-lymphocytes more than healthy ones. Leptin also increased healthy and HCV lymphocyte proliferations; increased their reactive-oxygen-species; decreased antioxidants (reduced-glutathione) levels while inhibited apoptosis only of HCV-lymphocytes. The leptin-treated HCV-lymphocytes activated HSCs, increase interleukin-4 while decreased their apoptosis.Leptin-receptor-deficient (db–db)-HSCs did not adhere lymphocytes. db/db-lymphocytes however showed fewer adherences to HSCs when compared to WT-counterparts.This study presents immune and oxidative modulatory effects of leptin on lymphocytes and their consequent interaction with HSCs.     

Toxicity of baited spinosad formulations to Ceratitis capitata: from the laboratory to the application

GF‐120, a fruit fly bait designed to attract and kill adult fruit flies, was tested in the laboratory and outdoors to determine effects of pre‐treatment diet and bait aging on mortality of Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae). Two spinosad‐based compounds, GF‐120 and Tracer^® Ultra, had generated two distinctive dose–mortality responds, with LC⁸⁰, LC⁹⁰, and LC⁹⁹ values of 2.4, 2.8, and 4.1 p.p.m., and 255, 479, and 1 143 p.p.m., respectively. The residues of GF‐120 drops, after feeding to the flies, generated 14.3% mortality. The droplet size of the baited spray plays an important role. The toxicity of large drops lasted more than that of small droplets. In the field, exposure to the sun further deteriorates the compound, which lost 50% of its toxicity within 6 days. Disappearance of the compound in the field, due to consumption by various insects, also played a role as 50% of the GF‐120 drops disappeared within 7 days. As mortality was directly related to the amount of insecticide eaten, the effect of GF‐120 depended on the feeding status of the flies: well‐fed flies were almost unaffected compared with starved ones.     

Identification of novel Xanthomonas euvesicatoria type III effector proteins by a machine‐learning approach

The Gram‐negative bacterium Xanthomonas euvesicatoria (Xcv) is the causal agent of bacterial spot disease in pepper and tomato. Xcv pathogenicity depends on a type III secretion (T3S) system that delivers effector proteins into host cells to suppress plant immunity and promote disease. The pool of known Xcv effectors includes approximately 30 proteins, most identified in the 85‐10 strain by various experimental and computational techniques. To identify additional Xcv 85‐10 effectors, we applied a genome‐wide machine‐learning approach, in which all open reading frames (ORFs) were scored according to their propensity to encode effectors. Scoring was based on a large set of features, including genomic organization, taxonomic dispersion, hypersensitive response and pathogenicity (hrp)‐dependent expression, 5′ regulatory sequences, amino acid composition bias and GC content. Thirty‐six predicted effectors were tested for translocation into plant cells using the hypersensitive response (HR)‐inducing domain of AvrBs2 as a reporter. Seven proteins (XopAU, XopAV, XopAW, XopAP, XopAX, XopAK and XopAD) harboured a functional translocation signal and their translocation relied on the HrpF translocon, indicating that they are bona fide T3S effectors. Remarkably, four belong to novel effector families. Inactivation of the xopAP gene reduced the severity of disease symptoms in infected plants. A decrease in cell death and chlorophyll content was observed in pepper leaves inoculated with the xopAP mutant when compared with the wild‐type strain. However, populations of the xopAP mutant in infected leaves were similar in size to those of wild‐type bacteria, suggesting that the reduction in virulence was not caused by impaired bacterial growth.