Activity-based protein profiling of the hepatitis C virus replication in Huh-7 hepatoma cells using a non-directed active site probe

Background  

Hepatitis C virus (HCV) poses a growing threat to global health as it often leads to serious liver diseases and is one of the primary causes for liver transplantation. Currently, no vaccines are available to prevent HCV infection and clinical treatments have limited success. Since HCV has a small proteome, it relies on many host cell proteins to complete its life cycle. In this study, we used a non-directed phenyl sulfonate ester probe (PS4≡) to selectively target a broad range of enzyme families that show differential activity during HCV replication in Huh-7 cells.     

Nutritional regulation of adipose tissue lipoprotein lipase is blunted in insulin resistant rats

Background  

Hypertriglyceridemia is a common lipid abnormality accompanying insulin resistance. This study was designed to assess the contribution of dysregulation of adipose tissue lipoprotein lipase (LPL) activity to the hypertriglyceridemia in a rat model of insulin resistance.     

Lipoprotein lipase in lungs, spleen, and liver: synthesis and distribution.

Lipoprotein lipase (LPL, E C 3.1.1.34) is the enzyme responsible for hydrolysis of triacylglycerols in plasma lipoproteins, making the fatty acids available for use by subjacent tissues. LPL is functional at the surface of endothelial cells, but it is not clear which cells synthesize the enzyme and what its distribution within tissues and vessels is. In previous studies we reported that in the major LPL-producing tissues (muscles, adipose tissue, and mammary gland) the enzyme is made by the major cell types. In the present work we have studied in adult guinea pigs some tissues that present LPL activity but in lower amounts (lung, spleen, and liver). On cryosections of these tissues we have searched for specific cell expression of the LPL gene (by in situ hybridization using a RNA probe) and for the corresponding protein distribution (by immunocytochemistry). Based on morphological criteria we can suggest that, contrary to the main LPL-producing tissues, in these tissues the enzyme is made by scattered cells, such as macrophages in the lung and spleen and Kupffer cells in the liver; endothelial cells present but do not synthesize the enzyme, indicating that the endothelial LPL originates in other cells. In the liver strong immunoreaction was detected in the sinusoid in contrast to the low level of mRNA expression, suggesting that liver takes up circulating LPL from blood.     

Extracellular degradation of lipoprotein lipase in rat adipose tissue

Background  

Recent studies in vivo indicate that short-term regulation of lipoprotein lipase (LPL) in rat adipose tissue is post-translational and occurs by a shift of the lipase protein towards an inactive form under the influence of another gene with short-lived message and product. It has not been possible to reproduce this process with isolated adipocytes suggesting that other cells are needed, and perhaps mediate the regulation. The objective of the present study was, therefore, to explore if explants of adipose tissue could be used for studies of the regulatory process.     

Synthesis of lipoprotein lipase in the liver of newborn rats and localization of the enzyme by immunofluorescence.

In newborn rats, lipoprotein lipase (LPL) activity was higher in the liver than in several other tissues, such as heart, diaphragm or lungs, and accounted for about 3% of total LPL activity in the body. There was no significant correlation between LPL activity in liver and in plasma. Thus transport of the enzyme from extrahepatic tissues was probably not the major source of LPL in liver. To study LPL biosynthesis directly, newborn rats were injected intraperitoneally with [35S]methionine, and LPL was isolated by immunoprecipitation and separation by SDS/polyacrylamide-gel electrophoresis. Radioactivity in LPL increased with a similar time course in all tissues studied, including the liver. Substantial synthesis of LPL was also demonstrated in isolated perfused livers from newborn rats, whereas synthesis was low in livers from adult rats. There was strong LPL immunofluorescence in livers from newborn rats, mainly within sinusoids and along the walls of larger vessels. This labelling disappeared after perfusion with heparin, which indicates that much of the enzyme is in contact with blood and can take part in lipoprotein metabolism.     

Human type 2 17beta-hydroxysteroid dehydrogenase mRNA and protein distribution in placental villi at mid and term pregnancy

Background  

During human pregnancy, the placental villi produces high amounts of estradiol. This steroid is secreted by the syncytium, which is directly in contact with maternal blood. Estradiol has to cross placental foetal vessels to reach foetal circulation. The enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2) was detected in placental endothelial cells of foetal vessels inside the villi. This enzyme catalyzes the conversion of estradiol to estrone, and of testosterone to androstenedione. It was proposed that estradiol level into foetal circulation could be regulated by 17beta-HSD2.     

Comparing Leishman and Giemsa staining for the assessment of peripheral blood smear preparations in a malaria-endemic region in India

Background

The first phase of malaria infection occurs in the liver and is clinically silent. Inside hepatocytes each Plasmodium sporozoite replicate into thousands of erythrocyte-infectious merozoites that when released into the blood stream result in clinical symptoms of the disease. The time between sporozoite inoculation and the appearance of parasites in the blood is defined as the pre-patent period, which is classically analysed by time-consuming and labor-intensive techniques, such as microscopy and PCR.

Methods

Luciferase-expressing Plasmodium berghei parasites were used to measure pre-patent period of malaria infection in rodents using a bioluminescence assay that requires only one microliter of blood collected from the tail-vein. The accuracy and sensitivity of this new method was compared with conventional microscopy and PCR based techniques, and its capacity to measure the impact of anti-malarial interventions against the liver evaluated.

Results

The described method is very sensitive allowing the detection of parasites during the first cycles of blood stage replication. It accurately translates differences in liver load due to inoculation of different sporozoite doses as well as a result of treatment with different primaquine regimens.

Conclusions

A novel, simple, fast, and sensitive method to measure pre-patent period of malaria infection in rodents is described here. The sensitivity and accuracy of this new method is comparable to standard PCR and microscopy-based techniques, respectively.     

PhiC31 integrase induces a DNA damage response and chromosomal rearrangements in human adult fibroblasts

Background  

PhiC31 integrase facilitates efficient integration of transgenes into human and mouse genomes and is considered for clinical gene therapy. However recent studies have shown that the enzyme can induce various chromosomal abnormalities in primary human embryonic cells and mammalian cell lines. The mechanisms involved are unknown, but it has been proposed that PhiC31 attachment sites in the host genome recombine leading to chromosomal translocations.     

Expression of thymidine phosphorylase in peripheral blood cells of breast cancer patients is not increased by paclitaxel

Background  

A synergistic cytotoxic effect has been hypothesized for taxanes and capecitabine, a prodrug of 5-fluorouracil. Based on preclinical studies, this synergism has been attributed to an up-regulation of the enzyme thymidine phosphorylase (TP). Beside tumour tissue, TP is highly expressed in white blood cells, possibly causing increased hematotoxicity, when taxanes are combined with capecitabine. So far, this hypothesis has not been investigated in humans.     

First evidence of bacterial translocation from the intestinal tract as a route of Helicobacter cinaedi bacteremia

Background

The route of Helicobacter cinaedi bacteremia has not yet been clarified. Although bacterial translocation from the intestinal tract into the circulation has been suggested, it has not been demonstrated thus far. The objective of this study was to investigate the port of entry of this bacterium.

Material and Methods

We conducted a retrospective study on patients with H. cinaedi bacteremia between March 2009 and May 2013. Records of patients in whom H. cinaedi was detected in both blood and stool cultures were extracted. H. cinaedi was identified using gyrB‐targeted PCR. Pulse‐field gel electrophoresis was used to investigate the consistency of the genotypes.

Results

Seventy‐one patients were diagnosed with H. cinaedi bacteremia during the study period. H. cinaedi was detected in both blood and stool samples of 21 patients. Pulse‐field gel electrophoresis was used to investigate the consistency of the genotypes in 18 evaluable strains (from 9 patients). The pulse‐field gel electrophoresis patterns of the stool‐ and blood‐derived strains of H. cinaedi were consistent among all 9 patients. Most of the 9 patients analyzed were immunocompromised and being treated with anticancer drugs or steroids, which suggests reduced intestinal immunity.

Conclusions

This is the first study to demonstrate that bacterial translocation from the intestinal tract could represent one route of H. cinaedi bacteremia.     

Influence of lipoprotein-lipase activity on plasma triacylglycerol concentration and lipid storage in three genotypes of ducks

The lipoprotein-lipase (LPL) hydrolyses the triacylglycerols (TG) secreted by the liver and, thus, allows the storage of lipids onto the extrahepatic tissues. The LPL activity has been studied by injection of LPL antibodies in three genotypes of ducks (Muscovy (Cairina moschat), Pekin (Anas plathyrhynchos) and Mule (hybrids of male Muscovy ducks and female Pekin ducks)) under overfeeding condition. The results show a similar weight gain between injected and control animals. A higher liver steatosis is observed in Mule ducks (616 ± 18 g; 8.79% of body mass (BW)) and Muscovy ducks (514 ± 13 g; 7.05% BW) compared to Pekin ducks (353 ± 21 g; 5.89% BW, p < 0.05). Pekin ducks showed a much marked extrahepatic fattening of abdominal and subcutaneous adipose tissues. The LPL activity was evaluated by comparing the evolution of the plasma TG concentrations after injections of saline (control animals) or injections of specific LPL-antibodies. Inhibition of LPL activity performed by intravenous injections of LPL-antibodies showed a spectacular increase in the plasma TG concentrations in the three genotypes. That increase was considerably higher in Pekin ducks (98 ± 10 g/L) compared to Muscovy ducks (35 ± 2 g/L, p < 0.01) and Mule ducks (30 ± 4 g/L, p < 0.01). Those data suggest that a high export of lipids synthesized in liver and a high LPL activity occur in overfed Pekin ducks, which can favour the extrahepatic fattening to the detriment of the liver steatosis, and conversely in overfed Muscovy and Mule ducks.     

Effect of chronic heparin administration on serum lipolytic activity and some aspects of lipid metabolism

Chronic heparin administration to rats for periods up to 8 days by i.p. implantation of mini pumps, increased serum total lipolytic activity in a dose-dependent manner up to infusion rates of 10 U/h per 100 g body weight. This augmentation was predominantly due to lipoprotein lipase (LPL). Synchronously, heart muscle demonstrated a dose-dependent reduction in LPL activity and adipose tissue showed a biphasic response, LPL activity decreasing at low doses and rising towards control levels at higher doses. Lipolytic activities of skeletal muscle and liver were unaffected. Increased serum LPL could not be attributed to altered enzyme clearance from the circulation in chronically heparinised rats, but was accompanied by a reduced response to i.v. high-dose heparin indicating reduction in the pool of endothelial-bound enzyme. Fasting serum concentrations of triacylglycerol and glycerol were unaffected in chronically heparinised animals although accelerated clearance of exogenous 14C-labelled VLDL was demonstrated, together with enhanced uptake of the isotope by liver and heart. Since de novo synthesis of fatty acids and triacylglycerol from 3H2O was not increased by heparin, we suggest that serum triacylglycerol concentrations were maintained by enhanced re-esterification of preformed fatty acids taken up by the liver. Hepatic cholesterol synthesis from 3H2O was augmented by heparin; this observation is consistent with reported increases in serum total and HDL-cholesterol mediated by chronic heparin administration in man and dog.     

Predicting the network of substrate-enzyme-product triads by combining compound similarity and functional domain composition

Background  

Metabolic pathway is a highly regulated network consisting of many metabolic reactions involving substrates, enzymes, and products, where substrates can be transformed into products with particular catalytic enzymes. Since experimental determination of the network of substrate-enzyme-product triad (whether the substrate can be transformed into the product with a given enzyme) is both time-consuming and expensive, it would be very useful to develop a computational approach for predicting the network of substrate-enzyme-product triads.     

Hfq stimulates the activity of the CCA-adding enzyme

Background  

The bacterial Sm-like protein Hfq is known as an important regulator involved in many reactions of RNA metabolism. A prominent function of Hfq is the stimulation of RNA polyadenylation catalyzed by E. coli poly(A) polymerase I (PAP). As a member of the nucleotidyltransferase superfamily, this enzyme shares a high sequence similarity with an other representative of this family, the tRNA nucleotidyltransferase that synthesizes the 3'-terminal sequence C-C-A to all tRNAs (CCA-adding enzyme). Therefore, it was assumed that Hfq might not only influence the poly(A) polymerase in its specific activity, but also other, similar enzymes like the CCA-adding enzyme.     

The effect of exercise training and water extract from propolis intake on the antioxidant enzymes activity of skeletal muscle and liver in rat

[Purpose]

In this study, the authors have intended to investigate the effects that the exercise training and the intake of the water extract from propolis have on the activity of antioxidant enzymes.

[Methods]

For this purpose, the exercise training (70% VO₂max treadmill running exercise for 60min)of 5 times per week for six weeks and the intake (50mg/kg/day) of the water extract from propolis were performed by separating the experimental animals (SD rats, n=32) into CON(n=8) group, CON+Ex(n=8), PA(n=8), and PA+Ex(n=8).

[Results]

As a result, the following conclusions were obtained: The concentration of the blood glucose and insulin of the CON+Ex group and PA+Ex group which are the exercise parallel group were significantly decreased in comparison with the control group, whereas if comparing the glycogen concentration in skeletal muscle and liver tissue between the exercise parallel group and the CON group, the former showed significantly high value in comparison with the latter (p < .05). In the case of the activity of the antioxidant enzyme in the skeletal muscle and the liver tissue, the activities of SOD, GPX and CAT in the gastrocnemius muscle tissue of the experimental animals showed significantly high value in PA+Ex group in comparison with other experimental groups (p < .05). In addition, the SOD activity in the liver tissue showed that only PA+Ex group was significantly increased, whereas GDX activity showed significantly higher value in CON+Ex group and PA group than CON group (p < .05). However, the activity of CAT in the liver tissue showed that there is no difference between the experimental groups. As a result that measured the concentration of MDA in order to evaluate the damage level of the tissue by oxygen free radicals, the difference between the groups in the liver tissue was not shown, while it was shown that only PA+Ex group in the skeletal muscle tissue was significantly decreased in comparison with other experimental groups (p < .05).

[Conclusion]

Taken together the above findings, it is considered that the parallel treatment of the exercise training and the water extract from propolis can not only increase the use of glycogen of the skeletal muscle and liver tissue, but also it can give the effect to suppress the creation of active oxygen by inducing the activity of the antioxidant enzyme in the body, and in the future, the possibility as the exercise supplements and the antioxidant of the water-soluble propolis are expected.     

Discovery of serum biomarkers of alcoholic fatty liver in a rodent model: C-reactive protein

Background  

Excessive consumption of alcohol contributes to alcoholic liver disease. Fatty liver is the early stage of alcohol-related liver disease. The aim of this study was to search for specific serological biomarkers of alcoholic fatty liver (AFL) compared to healthy controls, non-alcoholic fatty liver (NAFL) and liver fibrosis in a rodent model.     

Amelioration of hypertriglyceridemia with hypo-alpha-cholesterolemia in LPL deficient mice by hematopoietic cell-derived LPL

Background

Macrophage-derived lipoprotein lipase (LPL) has been shown uniformly to promote atherosclerotic lesion formation while the extent to which it affects plasma lipid and lipoprotein levels varies in wild-type and hypercholesterolemic mice. It is known that high levels of LPL in the bulk of adipose tissue and skeletal muscle would certainly mask the contribution of macrophage LPL to metabolism of plasma lipoprotein. Therefore, we chose LPL deficient (LPL^-/-) mice with severe hypertriglyceridemia as an alternative model to assess the role of macrophage LPL in plasma lipoprotein metabolism via bone marrow transplant, through which LPL will be produced mainly by hematopoietic cell-derived macrophages.

Methods and Results

Hypertriglyceridemic LPL^-/- mice were lethally irradiated, then transplanted with bone marrow from wild-type (LPL^+/+) or LPL^-/- mice, respectively. Sixteen weeks later, LPL^+/+ →LPL^-/- mice displayed significant reduction in plasma levels of triglyceride and cholesterol (408±44.9 vs. 2.7±0.5×10³ and 82.9±7.1 vs. 229.1±30.6 mg/dl, p<0.05, respectively), while a 2.7-fold increase in plasma high density lipoprotein- cholesterol (p<0.01) was observed, compared with LPL^-/-→LPL^-/- control mice. The clearance rate for the oral fat load test in LPL^+/+ →LPL^-/- mice was faster than that in LPL^-/-→LPL^-/- mice, but slower than that in wild-type mice. Liver triglyceride content in LPL^+/+→LPL^-/- mice was also significantly increased, compared with LPL^-/-→LPL^-/- mice (6.8±0.7 vs. 4.6±0.5 mg/g wet tissue, p<0.05, n = 6). However, no significant change was observed in the expression levels of genes involved in hepatic lipid metabolism between the two groups.

Conclusions

Hematopoietic cell-derived LPL could efficiently ameliorate severe hypertriglyceridemia and hypo-alpha-cholesterolemia at the compensation of increased triglyceride content of liver in LPL^-/- mice.     

Metabolic profiles in five high-producing Swedish dairy herds with a history of abomasal displacement and ketosis

Background  

Body condition score and blood profiles have been used to monitor management and herd health in dairy cows. The aim of this study was to examine BCS and extended metabolic profiles, reflecting both energy metabolism and liver status around calving in high-producing herds with a high incidence of abomasal displacement and ketosis and to evaluate if such profiles can be used at herd level to pinpoint specific herd problems.     

The small regulatory RNA molecule MicA is involved in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium biofilm formation

Background  

LuxS is the synthase enzyme of the quorum sensing signal AI-2. In Salmonella Typhimurium, it was previously shown that a luxS deletion mutant is impaired in biofilm formation. However, this phenotype could not be complemented by extracellular addition of quorum sensing signal molecules.     

Identification and characterization of the intracellular poly-3-hydroxybutyrate depolymerase enzyme PhaZ of Sinorhizobium meliloti

Background  

S. meliloti forms indeterminate nodules on the roots of its host plant alfalfa (Medicago sativa). Bacteroids of indeterminate nodules are terminally differentiated and, unlike their non-terminally differentiated counterparts in determinate nodules, do not accumulate large quantities of Poly-3-hydroxybutyrate (PHB) during symbiosis. PhaZ is in intracellular PHB depolymerase; it represents the first enzyme in the degradative arm of the PHB cycle in S. meliloti and is the only enzyme in this half of the PHB cycle that remains uncharacterized.