Expression of caveolin‐1 in hepatic cells increases oxidized LDL uptake and preserves the expression of lipoprotein receptors

Oxidized LDL (OxLDL) that are positively associated with the risk of developing cardiovascular diseases are ligands of scavenger receptor‐class B type I (SR‐BI) and cluster of differentiation‐36 (CD36) which can be found in caveolae. The contribution of these receptors in human hepatic cell is however unknown. The HepG2 cell, a human hepatic parenchymal cell model, expresses these receptors and is characterized by a very low level of caveolin‐1. Our aim was to define the contribution of human CD36, SR‐BI, and caveolin‐1 in the metabolism of OxLDL in HepG2 cells and conversely the effects of OxLDL on the levels/localization of these receptors. By comparing mildly (M)‐ and heavily (H)‐OxLDL metabolism between control HepG2 cells and HepG2 cells overexpressing CD36, SR‐BI, or caveolin‐1, we found that (1) CD36 increases M‐ and H‐OxLDL‐protein uptake; (2) SR‐BI drives M‐OxLDL through a degradation pathway at the expense of the cholesterol ester (CE) selective uptake pathway; (3) caveolin‐1 increases M‐ and H‐OxLDL‐protein uptake and decreases CE selective uptake from M‐OxLDL. Also, incubation with M‐ or H‐OxLDL decreases the levels of SR‐BI and LDL‐receptor in control HepG2 cells which can be overcome by caveolin‐1 expression. In addition, OxLDL move CD36 from low to high buoyant density membrane fractions, as well as caveolin‐1 in cells overexpressing this protein. Thus, hepatic caveolin‐1 expression has significant effects on OxLDL metabolism and on lipoprotein receptor levels. J. Cell. Biochem. 108: 906–915, 2009. © 2009 Wiley‐Liss, Inc.     

Phylogenetic analysis identifies many uncharacterized actin-like proteins (Alps) in bacteria: regulated polymerization, dynamic instability and treadmilling in Alp7A

Actin, one of the most abundant proteins in the eukaryotic cell, also has an abundance of relatives in the eukaryotic proteome. To date though, only five families of actins have been characterized in bacteria. We have conducted a phylogenetic search and uncovered more than 35 highly divergent families of actin-like proteins (Alps) in bacteria. Their genes are found primarily on phage genomes, on plasmids and on integrating conjugative elements, and are likely to be involved in a variety of functions. We characterize three Alps and find that all form filaments in the cell. The filaments of Alp7A, a plasmid partitioning protein and one of the most divergent of the Alps, display dynamic instability and also treadmill. Alp7A requires other elements from the plasmid to assemble into dynamic polymers in the cell. Our findings suggest that most if not all of the Alps are indeed actin relatives, and that actin is very well represented in bacteria.     

Accumulation of sesquiterpenes and polysaccharides in cells of zedoary (Curcuma zedoaria Roscoe) cultured in a 10 L bioreactor

We developed a cell suspension culture system for zedoary (Curcuma zedoaria Roscoe), using 100 g fresh weight inoculum in a batch culture. The maximum cell biomass of 68.46 g/L fresh weight was obtained after 14 days of culture in a 10 L bioreactor with a pitch-blade impeller maintained at an agitation speed of 150 rpm and an aeration rate of 2.5 L/min. The accumulation of sesquiterpenes and polysaccharide in zedoary cells from 2 to 18 days was measured by HPLC and a phenol-sulfuric acid assay, respectively. The total polysaccharide concentration increased between 2 to 10 days of culture and reached a maximum value of 6.55%. HPLC revealed several eluted peaks of sesquiterpenes, which increased in amplitude from days 2 to 10. Furthermore, our results indicated that biotransformation occurred in the cell suspension, transforming certain sesquiterpenes into other types during culture.     

The catch and trade of seahorses in Vietnam

Catch monitoring and surveys were used to assess the seahorse trade in Vietnam. Despite low daily catch rates, potentially 6.5 t of dried seahorses (~2.2 million seahorses) were taken annually as bycatch by trawlers operating out of five coastal provinces of Vietnam. Individual seahorse catches were collated by a few local buyers, who supplied wholesalers in three major markets: Ho Chi Minh City, Hai Phong City and Da Nang. Domestic consumption was small and most seahorses were exported, generally through unofficial and unregulated channels across the northern border into Guangxi province of China. Overall, the seahorse trade was of low economic value to Vietnam, but may constitute an important source of income to upper level buyers and exporters. Most fishers and buyers surveyed reported that seahorse catch had declined over time. This paper should help in meeting the new CITES requirements – through implementation of an Appendix II listing in 2004 – that all international trade in seahorses must be monitored and managed for sustainability.     

TGF-β1 induces an age-dependent inflammation of nerve ganglia and fibroplasia in the prostate gland stroma of a novel transgenic mouse

TGF-β1 is overexpressed in wound repair and in most proliferative disorders including benign prostatic hyperplasia and prostate cancer. The stromal microenvironment at these sites is reactive and typified by altered phenotype, matrix deposition, inflammatory responses, and alterations in nerve density and biology. TGF-β1 is known to modulate several stromal responses; however there are few transgenic models to study its integrated biology. To address the actions of TGF-β1 in prostate disorders, we targeted expression of an epitope tagged and constitutively active TGF-β1 via the enhanced probasin promoter to the murine prostate gland epithelium. Transgenic mice developed age-dependent lesions leading to severe, yet focal attenuation of epithelium, and a discontinuous basal lamina. These changes were associated with elevated fibroplasia and frequency of collagenous micronodules in collapsed acini, along with an induced inflammation in nerve ganglia and small vessels. Elevated recruitment of CD115+ myeloid cells but not mature macrophages was observed in nerve ganglia, also in an age-dependent manner. Similar phenotypic changes were observed using a human prostate epithelium tissue recombination xenograft model, where epithelial cells engineered to overexpress TGF-β1 induced fibrosis and altered matrix deposition concurrent with inflammation in the stromal compartment. Together, these data suggest that elevated TGF-β1 expression induces a fibroplasia stromal response associated with breach of epithelial wall structure and inflammatory involvement of nerve ganglia and vessels. The novel findings of ganglia and vessel inflammation associated with formation of collagenous micronodules in collapsed acini is important as each of these are observed in human prostate carcinoma and may play a role in disease progression.     

A Randomised Trial Evaluating the Safety and Immunogenicity of the Novel Single Oral Dose Typhoid Vaccine M01ZH09 in Healthy Vietnamese Children

Background

The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile.

Objectives

We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years.

Methods

Subjects were randomly assigned to receive either a nominal dose of 5×10⁹ CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA.

Principal Findings

One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18–5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12–36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI]  = 1.23 [0.550–2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC ⁻ ssaV ⁻) ZH9 was detected in 51 (51%; 95% CI, 41–61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92–99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7–29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88–100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested.

Conclusions

This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children.

Trial Registration

Controlled-trials.comISRCTN91111837     

Trace-Level Determination of Oxolinic Acid and Flumequine in Soil,River Bed Sediment,and River Water Using Microwave-Assisted Extraction and High-Performance Liquid Chromatography with Fluorimetric Detection

This work presents the optimization of analytical procedures for the determination of two antibiotics, oxolinic acid (OA) and flumequine (FL), in bed sediment, river water, and soil samples. Three extraction methods (microwave-assisted extraction (MAE), ultrasonication, and reflux) were tested, and the highest recoveries were obtained with MAE (94 ± 3% and 95 ± 3% for OA and FL, respectively). A solid-phase extraction (SPE) clean-up step was optimized by comparing two polymeric sorbents: Oasis HLB and Oasis MAX. The final extracts were analyzed by liquid chromatography with fluorimetric detection. Limits of detection (LOD) obtained for OA and FL in soil and sediment ranged from 0.3 to 0.5 µg kg^?1. Meanwhile, a novel SPE procedure was also implemented for OA and FL determination in river water samples. It also relied on the use of Oasis MAX, and recovery rates were in the range 90–94%; LODs were 2 ng L^?1 for both OA and FL. These methods were applied for the analysis of samples taken in the Seine River basin (France). The obtained results demonstrated the widespread occurrence of OA and FL, at ng L^?1 and µg kg^?1 levels in water and sediment/soil, respectively, and their persistence in the environment.     

Entropy-driven mechanism of an E3 ligase

Ubiquitin-like modifications are macromolecular chemistry for which our understanding of the enzymatic mechanisms is lacking. Most E3 ligases in ubiquitin-like modifications do not directly participate in chemistry but are thought to confer allosteric effects; however, the nature of the allosteric effects has been elusive. Recent molecular dynamics simulations suggested that an E3 binding enhances the population of the conformational states of the E2·SUMO thioester that favor reactions. In this study, we conducted the first temperature-dependent enzyme kinetic analysis to investigate the role of an E3 on activation entropy and enthalpy. The small ubiquitin-like modifier (SUMO) E3, RanBP2, confers unusually large, favorable activation entropy to lower the activation energy of the reaction. Mutants of RanBP2, designed to alter the flexibilities of the E2·SUMO thioester, showed a direct correlation of their favorable entropic effects with their ability to restrict the conformational flexibility of the E2·SUMO thioester. While the more favorable activation entropy is consistent with the previously suggested role of E3 in conformational selection, the large positive entropy suggests a significant role of solvent in catalysis. Indeed, molecular dynamics simulations in explicit water revealed that the more stable E2·SUMO thioester upon E3 binding results in stabilization of a large number of bound water molecules. Liberating such structured water at the transition state can result in large favorable activation entropy but unfavorable activation enthalpy. The entropy-driven mechanism of the E3 is consistent with the lack of structural conservation among E3s despite their similar functions. This study also illustrates how proteins that bind both SUMO and E2 can function as E3s and how intrinsically unstructured proteins can enhance macromolecular chemistry in addition to their known advantages in protein--protein interactions.