Non-cultured adipose-derived CD45- side population cells are enriched for progenitors that give rise to myofibres in vivo

Side population (SP) cells are highly able to exclude the Hoechst 33342 dye through membrane transporters, a feature associated with cell immaturity and therefore proposed as a marker of stem cells. Herein we demonstrate that the adipose tissue derived stromal vascular fraction (SVF) contains a novel population of non-haematopoietic “side population” (SPCD45⁻) cells. Simultaneous qRT-PCR of 64 genes revealed that the freshly isolated SPCD45⁻ was highly enriched for cells expressing genes related to stem cells, the Notch pathway, and early vascular precursors. Notably, the expression of smooth muscle actin, C-met and Cd34 together with Angpt2, Flk1, VE-cadherin, and Cd31 suggested a phenotypic resemblance to pericytes and aorta-derived mesoangioblasts. Recent evidence suggests that cells residing within the vascular niche may participate in regeneration of skeletal muscle and although skeletal muscle repair mainly relies on the satellitecell, several reports have shown that vessel-associated cells may adopt a myogenic phenotype when exposed to a muscle environment. In accordance with these findings, we also observed invitro myogenic specification of SPCD45⁻ cells when cocultured with myoblasts. Furthermore, immediate intramuscular engraftment of non-cultured SPCD45⁻ cells gave rise to myofibres andcells lining blood vessels, whereas the SVF only provided donor derived mononuclear cells. We therefore conclude that the SPCD45⁻ fraction of adipose-derived SVF is enriched for cells expressing vascular associated markers and that the myogenic differentiation potential of these cells does not depend on prior in vitro expansion.     

Conservation value of degraded habitats for forest birds in southern Peninsular Malaysia

Clearance of tropical forest for agricultural purposes is generally assumed to seriously threaten the survival of forest species. In this study, we quantified the conservation value, for forest bird species, of three degraded habitat types in Peninsular Malaysia, namely rubber tree plantations, oil palm plantations, and open areas. We surveyed these degraded habitats using point counts to estimate their forest bird species richness and abundance. We assessed whether richness, abundance, and activities of different avian dietary groups (i.e. insectivores and frugivores) varied among the habitats. We identified the critical habitat elements that accounted for the distribution of forest avifauna in these degraded habitats. Our results showed that these habitats harboured a moderate fraction of forest avifauna (approximately 46–76 species) and their functions were complementary (i.e. rubber tree plantations for moving; open habitats for perching; shrubs in oil palm plantations for foraging). In terms of species richness and abundance, rubber tree plantations were more important than oil palm plantations and open habitats. The relatively high species richness of this agricultural landscape was partly due to the contiguity of our study areas with extensive forest areas. Forecasts of forest-species presence under various canopy cover scenarios suggest that leaving isolated trees among non-arboreal crops could greatly attract relatively tolerant species that require tree canopy. The conservation value of degraded habitats in agricultural landscapes seems to depend on factors such as the type of crops planted and distance to primary forest remnants.     

Effects of decreased glutathione peroxidase activity on the pentose phosphate cycle in mouse lung

The effects of reducing glutathione peroxidase activity in the lung by changing dietary selenium intake has been investigated. In animals that were exposed to room air, selenium effects were confined to glutathione peroxidase activity, whereas under conditions of oxidant stress (ozone) the decrease in glutathione peroxidase activity prevented the stimulation of the pentose phosphate cycle (assayed by measuring glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities) which has been reported to increase in response to oxidant stress. The suppression of glutathione peroxidase activity was found to depend on dietary selenium concentration. The physiological significance of this observation may be related to the process of injury and repair in the lung.     

Prognostic significance of DNA ploidy and morphometric analyses of adenocarcinoma of the uterine cervix

In an effort to improve the prognostic accuracy of the histologic criteria used for cervical adenocarcinomas, the nuclear DNA ploidy levels, means and standard deviations of nuclear areas and amounts of lumen and neoplastic tissue were quantitated. Useful thresholds in discriminating recurrent disease, as identified by logistic regression analysis, included a DNA ploidy level of 3.0 N, a percent of lumen of 34.6% and nuclear area mean and standard deviation of 53.1 sq micron and 20.1 sq micron, respectively. These parameters should provide useful guidelines in the visual assessment of histologic features that have prognostic significance.     

p130/p107 expression distinguishes adipogenic potential in primary myoblasts based on age

Recent investigations have provided significant evidence that many mesodermally derived tissues contain stem cell-like precursors capable of being stimulated to undergo differentiation into a variety of cellular lineages. We have recently reported that primary myoblasts isolated from 23-month-old mice have an increased adipogenic potential when compared to their 8-month-old counterparts. To further characterize the degree of adipocyte differentiation in these myoblasts, we examined early and late markers of adipocyte differentiation. Within the first 24h of adipocyte differentiation, expression of p130 and p107, two members of the retinoblastoma tumor suppressor gene family, are regulated and this event is an important one early in adipogenesis. Consistent with the increased adipogenic potential of the older myoblasts and in contrast to the younger cells, the p130:p107 pattern of expression is very similar to that observed in adipogenesis where there is a transient increase in p107 expression accompanied by a decrease in p130 expression. Interestingly, while these older cells accumulated lipid and expressed genes associated with lipid metabolism, they failed to express adipsin and leptin, two well-established markers of terminal adipocyte differentiation. These results suggest that older myoblasts are capable of initiating and progressing through the adipogenic program to a point where they express genes associated with lipid metabolism, but do not reach a terminally differentiated state. This finding may have important metabolic implications in the aging population.     

Arthrobacter aurescens TC1 metabolizes diverse s-triazine ring compounds

Arthrobacter aurescens strain TC1 was isolated without enrichment by plating atrazine-contaminated soil directly onto atrazine-clearing plates. A. aurescens TC1 grew in liquid medium with atrazine as the sole source of nitrogen, carbon, and energy, consuming up to 3,000 mg of atrazine per liter. A. aurescens TC1 is metabolically diverse and grew on a wider range of s-triazine compounds than any bacterium previously characterized. The 23 s-triazine substrates serving as the sole nitrogen source included the herbicides ametryn, atratone, cyanazine, prometryn, and simazine. Moreover, atrazine substrate analogs containing fluorine, mercaptan, and cyano groups in place of the chlorine substituent were also growth substrates. Analogs containing hydrogen, azido, and amino functionalities in place of chlorine were not growth substrates. A. aurescens TC1 also metabolized compounds containing chlorine plus N-ethyl, N-propyl, N-butyl, N-s-butyl, N-isobutyl, or N-t-butyl substituents on the s-triazine ring. Atrazine was metabolized to alkylamines and cyanuric acid, the latter accumulating stoichiometrically. Ethylamine and isopropylamine each served as the source of carbon and nitrogen for growth. PCR experiments identified genes with high sequence identity to atzB and atzC, but not to atzA, from Pseudomonas sp. strain ADP.     

Using phylogeny to improve genome-wide distant homology recognition

The gap between the number of known protein sequences and structures continues to widen, particularly as a result of sequencing projects for entire genomes. Recently there have been many attempts to generate structural assignments to all genes on sets of completed genomes using fold-recognition methods. We developed a method that detects false positives made by these genome-wide structural assignment experiments by identifying isolated occurrences. The method was tested using two sets of assignments, generated by SUPERFAMILY and PSI-BLAST, on 150 completed genomes. A phylogeny of these genomes was built and a parsimony algorithm was used to identify isolated occurrences by detecting occurrences that cause a gain at leaf level. Isolated occurrences tend to have high e-values, and in both sets of assignments, a sudden increase in isolated occurrences is observed for e-values >10⁻⁸ for SUPERFAMILY and >10⁻⁴ for PSI-BLAST. Conditions to predict false positives are based on these results. Independent tests confirm that the predicted false positives are indeed more likely to be incorrectly assigned. Evaluation of the predicted false positives also showed that the accuracy of profile-based fold-recognition methods might depend on secondary structure content and sequence length. We show that false positives generated by fold-recognition methods can be identified by considering structural occurrence patterns on completed genomes; occurrences that are isolated within the phylogeny tend to be less reliable. The method provides a new independent way to examine the quality of fold assignments and may be used to improve the output of any genome-wide fold assignment method.     

Recent contributions of in vitro models to our understanding of hepatitis C virus life cycle

Hepatitis C virus is a human pathogen responsible for liver diseases including acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Its high prevalence, the absence of a prophylactic vaccine and the poor efficiency of current therapies are huge medical problems. Since the discovery of the hepatitis C virus, our knowledge of its biology has been largely punctuated by the development of original models of research. At the end of the 1980s, the chimpanzee model led to cloning of the viral genome and the definition of infectious molecular clones. In 1999, a breakthrough was achieved with the development of a robust in vitro replication model named 'replicon'. This system allowed intensive research into replication mechanisms and drug discovery. Later, in 2003, pseudotyped retroviruses harbouring surface proteins of hepatitis C virus were produced to specifically investigate the viral entry process. It was only in 2005 that infectious viruses were produced in vitro, enabling intensive investigations into the entire life cycle of the hepatitis C virus. This review describes the different in vitro models developed to study hepatitis C virus, their contribution to current knowledge of the virus biology and their future research applications.     

Cell cycle-dependent activity of the volume- and Ca2+-activated anion currents in Ehrlich lettre ascites cells

Recent evidence implicates the volume-regulated anion current (VRAC) and other anion currents in control or modulation of cell cycle progression; however, the precise involvement of anion channels in this process is unclear. Here, Cl- currents in Ehrlich Lettre Ascites (ELA) cells were monitored during cell cycle progression, under three conditions: (i) after osmotic swelling (i.e., VRAC), (ii) after an increase in the free intracellular Ca2+ concentration (i.e., the Ca2+-activated Cl- current, CaCC), and (iii) under steady-state isotonic conditions. The maximal swelling-activated VRAC current decreased in G1 and increased in early S phase, compared to that in G0. The isotonic steady-state current, which seems to be predominantly VRAC, also decreased in G1, and increased again in early S phase, to a level similar to that in G0. In contrast, the maximal CaCC current (500 nM free Ca2+ in the pipette), was unaltered from G0 to G1, but decreased in early S phase. A novel high-affinity anion channel inhibitor, the acidic di-aryl-urea NS3728, which inhibited both VRAC and CaCC, attenuated ELA cell growth, suggesting a possible mechanistic link between cell cycle progression and cell cycle-dependent changes in the capacity for conductive Cl- transport. It is suggested that in ELA cells, entrance into the S phase requires an increase in VRAC activity and/or an increased potential for regulatory volume decrease (RVD), and at the same time a decrease in CaCC magnitude.