Prothymosin alpha mRNA levels are invariant throughout the cell cycle.

Prothymosin alpha (PT-alpha) mRNA levels were evaluated at different stages during the cell cycle. NIH 3T3 cells were synchronized: (a) by serum deprivation, (b) by mitotic shake off after nocodazole arrest, and (c) by double thymidine block. Cell synchronism was estimated by flow cytometry. In cells grown in serum-free medium, PT-alpha mRNA levels were almost undetectable. 14 h after serum restoration PT-alpha mRNA was induced as had been described by others (Eschendfeldt, W. H., and Berger, S. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9403-9407). PT-alpha mRNA induction seems to require the synthesis of proteic factor(s) since PT-alpha mRNA response to serum restoration was abolished in the presence of cycloheximide. Interestingly, cycling cells that were synchronized at different stages of the cycle by means of mitotic shake off after nocodazole arrest or double thymidine block did not show variations in the levels of PT-alpha mRNA when progressed synchronously through the cycle. On the contrary, histone H4 mRNA was expressed only during the S phase. These data indicate that PT-alpha mRNA was present in roughly the same amount through all phases of the cell cycle, arguing against the concept that PT-alpha is a cell cycle-regulated gene.     

Viral fusion peptides and identification of membrane-interacting segments

Viral envelope glycoproteins promote infection by mediating fusion between viral and cellular membranes. Fusion occurs after dramatic conformational changes within fusion proteins, leading to the exposure of a short stretch of mostly apolar residues, termed the fusion peptide, which is presumed to insert into the membrane and initiate the fusion process. The typical global composition of fusion peptides, rich in hydrophobic but also in small amino acids such as alanine and glycine, was used here as bait to detect other peptidic segments that can insert into membranes. We so evidenced a similar composition in several cytotoxic peptides, which promote pore formation such as peptides involved in amyloidoses and hydrophobic alpha-hairpins of pore-forming toxins. It is suggested that the structural plasticity observed for several membrane active peptides can be conferred by this particular global amino acid composition, which could be thus used to predict such functional behavior from genome data.     

Inhibition of protein kinase C zeta subspecies blocks the activation of an NF-kappa B-like activity in Xenopus laevis oocytes.

Nuclear factor kappa B (NF-kappa B) plays a critical role in the regulation of a large variety of cellular genes. However, the mechanism whereby this nuclear factor is activated remains to be determined. In this report, we present evidence that in oocytes from Xenopus laevis, (i) ras p21- and phospholipase C (PLC)-mediated phosphatidylcholine (PC) hydrolysis activates NF-kappa B and (ii) protein kinase C zeta subspecies is involved in the activation of NF-kappa B in response to insulin/ras p21/PC-PLC. Thus, the microinjection of either ras p21 or PC-PLC, or the exposure of oocytes to insulin, promotes a significant translocation to the nucleus of an NF-kappa B-like activity. This effect is not observed when oocytes are incubated with phorbol myristate acetate or progesterone, both of which utilize a ras p21-independent pathway for oocyte activation. These data strongly suggest a critical role of the insulin/ras p21/PC-PLC/protein kinase C zeta pathway in the control of NF-kappa B activation.     

The ecology of chronic wasting disease in wildlife

Prions are misfolded infectious proteins responsible for a group of fatal neurodegenerative diseases termed transmissible spongiform encephalopathy or prion diseases. Chronic Wasting Disease (CWD) is the prion disease with the highest spillover potential, affecting at least seven Cervidae (deer) species. The zoonotic potential of CWD is inconclusive and cannot be ruled out. A risk of infection for other domestic and wildlife species is also plausible. Here, we review the current status of the knowledge with respect to CWD ecology in wildlife. Our current understanding of the geographic distribution of CWD lacks spatial and temporal detail, does not consider the biogeography of infectious diseases, and is largely biased by sampling based on hunters' cooperation and funding available for each region. Limitations of the methods used for data collection suggest that the extent and prevalence of CWD in wildlife is underestimated. If the zoonotic potential of CWD is confirmed in the short term, as suggested by recent results obtained in experimental animal models, there will be limited accurate epidemiological data to inform public health. Research gaps in CWD prion ecology include the need to identify specific biological characteristics of potential CWD reservoir species that better explain susceptibility to spillover, landscape and climate configurations that are suitable for CWD transmission, and the magnitude of sampling bias in our current understanding of CWD distribution and risk. Addressing these research gaps will help anticipate novel areas and species where CWD spillover is expected, which will inform control strategies. From an ecological perspective, control strategies could include assessing restoration of natural predators of CWD reservoirs, ultrasensitive CWD detection in biotic and abiotic reservoirs, and deer density and landscape modification to reduce CWD spread and prevalence.     

Susceptibility of heterochromatin to aphidicolin-induced chromosomal breakage

The distribution of aphidicolin-induced chromosomal lesions was analyzed to determine the relative breakage susceptibility of euchromatin and heterochromatin in the cactus mouse, Peromyscus eremicus. The observed breakage was tested against expected distributions corresponding to the karyotypic proportions of autosomal euchromatin, autosomal heterochromatin, X-chromosomal euchromatin, and X-chromosomal heterochromatin. The distribution of induced breakage was independent of sex but dependent on the individual. In all individuals, there was a highly significant (P0.0001) deficiency in the number of breaks observed as compared to expected in autosomal heterochromatin. Sparse observations in the X chromosome and the absence of breaks in the Y chromosome precluded valid statistical tests of the sex-chromosomal distribution of induced breakage. These data indicate that the autosomal heterochromatin of Peromyscus is resistant to aphidicolin-induced chromosomal breakage and argue against a simple relationship between late replication and a general mechanism for chromosomal fragility.     

The use of ROI overlays and a semi-automated method for measuring cortical area in ImageJ for histological analysis

A major part of histologic studies is the use of high resolution imaging for data collection and analysis. ImageJ, a freely available software from the NIH designed for image analysis, has many features that are not well-known among bone histologists and can be incredibly beneficial in terms of stream-lining data collection and maximizing limited resources. The aims of this technical note are twofold: (a) to describe methods for image annotation and measurement using region of interest overlays in ImageJ, and (b) to present a new code for a semi-automated method of measuring cortical bone areas from high resolution cross-sectional images also using ImageJ.     

Evaluation of the smooth muscle cell component and apoptosis in the varicose vein wall

This study was designed to evaluate the role of the smooth muscle cell and the apoptosis in the pathogenesis of the varicose vein. Segments of saphenous vein were obtained from healthy subjects and from those with varicose veins. The vein specimens were subdivided according to subject age (younger or older than 50 years) and according to the varicose vein source (distal or proximal). Morphological, ultrastructural, cell proliferation (anti-PCNA method) and cell death (TUNEL method) analysis were performed. The walls of healthy, control vein specimens acquired a more collagenous and papillomatous appearance with age. A slight increase in the number of TUNEL-positive cells was also observed in specimens from older subjects. The proportion of apoptotic cells was much greater in the varicose veins than in control specimens. Most cellular alterations were seen in proximal varicose segments obtained from young subjects. These specimens showed hypertrophic areas with a high degree of cellularity (both in the media and in the thickened intima). The highest proportion of apoptotic cells and collagenisation were also observed in these areas. The enhanced number of apoptotic cells in varicose veins observed mainly in proximal/young vein specimens could be responsible, at least in part, for the acceleration of the final fibrosclerotic process characteristic of the varicose vein wall.     

Regulation of membrane trafficking by a novel Cdc42-related protein in Caenorhabditis elegans epithelial cells

Rho GTPases are mainly known for their implication in cytoskeleton remodeling. They have also been recently shown to regulate various aspects of membrane trafficking. Here, we report the identification and the characterization of a novel Caenorhabditis elegans Cdc42-related protein, CRP-1, that shows atypical enzymatic characteristics in vitro. Expression in mouse fibroblasts revealed that, in contrast with CDC-42, CRP-1 was unable to reorganize the actin cytoskeleton and mainly localized to trans-Golgi network and recycling endosomes. This subcellular localization, as well as its expression profile restricted to a subset of epithelial-like cells in C. elegans, suggested a potential function for this protein in polarized membrane trafficking. Consistent with this hypothesis, alteration of CRP-1 expression affected the apical trafficking of CHE-14 in vulval and rectal epithelial cells and sphingolipids (C(6)-NBD-ceramide) uptake and/or trafficking in intestinal cells. However, it did not affect basolateral trafficking of myotactin in the pharynx and the targeting of IFB-2 and AJM-1, two cytosolic apical markers of intestine epithelial cells. Hence, our data demonstrate a function for CRP-1 in the regulation of membrane trafficking in a subset of cells with epithelial characteristics.     

4-dimethylamino-3',4'-dimethoxychalcone downregulates iNOS expression and exerts anti-inflammatory effects

Reactive oxygen and nitrogen species contribute to the pathophysiology of inflammatory conditions. We have studied the effects of a novel superoxide scavenger, 4-dimethylamino-3', 4'-dimethoxychalcone (CH11) in macrophages and in vivo. CH11 has been shown to inhibit the chemiluminescence induced by zymosan in mouse peritoneal macrophages and the cytotoxic effects of superoxide. In the same cells, the modulation by superoxide of nitric oxide (NO) production in response to zymosan was investigated. CH11 was more effective than the membrane-permeable scavenger Tiron for inhibition of inducible nitric oxide synthase (iNOS) protein expression and nitrite production. We have shown that CH11 inhibited chemiluminescence in vivo, as well as cell migration, and eicosanoid and tumor necrosis factor-alpha (TNF-alpha) levels in the mouse air pouch injected with zymosan. This chalcone derivative also exerted anti-inflammatory effects in the carrageenan paw oedema.