We have developed an improved vector for the stable expression of recombinant protein in mammalian cells. In this vector, designated pCIN, both the recombinant cDNA and the neomycin phosphotransferase selection marker are transcribed from a single promoter element. To facilitate translation of the second open reading frame, the encephalomyocarditis virus internal ribosome entry site has been inserted into the expression cassette immediately before the start codon of this sequence. We report the use of this vector to generate stable cell lines expressing the human 5-HT1Da serotonin receptor and show that following transfection and clonal selection, all ten cell lines characterized express similar and high levels of receptor (1.5-11.9 pmol receptor/mg protein). Use of pCIN should permit the rapid and efficient production of stable mammalian cell lines for the characterization of recombinant protein, as this vector appears to predispose all transfected cells to express such protein.

Periodontal disease is the most widespread oral disease in dogs which if left untreated results in significant pain to the pet and loss of dentition. The objective of this study was to identify bacterial species in canine plaque that are significantly associated with health, gingivitis and mild periodontitis (<25% attachment loss). In this survey subgingival plaque samples were collected from 223 dogs with healthy gingiva, gingivitis and mild periodontitis with 72 to 77 samples per health status. DNA was extracted from the plaque samples and subjected to PCR amplification of the V1-V3 region of the 16S rDNA. Pyrosequencing of the PCR amplicons identified a total of 274 operational taxonomic units after bioinformatic and statistical analysis. Porphyromonas was the most abundant genus in all disease stages, particularly in health along with Moraxella and Bergeyella. Peptostreptococcus, Actinomyces, and Peptostreptococcaceae were the most abundant genera in mild periodontitis. Logistic regression analysis identified species from each of these genera that were significantly associated with health, gingivitis or mild periodontitis. Principal component analysis showed distinct community profiles in health and disease. The species identified show some similarities with health and periodontal disease in humans but also major differences. In contrast to human, healthy canine plaque was found to be dominated by Gram negative bacterial species whereas Gram positive anaerobic species predominate in disease. The scale of this study surpasses previously published research and enhances our understanding of the bacterial species present in canine subgingival plaque and their associations with health and early periodontal disease.

Smooth muscle cells (SMCs) display remarkable phenotypic diversity and plasticity and can readily switch between proliferative and differentiated states in response to extracellular cues. In an effort to identify novel transcriptional regulators of smooth muscle phenotypes, we compared the gene expression profiles of arterial and venous SMCs by microarray-based transcriptional profiling. Among numerous genes displaying distinct expression patterns in these two SMC types, we discovered an expressed sequence tag encoding a previously uncharacterized zinc finger protein belonging to the PRDM (PRDI-BF1 and RIZ homology domain) family of chromatin-remodeling proteins and named it PRISM (PR domain in smooth muscle). PRISM is expressed in a variety of smooth muscle-containing tissues and displays especially robust expression in the cardiac outflow tract and descending aorta during embryogenesis. PRISM is localized to the nucleus and contains an aminoterminal PR domain and four Krüppel-like zinc fingers at the carboxy terminus. We show that PRISM acts as a transcriptional repressor by interacting with class I histone deacetylases and the G9a histone methyltransferase, thereby identifying PRISM as a novel SMC-restricted epigenetic regulator. Overexpression of PRISM in cultured primary SMCs induces genes associated with the proliferative smooth muscle phenotype while repressing regulators of differentiation, including myocardin and GATA-6. Conversely, small interfering RNA-mediated knockdown of PRISM slows cell growth and induces myocardin, GATA-6, and markers of SMC differentiation. We conclude that PRISM acts as a novel epigenetic regulator of SMC phenotypic plasticity by suppressing differentiation and maintaining the proliferative potential of vascular SMCs.Smooth muscle (SM) cells (SMCs) display remarkable phenotypic diversity and plasticity. SMCs of the venous and arterial systems, for example, can be distinguished by their contractile properties, morphologies, and patterns of gene expression (34). Similarly, visceral SMCs of the lungs, bladder, intestine, and other internal organs display distinct properties that reflect their unique functions. SMCs also modulate their phenotypes in response to extracellular cues. In the absence of growth signals, arterial SMCs withdraw from the cell cycle and express a set of contractile protein genes. Conversely, growth factor signaling provokes SMCs to reenter the cell cycle with a consequent activation of genes involved in proliferation and a concomitant down-regulation of contractile transcripts (35). Such phenotypic plasticity is responsible for abnormalities in SMC growth and differentiation in a variety of disorders, including atherosclerosis, vascular restenosis following angioplasty, hypertension, and smooth muscle tumors (10,36,45,51).Several transcription factors have been implicated in the control of SMC differentiation (35). The best-characterized regulator of smooth muscle gene expression is serum response factor (SRF), which binds a DNA sequence known as a CArG box ...

Society. Effect of exercise on cerebral perfusion in humans at high altitude. J Appl Physiol 99: 699 -706, 2005. First published May 26, 2005 doi:10.1152/japplphysiol.00973.2004.-The effects of submaximal and maximal exercise on cerebral perfusion were assessed using a portable, recumbent cycle ergometer in nine unacclimatized subjects ascending to 5,260 m. At 150 m, mean (SD) cerebral oxygenation (rSO2%) increased during submaximal exercise from 68.4 (SD 2.1) to 70.9 (SD 3.8) (P Ͻ 0.0001) and at maximal oxygen uptake (V O2 max) to 69.8 (SD 3.1) (P Ͻ 0.02). In contrast, at each of the high altitudes studied, rSO2 was reduced during submaximal exercise from 66.2 (SD 2.5) to 62.6 (SD 2.1) at 3,610 m (P Ͻ 0.0001), 63.0 (SD 2.1) to 58.9 (SD 2.1) at 4,750 m (P Ͻ 0.0001), and 62.4 (SD 3.6) to 61.2 (SD 3.9) at 5,260 m (P Ͻ 0.01), and at V O2 max to 61.2 (SD 3.3) at 3,610 m (P Ͻ 0.0001), to 59.4 (SD 2.6) at 4,750 m (P Ͻ 0.0001), and to 58.0 (SD 3.0) at 5,260 m (P Ͻ 0.0001). Cerebrovascular resistance tended to fall during submaximal exercise (P ϭ not significant) and rise at V O2 max, following the changes in arterial oxygen saturation and end-tidal CO 2. Cerebral oxygen delivery was maintained during submaximal exercise at 150 m with a nonsignificant fall at V O2 max, but at high altitude peaked at 30% of V O2 max and then fell progressively at higher levels of exercise. The fall in rSO2 and oxygen delivery during exercise may limit exercise at altitude and is likely to contribute to the problems of acute mountain sickness and highaltitude cerebral edema. maximal oxygen uptake; cerebral oxygenation; cerebral blood flow; cerebrovascular resistance; cerebral oxygen delivery ALTERED CEREBRAL FUNCTION on ascent to altitude was part of the first description of mountain sickness in 1913 (40), and acute mountain sickness (AMS) and high-altitude cerebral edema (HACE) have been shown to be potentially serious clinical conditions that may occur on acute exposure to altitudes above 2,500 -3,000 m. Exercise at altitude causes further decreases in arterial oxygenation and so may exacerbate cerebral hypoxia. However, the fall in arterial saturation that occurs during exercise at high altitude (14) might not affect cerebral oxygenation to the same extent as it does in peripheral tissues due to a compensatory increase in cerebral blood flow (24). Nevertheless, avoidance of strenuous exercise during ascent, and on arrival at altitude, is standard advice for reducing the risk of AMS and HACE. Evidence from clinical studies is conflicting. Higher AMS symptom scores were found in subjects exercising four times a day for 30 min at 50% of their altitude-specific maximal oxygen consumption (V O 2 max ), compared with no exercise, in a chamber study at simulated altitude of 4,800 m (41). Another study of mountaineers, however, showed that physical fitness and exercise intensity during ascent to 4,559 m were of minor importance for the development of AMS (3). It is also possible that other neurological conditions falling outside the usual def...

Ty virus-like particles consist of a single protein species that can be produced in yeast. Recombinant Ty-VLPs carrying a string of up to 15 defined cytotoxic T lymphocyte (CTL) epitopes from Plasmodium species prime protective CTL responses in mice following a single administration without adjuvant. Effective processing of epitopes from the string was demonstrated in vitro and in vivo and was not affected by flanking sequences.