Noninfectious human immunodeficiency virus type 1 mutants deficient in genomic RNA.

All retroviruses contain, in the nucleocapsid domain of the Gag protein, one or two copies of the sequence Cys-X2-Cys-X4-His-X4-Cys. We have generated a series of mutants in the two copies of this motif present in human immunodeficiency virus type 1. These mutants encoded virus particles that were apparently composed of the normal complement of viral proteins but contained only 2 to 20% of the normal level of genomic RNA. No infectivity could be detected in the mutant particles, while 10(5) infectious U were present in an equivalent amount of wild-type particles. Thus, the mutants have another defect in addition to the inefficiency with which they encapsidate genomic RNA. Our results show that both copies of the motif are required for normal RNA packaging and for infectivity. Mutants of this type may have important applications, including nonhazardous materials for research, immunogens in vaccine and immunotherapy studies, and diagnostic reagents.     

Micropropagation of Cowania subintegra and Cowania stansburiana

Both Cowania subintegra Kearney and C. stansburiana Torr. were successfully propagated in vitro. Shoot proliferation occurred from shoot tips of green-house grown C. subintegra using a modified Murashige and Skoog medium supplemented with 4.4 M 6-benzyladenine and 0.5 M indole butyric acid. Excised microshoots (1.5–3.0 cm long) of both species were rooted using a two-step process in which they were cultured for 3 days in a root initiation medium with 2.7 M naphthaleneacetic acid and then transferred to a low nitrogen root elongation medium without auxin. Plantlets were successfully transferred to soilless potting mix.     

Fingerprinting Diazotroph Communities in the Chesapeake Bay by Using a DNA Macroarray

Investigations of the distribution and diversity of nitrogen-fixing microorganisms in natural environments have often relied on PCR amplification and sequence analysis of a portion of one of the key enzymes in nitrogen fixation, dinitrogenase reductase, encoded by nifH. Recent work has suggested that DNA macroarrays provide semiquantitative fingerprints of diversity within mixtures of nifH amplicons (G. F. Steward, B. D. Jenkins, B. B. Ward, and J. P. Zehr, Appl. Environ. Microbiol. 70:1455-1465, 2004). Here we report the application of macroarrays for a study in the Chesapeake Bay. Samples from different locations in the bay yielded distinct fingerprints. Analysis of replicates and samples from different locations by cluster analysis showed that replicates clustered together, whereas different samples formed distinct clusters. There was a correspondence between the hybridization pattern observed and that predicted from the distribution of sequence types in a corresponding clone library. Some discrepancies between the methods were observed which are likely a result of the high nifH sequence diversity in the Chesapeake Bay and the limited number of sequences represented on this version of the array. Analyses of sequences in the clone library indicate that the Chesapeake Bay harbors unique, phylogenetically diverse diazotrophs. The macroarray hybridization patterns suggest that there are spatially variable communities of diazotrophs, which have been confirmed by quantitative PCR methods (S. M. Short, B. D. Jenkins, and J. P. Zehr, Appl. Environ. Microbiol., in press). The results show that DNA macroarrays have great potential for mapping the spatial and temporal variability of functional gene diversity in the environment.     

Alteration of surfactant protein A expression in renal cell carcinoma

Surfactant protein-A (SP-A) belongs to a family of collagen-containing C-type lectins called collectins. SP-A is expressed by renal tubule epithelial cells. We investigated the distribution of SP-A in renal cell carcinomas (RCC) using immunohistochemical techniques and western blotting. We used 35 formalin fixed, paraffin embedded (FFPE) RCC tissue samples. We compared results with clinico-pathological parameters of RCC including age, sex, Fuhrman grade, tumor volume, tumor node metastasis (TNM) and clinical stage. SP-A was localized in the glomerulus and renal tubule epithelium in nontumor tissue and strong SP-A immunoreactivity was observed in tumor tissue. SP-A was expressed in the RCC tumor cells (64%) and nontumor cells (34%) in males and RCC tumor cells (90%) and nontumor cells (30%) in females. There was a significant correlation between SP-A immunoreactivity in tumor cells and gender, age, tumor diameter, Fuhrman grade and tumor diameter. Western blot analysis supported the immunohistochemical findings. We present evidence for involvement of SP-A in RCC and suggest that increased SP-A expression in RCC is associated with favorable prognosis.     

Sphingosine Kinase 1 Is Regulated by Peroxisome Proliferator-activated Receptor α in Response to Free Fatty Acids and Is Essential for Skeletal Muscle Interleukin-6 Production and Signaling in Diet-induced Obesity

We previously demonstrated that sphingosine kinase 1 (Sphk1) expression and activity are up-regulated by exogenous palmitate (PAL) in a skeletal muscle model system and in diet-induced obesity in mice; however, potential functions and in vivo relevance of this have not been addressed. Here, we aimed to determine the mechanism by which PAL regulates SphK1 in muscle, and to determine potential roles for its product, sphingosine-1-phosphate (S1P), in muscle biology in the context of obesity. Cloning and analysis of the mouse Sphk1 promoter revealed a peroxisome proliferator-activated receptor (PPAR) α cis-element that mediated activation of a reporter under control of the Sphk1 promoter; direct interaction of PPARα was demonstrated by chromatin immunoprecipitation. PAL treatment induced the proinflammatory cytokine interleukin (IL)-6 in a manner dependent on SphK1, and this was attenuated by inhibition of the sphingosine-1-phosphate receptor 3 (S1PR3)_. Diet-induced obesity in mice demonstrated that IL-6 expression in muscle, but not adipose tissue, increased in obesity, but this was attenuated in Sphk1^−/− mice. Moreover, plasma IL-6 levels were significantly decreased in obese Sphk1^−/− mice relative to obese wild type mice, and muscle, but not adipose tissue IL-6 signaling was activated. These data indicate that PPARα regulates Sphk1 expression in the context of fatty acid oversupply and links PAL to muscle IL-6 production. Moreover, this function of SphK1 in diet-induced obesity suggests a potential role for SphK1 in obesity-associated pathological outcomes.     

Exercise effect on weight and body fat in men and women

Objectives: The effect of national exercise recommendations on adiposity is unknown and may differ by sex. We examined long‐term effects of aerobic exercise on adiposity in women and men. Research Methods and Procedures: This was a 12‐month randomized, controlled clinical trial testing exercise effect on weight and body composition in men (N = 102) and women (N = 100). Sedentary/unfit persons, 40 to 75 years old, were recruited through physician practices and media. The intervention was facility‐ and home‐based moderate‐to‐vigorous intensity aerobic activity, 60 min/d, 6 days/wk vs. controls (no intervention). Results: Exercisers exercised a mean 370 min/wk (men) and 295 min/wk (women), and seven dropped the intervention. Exercisers lost weight (women, ?1.4 vs. +0.7 kg in controls, p = 0.008; men, ?1.8 vs. ?0.1 kg in controls, p = 0.03), BMI (women, ?0.6 vs. +0.3 kg/m² in controls, p = 0.006; men, ?0.5 kg/m² vs. no change in controls, p = 0.03), waist circumference (women, ?1.4 vs. +2.2 cm in controls, p < 0.001; men, ?3.3 vs. ?0.4 cm in controls, p = 0.003), and total fat mass (women, ?1.9 vs. +0.2 kg in controls, p = 0.001; men, ?3.0 vs. +0.2 kg in controls, p < 0.001). Exercisers with greater increases in pedometer‐measured steps per day had greater decreases in weight, BMI, body fat, and intra‐abdominal fat (all p trend < 0.05 in both men and women). Similar trends were observed for increased minutes per day of exercise and for increases in maximal oxygen consumption. Discussion: These data support the U.S. Department of Agriculture and Institute of Medicine guidelines of 60 min/d of moderate‐to‐vigorous physical activity.     

Dominant Microbial Populations in Limestone-Corroding Stream Biofilms, Frasassi Cave System, Italy

Waters from an extensive sulfide-rich aquifer emerge in the Frasassi cave system, where they mix with oxygen-rich percolating water and cave air over a large surface area. The actively forming cave complex hosts a microbial community, including conspicuous white biofilms coating surfaces in cave streams, that is isolated from surface sources of C and N. Two distinct biofilm morphologies were observed in the streams over a 4-year period. Bacterial 16S rDNA libraries were constructed from samples of each biofilm type collected from Grotta Sulfurea in 2002. β-, γ-, δ-, and -proteobacteria in sulfur-cycling clades accounted for ≥75% of clones in both biofilms. Sulfate-reducing and sulfur-disproportionating δ-proteobacterial sequences in the clone libraries were abundant and diverse (34% of phylotypes). Biofilm samples of both types were later collected at the same location and at an additional sample site in Ramo Sulfureo and examined, using fluorescence in situ hybridization (FISH). The biomass of all six stream biofilms was dominated by filamentous γ-proteobacteria with Beggiatoa-like and/or Thiothrix-like cells containing abundant sulfur inclusions. The biomass of -proteobacteria detected using FISH was consistently small, ranging from 0 to less than 15% of the total biomass. Our results suggest that S cycling within the stream biofilms is an important feature of the cave biogeochemistry. Such cycling represents positive biological feedback to sulfuric acid speleogenesis and related processes that create subsurface porosity in carbonate rocks.     

三氧化二砷对食管癌细胞增殖和热休克蛋白70表达的影响

目的：研究三氧化二砷（As2O3）对食管癌细胞增殖和热休克蛋白70（HSP70）表达的影响。方法：通过相差显微镜、流式细胞术、免疫细胞化学染色和免疫印迹分析等方法观察As2O3对人食管癌细胞株EC1的作用效果和作用机制。结果：与对照组相比,经2μmol/L和5μmol/Las2O3作用的细胞出现明显的生长抑制,G2/M期细胞比例增加;2μmol/Las2O3作用48h后经Ecl细胞HSP70（heat shock protein70）及HSC70(heat shock cognate protein70)表达均增加。结论：As2O3诱导食管癌细胞G2/M期阻滞抑制细胞增殖和生长;HSP70的升高是细胞对As2O3作用后出现的应激反应,并与细胞周期阻滞相关。     

Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.