The envelope gp120 gene of human immunodeficiency virus type 1 determines the rate of CD4-positive T-cell depletion in SCID mice engrafted with human peripheral blood leukocytes.

We have used envelope recombinant viruses generated between two molecular clones of human immunodeficiency virus type 1 (HIV-1), T-cell-tropic HIV-1SF2 and macrophage-tropic HIV-1SF162, to assess pathogenic potential in the human peripheral blood leukocyte-reconstituted severe combined immune deficiency mouse model. Recombinant HIV-1SF2 viruses expressing the envelope gp120 gene of HIV-ISF162 caused as rapid a CD4+ T-cell depletion as did HIV-1SF162. The reciprocal HIV-1SF162 recombinant virus with the HIV-1SF2 envelope caused slower CD4+ T-cell loss. Although changing the V3 loop sequence of HIV-1SF162 to that of HIV-1SF2 did not change the rate of CD4+ T-cell depletion, replacing the V3 of HIV-1SF2 with the sequence of HIV-1SF162 resulted in virus that was poorly infectious in vivo but not in vitro. These studies suggest that the envelope gene determines properties important for pathogenesis in vivo as well as for cell tropism in vitro. HIV-1 infection in vivo may have more stringent requirements for envelope conformation.     

Microbiota within the perennial ice cover of Lake Vida, Antarctica

Lake Vida, located in the McMurdo Dry Valleys, Antarctica, is an 'ice-sealed' lake with approximately 19 m of ice covering a highly saline water column (approximately 245 ppt). The lower portions of the ice cover and the lake beneath have been isolated from the atmosphere and land for circa 2800 years. Analysis of microbial assemblages within the perennial ice cover of the lake revealed a diverse array of bacteria and eukarya. Bacterial and eukaryal denaturing gradient gel electrophoresis phylotype profile similarities were low (<59%) between all of the depths compared (five depths spanning 11 m of the ice cover), with the greatest differences occurring between surface and deep ice. The majority of bacterial 16S rRNA gene sequences in the surface ice were related to Actinobacteria (42%) while Gammaproteobacteria (52%) dominated the deep ice community. Comparisons of assemblage composition suggest differences in ice habitability and organismal origin in the upper and lower portions of ice cover. Specifically, the upper ice cover microbiota likely reflect the modern day transport and colonization of biota from the terrestrial landscape, whereas assemblages in the deeper ice are more likely to be persistent remnant biota that originated from the ancient liquid water column of the lake that froze.     

Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion

Proteases within secretory vesicles are required for conversion of neuropeptide precursors into active peptide neurotransmitters and hormones. This study demonstrates the novel cellular role of the cysteine protease cathepsin L for producing the (Met)enkephalin peptide neurotransmitter from proenkephalin (PE) in the regulated secretory pathway of neuroendocrine PC12 cells. These findings were achieved by coexpression of PE and cathepsin L cDNAs in PC12 cells with analyses of PE-derived peptide products. Expression of cathepsin L resulted in highly increased cellular levels of (Met)enkephalin, resulting from the conversion of PE to enkephalin-containing intermediates of 23, 18-19, 8-9, and 4.5 kDa that were similar to those present in vivo. Furthermore, expression of cathepsin L with PE resulted in increased amounts of nicotine-induced secretion of (Met)enkephalin. These results indicate increased levels of (Met)enkephalin within secretory vesicles of the regulated secretory pathway. Importantly, cathespin L expression was directed to secretory vesicles, demonstrated by colocalization of cathepsin L-DsRed fusion protein with enkephalin and chromogranin A neuropeptides that are present in secretory vesicles. In vivo studies also showed that cathepsin L in vivo was colocalized with enkephalin. The newly defined secretory vesicle function of cathepsin L for biosynthesis of active enkephalin opioid peptide contrasts with its function in lysosomes for protein degradation. These findings demonstrate cathepsin L as a distinct cysteine protease pathway for producing the enkephalin member of neuropeptides.     

Conserved changes in envelope function during human immunodeficiency virus type 1 coreceptor switching

We studied the evolution of human immunodeficiency virus type 1 (HIV-1) envelope function during the process of coreceptor switching from CCR5 to CXCR4. Site-directed mutagenesis was used to introduce most of the possible intermediate mutations in the envelope for four distinct coreceptor switch mutants, each with a unique pattern of CCR5 and CXCR4 utilization that extended from highly efficient use of both coreceptors to sole use of CXCR4. Mutated envelopes with some preservation of entry function on either CCR5- or CXCR4-expressing target cells were further characterized for their sensitivity to CCR5 or CXCR4 inhibitors, soluble CD4, and the neutralizing antibodies b12-IgG and 4E10. A subset of mutated envelopes was also studied in direct CD4 or CCR5 binding assays and in envelope-mediated fusion reactions. Coreceptor switch intermediates displayed increased sensitivity to CCR5 inhibitors (except for a few envelopes with mutations in V2 or C2) that correlated with a loss in CCR5 binding. As use of CXCR4 improved, infection mediated by the mutated envelopes became more resistant to soluble CD4 inhibition and direct binding to CD4 increased. These changes were accompanied by increasing resistance to the CXCR4 inhibitor AMD3100. Sensitivity to neutralizing antibody was more variable, although infection of CXCR4-expressing targets was generally more sensitive to neutralization by both b12-IgG and 4E10 than infection of CCR5-expressing target cells. These changes in envelope function were uniform in all four series of envelope mutations and thus were independent of the final use of CCR5 and CXCR4. Decreased CCR5 and increased CD4 binding appear to be common features of coreceptor switch intermediates.     

Core and intact polar glycerol dibiphytanyl glycerol tetraether lipids of ammonia-oxidizing archaea enriched from marine and estuarine sediments

Glycerol dibiphytanyl glycerol tetraether (GDGT)-based intact membrane lipids are increasingly being used as complements to conventional molecular methods in ecological studies of ammonia-oxidizing archaea (AOA) in the marine environment. However, the few studies that have been done on the detailed lipid structures synthesized by AOA in (enrichment) culture are based on species enriched from nonmarine environments, i.e., a hot spring, an aquarium filter, and a sponge. Here we have analyzed core and intact polar lipid (IPL)-GDGTs synthesized by three newly available AOA enriched directly from marine sediments taken from the San Francisco Bay estuary ("Candidatus Nitrosoarchaeum limnia"), and coastal marine sediments from Svalbard, Norway, and South Korea. Like previously screened AOA, the sedimentary AOA all synthesize crenarchaeol (a GDGT containing a cyclohexane moiety and four cyclopentane moieties) as a major core GDGT, thereby supporting the hypothesis that crenarchaeol is a biomarker lipid for AOA. The IPL headgroups synthesized by sedimentary AOA comprised mainly monohexose, dihexose, phosphohexose, and hexose-phosphohexose moieties. The hexose-phosphohexose headgroup bound to crenarchaeol was common to all enrichments and, in fact, the only IPL common to every AOA enrichment analyzed to date. This apparent specificity, in combination with its inferred lability, suggests that it may be the most suitable biomarker lipid to trace living AOA. GDGTs bound to headgroups with a mass of 180 Da of unknown structure appear to be specific to the marine group I.1a AOA: they were synthesized by all three sedimentary AOA and "Candidatus Nitrosopumilus maritimus"; however, they were absent in the group I.1b AOA "Candidatus Nitrososphaera gargensis."     

Enzyme hydrolysis and ethanol fermentation of liquid hot water and AFEX pretreated distillers' grains at high-solids loadings

The dry milling ethanol industry produces distiller's grains as major co-products, which are composed of unhydrolyzed and unfermented polymeric sugars. Utilization of the distiller's grains as an additional source of fermentable sugars has the potential to increase overall ethanol yields in current dry grind processes. In this study, controlled pH liquid hot water pretreatment (LHW) and ammonia fiber expansion (AFEX) treatment have been applied to enhance enzymatic digestibility of the distiller's grains. Both pretreatment methods significantly increased the hydrolysis rate of distiller's dried grains with solubles (DDGS) over unpretreated material, resulting in 90% cellulose conversion to glucose within 24h of hydrolysis at an enzyme loading of 15FPU cellulase and 40 IU beta-glucosidase per gram of glucan and a solids loading of 5% DDGS. Hydrolysis of the pretreated wet distiller's grains at 13-15% (wt of dry distiller's grains per wt of total mixture) solids loading at the same enzyme reduced cellulose conversion to 70% and increased conversion time to 72h for both LHW and AFEX pretreatments. However, when the cellulase was supplemented with xylanase and feruloyl esterase, the pretreated wet distiller's grains at 15% or 20% solids (w/w) gave 80% glucose and 50% xylose yields. The rationale for supplementation of cellulases with non-cellulolytic enzymes is given by Dien et al., later in this journal volume. Fermentation of the hydrolyzed wet distiller's grains by glucose fermenting Saccharomyces cerevisiae ATCC 4124 strain resulted in 100% theoretical ethanol yields for both LHW and AFEX pretreated wet distiller's grains. The solids remaining after fermentation had significantly higher protein content and are representative of a protein-enhanced wet DG that would result in enhanced DDGS. Enhanced DDGS refers to the solid product of a modified dry grind process in which the distiller's grains are recycled and processed further to extract the unutilized polymeric sugars. Compositional changes of the laboratory generated enhanced DDGS are also presented and discussed.     

Donor- and ligand-dependent differences in C-C chemokine receptor 5 reexpression

N-terminal modifications of the chemokine RANTES bind to C-C chemokine receptor 5 (CCR5) and block human immunodeficiency virus type 1 (HIV-1) infection with greater efficacy than native RANTES. Modified RANTES compounds induce rapid CCR5 internalization and much slower receptor reexpression than native RANTES, suggesting that receptor sequestration is one mode of anti-HIV activity. The rates of CCR5 internalization and reexpression were compared using the potent n-nonanoyl (NNY)-RANTES derivative and CD4(+) T cells derived from donors with different CCR5 gene polymorphisms. NNY-RANTES caused even more rapid receptor internalization and slower reexpression than aminooxypentane (AOP)-RANTES. Polymorphisms in the promoter and coding regions of CCR5 significantly affected the receptor reexpression rate after exposure of cells to NNY-RANTES. These observations may be relevant for understanding the protective effects of different CCR5 genotypes against HIV-1 disease progression.     

Optimizing nitrogen management in food and energy production and environmental protection: summary statement from the Second International Nitrogen Conference

Human efforts to produce food and energy are changing the nitrogen (N) cycle of the Earth. Many of these changes are highly beneficial for humans, while others are detrimental to people and the environment. These changes transcend scientific disciplines, geographical boundaries, and political structures. They challenge the creative minds of natural and social scientists, economists, engineers, business leaders, and decision makers. The Second International Nitrogen Conference was designed to facilitate communications among all stakeholders in the "nitrogen community" of the world. The Conference participants" goal in the years and decades ahead is to encourage every country to make optimal choices about N management in food production and consumption, energy production and use, and environmental protection. Scientific findings and recommendations for decision makers that emerged from the Conference are presented.