Metabolic profiles and genetic diversity of denitrifying communities in activated sludge after addition of methanol or ethanol

External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.     

Survival of yogurt bacteria in the human gut

Whether Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus can be recovered after passage through the human gut was tested by feeding 20 healthy volunteers commercial yogurt. Yogurt bacteria were found in human feces, suggesting that they can survive transit in the gastrointestinal tract.     

Soluble factors with inhibitory activity against type 1 Human Immunodeficiency Virus

Soluble factors with inhibitory activity against type 1 Human Immunodeficiency Virus The pathogenesis of HIV-1 infection is a complex process that depends on multiple factors, including viral and host immune and genetic characteristics. This leads to a variable pattern of disease progression among those HIV-1-exposed individuals who become infected, while there are a number of individuals who remain healthy and HIV-1 seronegative despite being serially exposed to HIV-1. These variable outcomes of HIV-1 exposure suggest that there are mechanisms of natural resistance to HIV-1 infection. Although several genetic and adaptive immune mechanisms of resistance have been reported in some exposed seronegative and long-term non-progressor individuals, the mechanisms involved in controlling the establishment and progression of HIV-1 infection are not fully understood. Several soluble factors, such as defensins, chemokines, interferons and ribonucleases, among others, produced by cells of the immune system and epithelial tissues, have a broad anti-viral activity that might play a role as protective mechanisms during HIV-1 exposure. A better understanding of the mechanisms and role of these soluble factors during the natural resistance to HIV-1 infection may have important implications for the design of novel therapeutic strategies to combat the morbidity and mortality associated with the HIV-1 pandemic.     

Conjugation to Nedd8 instigates ubiquitylation and down-regulation of activated receptor tyrosine kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.     

A requirement for dimerization of HP1Hsalpha in suppression of breast cancer invasion

The development and progression of cancer is controlled by gene expression, often regulated through chromatin packaging. Heterochromatin protein 1(Hsalpha) (HP1(Hsalpha)), one of three human HP1 family members, participates in heterochromatin formation and gene regulation. HP1(Hsalpha) possesses an amino-terminal chromodomain, which binds methylated lysine 9 of histone H3 (meK9 H3), and a carboxyl-terminal chromoshadow domain (CSD) that is required for dimerization and interaction with partner proteins. HP1(Hsalpha) is down-regulated in invasive metastatic breast cancer cells compared with poorly invasive nonmetastatic breast cancer cells. Expression of EGFP-HP1(Hsalpha) in highly invasive MDA-MB-231 cells causes a reduction in in vitro invasion, without affecting cell growth. Conversely, knock-down of HP1(Hsalpha) levels in the poorly invasive breast cancer cell line MCF-7 increased invasion, without affecting cell growth. To determine whether functions of the CSD were required for the regulation of invasion, mutant forms of HP1(Hsalpha) were expressed in MDA-MB-231 cells. A W174A mutation that disrupts interactions between HP1(Hsalpha) and PXVXL-containing partner proteins reduced invasion similar to that of the wild type protein. In contrast, an I165E mutation that disrupts dimerization of HP1(Hsalpha) did not decrease invasion. No gross changes in localization and abundance of HP1(Hsbeta), HP1(Hsgamma), and meK9 H3 were observed upon expression of wild type and mutant forms of HP1(Hsalpha) in MDA-MB-231 cells. Taken together, these data demonstrate that modulation of HP1(Hsalpha) alters the invasive potential of breast cancer cells through mechanisms requiring HP1 dimerization, but not interactions with PXVXL-containing proteins.     

Modeling H3 histone N-terminal tail and linker DNA interactions

Molecular dynamics computer simulations were performed for the 25-residue N-terminal tail of the H3 histone protein in the proximity of a DNA segment of 10 base pairs (bp), representing a model for the linker DNA in chromatin. Several least biased configurations were used as initial configurations. The secondary structure content of the protein was increased by the presence of DNA close to it, but the locations of the secondary motifs were different for different initial orientations of the DNA grooves with respect to the protein. As a common feature to all simulations, the electrostatic attraction between negatively charged DNA and positively charged protein was screened by the water solvent and counterbalanced by the intrinsic compaction of the protein due to hydrophobic effects. The protein secondary structure limited the covering of DNA by the protein to 4-5 bp. The degree of compaction and charge density of the bound protein suggests a possible role of H3 tail in a nonspecific bending and plasticity of the linker DNA when the protein is located in the crowded dense chromatin.     

Amino acids 15-28 in the ectodomain of SARS coronavirus 3a protein induces neutralizing antibodies

A synthetic peptide corresponding to amino acids (aa) 15-28 of the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein was used to raise polyclonal antibodies in rabbits. This anti-3a N-terminal antibody could detect 3a protein in infected cells, as did an anti-3a C-terminal antibody previously described. The latter targeted the C-terminal cytoplasmic domain of 3a (aa 134-274). The anti-3a N-terminal antibody could detect intracellular 3a as well as 3a expressed on the cell surface. Interestingly, only the anti-3a N-terminal antibody can inhibit SARS-CoV propagation in Vero E6 culture although the binding affinity of the anti-3a N-terminal antibody was lower than the anti-3a C-terminal antibody.