Anti-human immunodeficiency virus type 1 humoral immune response and highly active antiretroviral treatment

Highly active antiretroviral treatment (HAART) of human immunodeficiency type 1 (HIV-1) infection is very effective in controlling infection, but elimination of viral infection has not been achieved as yet, and upon treatment interruption an immediate rebound of viremia is observed. A combination of HAART with an immune stimulation might allow treatment interruption without this rebounding viremia, as the very low viremias observed with successful HAART may be insufficient to permit maintenance of a specific anti-HIV-1 immune response. The objective of this study was to compare the humoral immune response of individuals undergoing successful HAART (NF=no failure) with that of individuals with evidence of failure of therapy (FT) and to verify if the viremia peaks observed in individuals with therapy failure would act as a specific stimulus for the humoral anti-HIV-1 immune response. Antibodies binding to gp120 V3 genotype consensus peptides were more frequently observed for FT, mainly against peptides corresponding to sequences of genotypes prevalent in the Rio de Janeiro city area, B and F. HIV-1 neutralization of HIV-1 IIIB and of four primary isolates from Rio de Janeiro was less frequently observed for plasma from the NF than the FT group, but this difference was more expressive when plasma from individuals with detectable viremia were compared to that of individuals with undetectable viral loads in the year before sample collection. Although statistically significant differences were observed only in some specific comparisons, the study indicates that presence of detectable viremia may contribute to the maintenance of a specific anti-HIV-1 humoral immune response.     

Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite

The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFkappaB nuclear import, thus downregulating the expression of many genes controlled by NFkappaB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite.     

Comparative high-density microarray analysis of gene expression during growth of Lactobacillus helveticus in milk versus rich culture medium

Lactobacillus helveticus CNRZ32 is used by the dairy industry to modulate cheese flavor. The compilation of a draft genome sequence for this strain allowed us to identify and completely sequence 168 genes potentially important for the growth of this organism in milk or for cheese flavor development. The primary aim of this study was to investigate the expression of these genes during growth in milk and MRS medium by using microarrays. Oligonucleotide probes against each of the completely sequenced genes were compiled on maskless photolithography-based DNA microarrays. Additionally, the entire draft genome sequence was used to produce tiled microarrays in which noninterrupted sequence contigs were covered by consecutive 24-mer probes and associated mismatch probe sets. Total RNA isolated from cells grown in skim milk or in MRS to mid-log phase was used as a template to synthesize cDNA, followed by Cy3 labeling and hybridization. An analysis of data from annotated gene probes identified 42 genes that were upregulated during the growth of CNRZ32 in milk (P < 0.05), and 25 of these genes showed upregulation after applying Bonferroni's adjustment. The tiled microarrays identified numerous additional genes that were upregulated in milk versus MRS. Collectively, array data showed the growth of CNRZ32 in milk-induced genes encoding cell-envelope proteinases, oligopeptide transporters, and endopeptidases as well as enzymes for lactose and cysteine pathways, de novo synthesis, and/or salvage pathways for purines and pyrimidines and other functions. Genes for a hypothetical phosphoserine utilization pathway were also differentially expressed. Preliminary experiments indicate that cheese-derived, phosphoserine-containing peptides increase growth rates of CNRZ32 in a chemically defined medium. These results suggest that phosphoserine is used as an energy source during the growth of L. helveticus CNRZ32.     

Protein-flexible chain polymer interactions to explain protein partition in aqueous two-phase systems and the protein-polyelectrolyte complex formation

Complexes formation between two model proteins (catalase and chymotrypsin) and polyelectrolytes (polyvinyl sulphonate and polyacrilic acid) and a non-charged flexible chain polymer (PCF) as polyethylene propylene oxide (molecular mass 8400) was studied by a spectroscopy technique combination: UV absorption, fluorescence emission and circular dichroism. All the polymers increase the protein surface hydrophobicity (S(0)) parameter value as a proof of the modification of the protein surface exposed to the solvent. Chymotrypsin showed an increase in its biological activity in polymer presence, which suggests a change in the superficial microenvironment. The decrease in the biological activity of catalase might be due to a competition between the polymer and the substrate. This result agrees with the polymer effect on the catalase superficial hydrophobic area. It was found that, when flexible chain polymers increase protein stability and the enzymatic activity they could be used to isolate this enzyme without inducing loss of protein enzymatic activity. Our findings suggest that the interactions are dependent on the protein physico-chemical parameters such as: isoelectric pH, hydrophobic surface area, etc.     

Vasoactive intestinal peptide (VIP) increases vascular endothelial growth factor (VEGF) expression and secretion in human breast cancer cells

Previous studies have shown that vasoactive intestinal peptide (VIP) and its receptors (VPAC(1) and VPAC(2) receptors) are involved in promotion and growth of many human tumours including breast cancer. Here we investigated whether VIP regulates the expression of the main angiogenic factor, vascular endothelial cell growth factor (VEGF) in human oestrogen-dependent (T47D) and oestrogen-independent (MDA-MB-4687) breast cancer cells. Semiquantitative and quantitative real-time RT-PCRs were used at mRNA level whereas enzyme immunoanalysis was performed at protein level. Both cancer cell lines expressed VIP and VPAC(1) (but not VPAC(2)) receptors that were functional as shown by VIP stimulation of adenylate cyclase activity. VIP induced VEGF expression at both mRNA and protein levels following a time-dependent pattern. The responses were faster in T47D than in MDA-MB-468 cells. The observed VIP regulation of VEGF expression appears to be modulated at least by the cAMP/protein kinase A (PKA) and the phosphoinositide 3-kinase (PI3-K) signalling systems as shown by studies of adenylate cyclase stimulation and using specific kinase inhibitors such as H89 and wortmannin. These actions suggest a proangiogenic potential of VIP in breast cancer.     

Antimitotic agents increase the production of doubled-haploid embryos from cork oak anther culture

The objective of this study is to induce the nuclear DNA duplication of anther-derived embryos of cork oak (Quercus suber L.) to obtain doubled-haploid plants. Anther culture of this species produces a low percentage (7.78%) of spontaneous diploids, as assessed by flow cytometry. Therefore, three antimitotic agents, colchicine, oryzalin and amiprophos-methyl (APM), were applied in vitro to anther-derived cork oak haploid embryos from six genotypes at different concentrations and for different treatment durations. Antimitotic toxicity was determined by embryo survival. Efficiency in inducing chromosome doubling of haploid embryos was evaluated by flow cytometry measurements and differences were observed between treatments. Nuclear DNA duplication and embryo survival of cork oak haploid embryos was most efficiently induced with oryzalin 0.01 mM for 48 h. Around 50% diploid embryos were obtained. The rate of chromosome duplication induced by APM 0.01 mM was also acceptable but lower than that induced by oryzalin, regardless of the duration of the treatment. Colchicine 1.3 or 8.8 mM was the least efficient, with the induction of necrosis and only a small rate of nuclear DNA duplication.     

Influence of culture conditions on glutathione production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20–30°C), agitation rate (100–300 rpm), initial pH (5.0–7.0), inoculum concentration (5–15%), and glucose concentration (30–70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2^5–2), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2²) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20°C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.     

TLR4-mediated survival of macrophages is MyD88 dependent and requires TNF-alpha autocrine signalling

Modulation of macrophage survival is a critical factor in the resolution of inflammatory responses. Exposure to LPS protects innate immune cells against apoptosis, although the precise pathways responsible for prolongation of macrophage survival remain to be fully established. The goal of this study was to characterize the mechanism of TLR4-mediated survival of murine bone marrow-derived macrophages upon M-CSF withdrawal in more detail. Using a combination of knockout mice and pharmacological inhibitors allowed us to show that TLR4 and TLR2 stimulation promotes long-term survival of macrophages in a MyD88-, PI3K-, ERK-, and NF-kappaB-dependent manner. LPS-induced long-term, but not short-term, survival requires autocrine signaling via TNF-alpha and is facilitated by a general cytoprotective program, similar to that mediated by M-CSF. TLR4-mediated macrophage survival is accompanied by a remarkable up-regulation of specific cell surface markers, suggesting that LPS stimulation leads to the differentiation of macrophages toward a mixed macrophage/dendritic cell-like phenotype.     

Cells migrating to sites of tissue damage in response to the danger signal HMGB1 require NF-kappaB activation

Tissue damage is usually followed by healing, as both differentiated and stem cells migrate to replace dead or damaged cells. Mesoangioblasts (vessel-associated stem cells that can repair muscles) and fibroblasts migrate toward soluble factors released by damaged tissue. Two such factors are high mobility group box 1 (HMGB1), a nuclear protein that is released by cells undergoing unscheduled death (necrosis) but not by apoptotic cells, and stromal derived factor (SDF)-1/CXCL12. We find that HMGB1 activates the canonical nuclear factor kappaB (NF-kappaB) pathway via extracellular signal-regulated kinase phosphorylation. NF-kappaB signaling is necessary for chemotaxis toward HMGB1 and SDF-1/CXCL12, but not toward growth factor platelet-derived growth factor, formyl-met-leu-phe (a peptide that mimics bacterial invasion), or the archetypal NF-kappaB-activating signal tumor necrosis factor alpha. In dystrophic mice, mesoangioblasts injected into the general circulation ingress inefficiently into muscles if their NF-kappaB signaling pathway is disabled. These findings suggest that NF-kappaB signaling controls tissue regeneration in addition to early events in inflammation.