Interplay between Basic Residues of Hepatitis C Virus Glycoprotein E2 with Viral Receptors,Neutralizing Antibodies and Lipoproteins

Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H³⁸⁶ and R⁴⁰⁸), and the two highly conserved basic residues H⁴⁸⁸ and R⁶⁴⁸ contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions.     

Phylogenetic analysis of Bolivian bat trypanosomes of the subgenus schizotrypanum based on cytochrome B sequence and minicircle analyses

The aim of this study was to establish the phylogenetic relationships of trypanosomes present in blood samples of Bolivian Carollia bats. Eighteen cloned stocks were isolated from 115 bats belonging to Carollia perspicillata (Phyllostomidae) from three Amazonian areas of the Chapare Province of Bolivia and studied by xenodiagnosis using the vectors Rhodnius robustus and Triatoma infestans (Trypanosoma cruzi marenkellei) or haemoculture (Trypanosoma dionisii). The PCR DNA amplified was analyzed by nucleotide sequences of maxicircles encoding cytochrome b and by means of the molecular size of hyper variable regions of minicircles. Ten samples were classified as Trypanosoma cruzi marinkellei and 8 samples as Trypanosoma dionisii. The two species have a different molecular size profile with respect to the amplified regions of minicircles and also with respect to Trypanosoma cruzi and Trypanosoma rangeli used for comparative purpose. We conclude the presence of two species of bat trypanosomes in these samples, which can clearly be identified by the methods used in this study. The presence of these trypanosomes in Amazonian bats is discussed.     

HIV-1 Genetic Diversity and Transmitted Drug Resistance Mutations among Patients from the North, Central and South Regions of Angola

Background

Angola presents a very complex HIV-1 epidemic characterized by the co-circulation of several HIV-1 group M subtypes, intersubtype recombinants and unclassified (U) variants. The viral diversity outside the major metropolitan regions (Luanda and Cabinda) and the prevalence of transmitted drug resistance mutations (DRM) since the introduction of HAART in 2004, however, has been barely studied.

Methods

One hundred and one individuals from the Central (n = 44), North (n = 35), and South (n = 22) regions of Angola were diagnosed as HIV-1 positive and had their blood collected between 2008 and 2010, at one of the National Referral Centers for HIV diagnosis, the Kifangondo Medical Center, located in the border between the Luanda and Bengo provinces. Angolan samples were genotyped based on phylogenetic and bootscanning analyses of the pol (PR/RT) gene and their drug resistance profile was analyzed.

Results

Among the 101 samples analyzed, 51% clustered within a pure group M subtype, 42% were classified as intersubtype recombinants, and 7% were denoted as U. We observed an important variation in the prevalence of different HIV-1 genetic variants among country regions, with high frequency of subtype F1 in the North (20%), intersubtype recombinants in the Central (42%), and subtype C in the South (45%). Statistically significant difference in HIV-1 clade distribution was only observed in subtype C prevalence between North vs South (p = 0.0005) and Central vs South (p = 0.0012) regions. DRM to NRTI and/or NNRTI were detected in 16.3% of patients analyzed.

Conclusions

These results demonstrate a heterogeneous distribution of HIV-1 genetic variants across different regions in Angola and also revealed an unexpected high frequency of DRM to RT inhibitors in patients that have reported no antiretroviral usage, which may decrease the efficiency of the standard first-line antiretroviral regimens currently used in the country.     

Tagging the next generation: validation of trans-generational chemical tagging for an endangered fish

In this study we validated marking offspring through peritoneal injection of ripe females using two concentrations of strontium (strontium chloride hexahydrate). Larvae from treatments were monitored for condition morphometrics and tested for chemical mark incorporation in their otoliths via laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) to quantify the strontium concentration (Sr/Ca) and laser ablation multi-collector inductively coupled plasma mass spectrometry (LA-MC-ICPMS) to measure the strontium isotope ratios (⁸⁷Sr:⁸⁶Sr) of otoliths. Otolith strontium concentrations and strontium isotopes ratios were elevated in the high-concentration treatment, while the low-concentration and control treatments were not significantly different from each other. Larval size and eye diameter at hatch were similar among treatments; however, yolk and oil globule diameters were significantly reduced in the high-concentration treatment. Moreover, growth rates after 60?days post-hatch were significantly reduced in the high-concentration treatment relative to the low-concentration and control treatments, suggesting trans-generational tagging can have deleterious effects on offspring. Our study provides evidence for the efficacy of artificially marking offspring via injection of strontium into ripe females and could provide new tools for managing endangered fish populations; however, careful consideration of chemical concentrations and dosages may be required prior to its application in a fisheries management experiment.     

Presynaptic kainate receptor-mediated facilitation of glutamate release involves Ca(2+) -calmodulin at mossy fiber-CA3 synapses

J. Neurochem. (2012) 122, 891-899. ABSTRACT: Presynaptic kainate receptors (KARs) modulate the release of glutamate at synapses established between mossy fibers (MF) and CA3 pyramidal cells in the hippocampus. The activation of KAR by low, nanomolar, kainate concentrations facilitates glutamate release. KAR-mediated facilitation of glutamate release involves the activation of an adenylate cyclase/cyclic adenosine monophosphate/protein kinase A cascade at MF-CA3 synapses. Here, we studied the mechanisms by which KAR activation produces this facilitation of glutamate release in slices and synaptosomes. We find that the facilitation of glutamate release mediated by KAR activation requires an increase in Ca(2+) levels in the cytosol and the formation of a Ca(2+) -calmodulin complex to activate adenylate cyclase. The increase in cytosolic Ca(2+) underpinning this modulation is achieved, both, by Ca(2+) entering via Ca(2+) -permeable KARs and, by the mobilization of intraterminal Ca(2+) stores. Finally, we find that, congruent with the Ca(2+) -calmodulin support of KAR-mediated facilitation of glutamate release, induction of long-term potentiation at MF-CA3 synapses has an obligate requirement for Ca(2+) -calmodulin activity.     

Src kinases catalytic activity regulates proliferation, migration and invasiveness of MDA-MB-231 breast cancer cells

SFKs are frequently deregulated in cancer where they control cellular proliferation, migration, survival and metastasis. Here we study the role of SFKs catalytic activity in triple-negative/basal-like and metastatic human breast cancer MDA-MB-231 cells employing three well-established inhibitors: Dasatinib, PP2 and SU6656. These compounds inhibited migration and invasion. Concomitantly, they reduced Fak, paxillin, p130CAS, caveolin-1 phosphorylation and altered cytoskeletal structures. They also inhibited cell proliferation, but in different manners. Dasatinib and PP2 increased p27(Kip1) expression and reduced c-Myc levels, restraining G1–S transition. In contrast, SU6656 did not modify p27(Kip1) expression, slightly altered c-Myc levels and generated polyploid multinucleated cells, indicating inhibition of cytokinesis. These later effects were also observed in SYF fibroblasts, suggesting a SFKs-independent action. ZM447439, an Aurora B kinase inhibitor, produced similar cell cycle and morphological alterations in MDA-MB-231 cells, indicating that SU6656 blocked Aurora B kinase. This was confirmed by inhibition of histone H3 phosphorylation, the canonical Aurora B kinase substrate. Furthermore, hierarchical clustering analysis of gene expression profiles showed that SU6656 defined a set of genes that differed from Dasatinib and PP2. Additionally, Gene Set Enrichment Analyses revealed that SU6656 significantly reduces the Src pathway. Together, these results show the importance of SFKs catalytic activity for MDA-MB-231 proliferation, migration and invasiveness. They also illustrate that SU6656 acts as dual SFKs and Aurora B kinase inhibitor, suggesting its possible use as a therapeutic agent in breast cancer.     

Enantioselective copper-aminopyridine-catalyzed aza-Henry reaction with chelating N-(2-pyridyl)sulfonyl imines

This article describes a copper-catalyzed aza-Henry reaction. Copper complexes of camphor-derived aminopyridines catalyze the addition of nitromethane to N-(2-pyridyl)sulfonyl aldimines to give the corresponding β-nitrosulfonamides with good yields and variable enantiomeric excesses (up to 83%). An example of transformation of these compounds into N-(2-pyridyl)sulfonyl-α-amino acids and deprotection of the sulfonamide with Mg-MeOH is provided.     

Genome size and base composition variation in natural and experimental Narcissus (Amaryllidaceae) hybrids

Background and Aims

Although there is evidence that both allopolyploid and homoploid hybridization lead to rapid genomic changes, much less is known about hybrids from parents with different basic numbers without further chromosome doubling. Two natural hybrids, Narcissus × alentejanus (2n = 19) and N. × perezlarae (2n = 29), originated by one progenitor (N. cavanillesii, 2n = 28) and two others (N. serotinus, 2n = 10 and N. miniatus, 2n = 30, respectively) allow us to study how DNA content and composition varies in such hybrids.

Methods

Flow cytometry measurements with two staining techniques, PI and DAPI, were used to estimate 2C values and base composition (AT/GC ratio) in 390 samples from 54 wild populations of the two natural hybrids and their parental species. In addition, 20 synthetic F₁ hybrid individuals were also studied for comparison.

Key Results

Natural hybrids presented 2C values intermediate between those found in their parental species, although intra-population variance was very high in both hybrids, particularly for PI. Genome size estimated from DAPI was higher in synthetic hybrids than in hybrids from natural populations. In addition, differences for PI 2C values were detected between synthetic reciprocal crosses, attributable to maternal effects, as well as between natural hybrids and those synthetic F₁ hybrids in which N. cavanillesii acted as a mother.

Conclusions

Our results suggest that natural hybrid populations are composed of a mixture of markedly different hybrid genotypes produced either by structural chromosome changes, consistent with classic cytogenetic studies in Narcissus, or by transposon-mediated events.