TIA proteins are necessary but not sufficient for the tissue-specific splicing of the myosin phosphatase targeting subunit 1

We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. We previously identified a U-rich intronic enhancer flanking the 5' splice site (IE1), and a bipartite exonic enhancer/suppressor, that regulate splicing of the MYPT1 central alternative exon. Here we show that T-cell inhibitor of apoptosis (TIA-1) and T-cell inhibitor of apoptosis-related (TIAR) proteins bind to the IE1. Co-transfection of TIA expression vectors with a MYPT1 mini-gene construct increase splicing of the central alternative exon. TIA proteins do not enhance splicing when the palindromic exonic splicing enhancer (ESE) is mutated, indicating that TIAs are necessary but not sufficient for splicing. The ESE specifically binds SRp55 and SRp20 proteins, supporting a model in which both SR and TIA proteins binding to their cis-elements are required for the recruitment of the splicing complex to a weak 5' splice site. Inactivation of TIA proteins in the DT40 cell line (TIA-1(-/-)TIAR(+/-)) reduced the splicing of the central alternative exon of the endogenous MYPT1 as well as stably transfected MYPT1 minigene constructs. Splicing of the MYPT1 3' alternative exon and the MLC(17) alternative exon were unaffected, suggesting that TIA proteins regulate a subset of smooth muscle/nonmuscle alternative splicing reactions. Finally, reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.     

Integration of clinical and gene expression endpoints to explore furan-mediated hepatotoxicity

Molecular techniques, such as cDNA microarrays, are being used to aid in the elucidation of the mechanisms of toxicity of a variety of compounds. In this study, we evaluate the molecular effects of furan in the rat liver. Sprague-Dawley rats were exposed to 4 or 40 mg/kg furan for up to 14 days. Furan induced an initial degenerative and necrotic phenotype that was followed by inflammation and fibrosis, consistent with previous observations for this compound. RNA was harvested from each lobe of the liver at several time points to observe whether lobe-specific gene expression effects occurred. Similar gene expression changes were observed in all lobes, however the magnitude of gene expression change was more pronounced in the right lobe. Finally, to help determine the correlation between gene expression changes and liver pathology, we applied traditional microarray visualization tools to the assessment of clinical chemistry and pathology parameters.     

Dynamic and cellular interactions of nanoparticles in vascular-targeted drug delivery

Vascular-targeted drug delivery systems could provide more efficient and effective pharmaceutical interventions for treating a variety of diseases including cardiovascular, pulmonary, inflammatory, and malignant disorders. However, several factors must be taken into account when designing these systems. The diverse blood hemodynamics and rheology, and the natural clearance process that tend to decrease the circulation time of foreign particles all lessen the probability of successful carrier interaction with the vascular wall. An effective vascular-targeted drug delivery system must be able to navigate through the bloodstream while avoiding immune clearance, attach to the vascular wall, and release its therapeutic cargo at the intended location. This review will summarize and analyze current literature reporting on (1) nanocarrier fabrication methods and materials that allow for optimum therapeutic encapsulation, protection, and release; (2) localization and binding dynamics of nanocarriers as influenced by hemodynamics and blood rheology in medium-to-large vessels; (3) blood cells' responses to various types of nanocarrier compositions and its effects on particle circulation time; and (4) properties that affect nanocarrier internalization at the target site.     

Extracellular β‐nicotinamide adenine dinucleotide (β‐NAD) promotes the endothelial cell barrier integrity via PKA‐ and EPAC1/Rac1‐dependent actin cytoskeleton rearrangement

Extracellular β‐NAD is known to elevate intracellular levels of calcium ions, inositol 1,4,5‐trisphate and cAMP. Recently, β‐NAD was identified as an agonist for P2Y1 and P2Y11 purinergic receptors. Since β‐NAD can be released extracellularly from endothelial cells (EC), we have proposed its involvement in the regulation of EC permeability. Here we show, for the first time, that endothelial integrity can be enhanced in EC endogenously expressing β‐NAD‐activated purinergic receptors upon β‐NAD stimulation. Our data demonstrate that extracellular β‐NAD increases the transendothelial electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration‐dependent manner indicating endothelial barrier enhancement. Importantly, β‐NAD significantly attenuated thrombin‐induced EC permeability as well as the barrier‐compromising effects of Gram‐negative and Gram‐positive bacterial toxins representing the barrier‐protective function of β‐NAD. Immunofluorescence microscopy reveals more pronounced staining of cell–cell junctional protein VE‐cadherin at the cellular periphery signifying increased tightness of the cell‐cell contacts after β‐NAD stimulation. Interestingly, inhibitory analysis (pharmacological antagonists and receptor sequence specific siRNAs) indicates the participation of both P2Y1 and P2Y11 receptors in β‐NAD‐induced TER increase. β‐NAD‐treatment attenuates the lipopolysaccharide (LPS)‐induced phosphorylation of myosin light chain (MLC) indicating its involvement in barrier protection. Our studies also show the involvement of cAMP‐dependent protein kinase A and EPAC1 pathways as well as small GTPase Rac1 in β‐NAD‐induced EC barrier enhancement. With these results, we conclude that β‐NAD regulates the pulmonary EC barrier integrity via small GTPase Rac1‐ and MLCP‐ dependent signaling pathways. J. Cell. Physiol. 223: 215–223, 2010. © 2010 Wiley‐Liss, Inc.     

Leishmania donovani exploits host deubiquitinating enzyme A20, a negative regulator of TLR signaling, to subvert host immune response

TLRs, which form an interface between mammalian host and microbe, play a key role in pathogen recognition and initiation of proinflammatory response thus stimulating antimicrobial activity and host survival. However, certain intracellular pathogens such as Leishmania can successfully manipulate the TLR signaling, thus hijacking the defensive strategies of the host. Despite the presence of lipophosphoglycan, a TLR2 ligand capable of eliciting host-defensive cytokine response, on the surface of Leishmania, the strategies adopted by the parasite to silence the TLR2-mediated proinflammatory response is not understood. In this study, we showed that Leishmania donovani modulates the TLR2-mediated pathway in macrophages through inhibition of the IKK-NF-κB cascade and suppression of IL-12 and TNF-α production. This may be due to impairment of the association of TRAF6 with the TAK-TAB complex, thus inhibiting the recruitment of TRAF6 in TLR2 signaling. L. donovani infection drastically reduced Lys 63-linked ubiquitination of TRAF6, and the deubiquitinating enzyme A20 was found to be significantly upregulated in infected macrophages. Small interfering RNA-mediated silencing of A20 restored the Lys 63-linked ubiquitination of TRAF6 as well as IL-12 and TNF-α levels with a concomitant decrease in IL-10 and TGF-β synthesis in infected macrophages. Knockdown of A20 led to lower parasite survival within macrophages. Moreover, in vivo silencing of A20 by short hairpin RNA in BALB/c mice led to increased NF-κB DNA binding and host-protective proinflammatory cytokine response resulting in effective parasite clearance. These results suggest that L. donovani might exploit host A20 to inhibit the TLR2-mediated proinflammatory gene expression, thus escaping the immune responses of the host.     

Morphine and galectin-1 modulate HIV-1 infection of human monocyte-derived macrophages

Morphine is a widely abused, addictive drug that modulates immune function. Macrophages are a primary reservoir of HIV-1; therefore, they play a role in the development of this disease, as well as impact the overall course of disease progression. Galectin-1 is a member of a family of β-galactoside-binding lectins that are soluble adhesion molecules and that mediate direct cell-pathogen interactions during HIV-1 viral adhesion. Because the drug abuse epidemic and the HIV-1 epidemic are closely interrelated, we propose that increased expression of galectin-1 induced by morphine may modulate HIV-1 infection of human monocyte-derived macrophages (MDMs). In this article, we show that galectin-1 gene and protein expression are potentiated by incubation with morphine. Confirming previous studies, morphine alone or galectin-1 alone enhance HIV-1 infection of MDMs. Concomitant incubation with exogenous galectin-1 and morphine potentiated HIV-1 infection of MDMs. We used a nanotechnology approach that uses gold nanorod-galectin-1 small interfering RNA complexes (nanoplexes) to inhibit gene expression for galectin-1. We found that nanoplexes silenced gene expression for galectin-1, and they reversed the effects of morphine on galectin-1 expression. Furthermore, the effects of morphine on HIV-1 infection were reduced in the presence of the nanoplex.     

Immunogenicity and cross-reactivity of a representative ancestral sequence in hepatitis C virus infection

Vaccines designed to prevent or to treat hepatitis C viral infection must achieve maximum cross-reactivity against widely divergent circulating strains. Rational approaches for sequence selection to maximize immunogenicity and minimize genetic distance across circulating strains may enhance vaccine induction of optimal cytotoxic T cell responses. We assessed T cell recognition of potential hepatitis C virus (HCV) vaccine sequences generated using three rational approaches: combining epitopes with predicted tight binding to the MHC, consensus sequence (most common amino acid at each position), and representative ancestral sequence that had been derived using bayesian phylogenetic tools. No correlation was seen between peptide-MHC binding affinity and frequency of recognition, as measured by an IFN-γ T cell response in HLA-matched HCV-infected individuals. Peptides encoding representative, consensus, and natural variant sequences were then tested for the capacity to expand CD8 T cell populations and to elicit cross-reactive CD8 T cell responses. CD8(+) T cells expanded with representative sequence HCV generally more broadly and robustly recognized highly diverse circulating HCV strains than did T cells expanded with either consensus sequence or naturally occurring sequence variants. These data support the use of representative sequence in HCV vaccine design.     

Constraints on Viral Evolution during Chronic Hepatitis C Virus Infection Arising from a Common-Source Exposure

Extraordinary viral sequence diversity and rapid viral genetic evolution are hallmarks of hepatitis C virus (HCV) infection. Viral sequence evolution has previously been shown to mediate escape from cytotoxic T-lymphocyte (CTL) and neutralizing antibody responses in acute HCV infection. HCV evolution continues during chronic infection, but the pressures driving these changes are poorly defined. We analyzed plasma virus sequence evolution in 5.2-kb hemigenomes from multiple longitudinal time points isolated from individuals in the Irish anti-D cohort, who were infected with HCV from a common source in 1977 to 1978. We found phylogenetically distinct quasispecies populations at different plasma time points isolated late in chronic infection, suggesting ongoing viral evolution and quasispecies replacement over time. We saw evidence of early pressure driving net evolution away from a computationally reconstructed common ancestor, known as Bole1b, in predicted CTL epitopes and E1E2, with balanced evolution toward and away from the Bole1b amino acid sequence in the remainder of the genome. Late in chronic infection, the rate of evolution toward the Bole1b sequence increased, resulting in net neutral evolution relative to Bole1b across the entire 5.2-kb hemigenome. Surprisingly, even late in chronic infection, net amino acid evolution away from the infecting inoculum sequence still could be observed. These data suggest that, late in chronic infection, ongoing HCV evolution is not random genetic drift but rather the product of strong pressure toward a common ancestor and concurrent net ongoing evolution away from the inoculum virus sequence, likely balancing replicative fitness and ongoing immune escape.     

Diversity and antagonistic potential of Bacillus spp. associated to the rhizosphere of tomato for the management of Rhizoctonia solani

Bacillus spp. has emerged as the most effective alternative to synthetic chemical fungicides. To get a better insight in the antagonistic potential of Bacillus strains, rhizospheric soil samples of healthy tomato plants from Indo-gangetic plain regions of India were analysed. A total of 108 Bacillus strains were obtained from preliminary screening. Potent strains identified on the basis of in vitro antagonistic and biochemical assays were subjected to diversity analysis using 16S-rDNA, BOX and ERIC-PCR. Furthermore, the four best performing antagonistic Bacillus strains under in vitro plant growth promotion and antagonistic assay were selected for pot experiment. In field study, Bacillus amyloliquefaciens MB101 and Bacillus subtilis MB14 showed drastic reduction in disease index by 55.7 and 41.74% with significant elevation in fruit yield up to 220 and 184 qha^–1, respectively. The present study was successful in selecting effective Bacillus strains by performing phenotypic and genotypic characterisation of Bacillus strains that can be used as an integral component of integrated disease management of tomato root rot and damping-off.