Upregulation of surface proteins in Leishmania donovani isolated from patients of post kala-azar dermal leishmaniasis

Five to fifteen percent of visceral leishmaniasis (VL) patients in India develop post kala-azar dermal leishmaniasis (PKDL), usually 1-2 years after apparent clinical cure. There is evidence pointing to a role played by the host immune responses in the disease pathogenesis, however, the contribution of changes in parasite gene expression has not been explored. Highly sensitive gene expression microarray technology was employed to identify genes that are differentially expressed in Leishmania parasites isolated from PKDL patients in comparison with those from VL. Hybridization on Leishmania donovani genomic microarray comprised of unique clones allowed us to identify 46/2268 (2%) clones that showed statistically significant (P<0.05) changes in expression (1.5-3.5-fold) in parasites of PKDL origin compared to those of VL origin. Sequence analysis of six genomic clones, consistently showing approximately 2-fold higher expression in PKDL parasites, revealed significant homology with gp63, gp46, putative amastin, a putative reductase and a possible calpain-like protein. The gene products showing upregulated expression in PKDL isolates may be candidates playing a role in the altered clinical manifestation in PKDL. Such differentially expressed genes hold the key to understanding the parasite genetic factors that contribute to the persistence after clinical cure of VL.     

PAN1/NALP2/PYPAF2, an inducible inflammatory mediator that regulates NF-kappaB and caspase-1 activation in macrophages

Genes encoding proteins with PYRIN/PAAD/DAPIN domains, a nucleotide binding fold (NACHT), and leucine rich repeats have recently been recognized as important mediators in autoimmune inflammatory disorders. Here we characterize the expression and function of a member of the PYRIN and NACHT domain (PAN) family, PAN1 (also known as NALP2 and PYPAF2). PAN1 protein expression is regulated by lipopolysaccharide (LPS) and interferons (IFNbeta and IFNgamma) in THP-1 macrophage cells. In gene transfection studies PAN1 manifests an inhibitory influence on NF-kappaB activation induced by various pro-inflammatory stimuli, including tumor necrosis factor TNFalpha and interleukin-1beta (IL-1beta). Gene transfer-mediated elevations in PAN1 protein also suppressed activation of IkappaB kinases induced by inflammatory cytokines. Conversely, reducing endogenous levels of PAN1 using small interfering RNA enhanced LPS-induced production of ICAM-1 (intercellular adhesion molecule 1), an NF-kappaB-dependent gene. We also show here that PAN1 binds via its PYRIN domain to ASC, an adapter protein involved in caspase-1 activation. This binding is disrupted by mutation of the alpha1 helix of ASC. In gene transfer experiments PAN1 enhances caspase-1 activation and IL-1beta secretion in collaboration with ASC. Conversely, reducing endogenous levels of PAN1 using small interfering RNA significantly reduced LPS-induced secretion of IL-1beta in monocytes. We propose that PAN1 functions as a modulator of the activation of NF-kappaB and pro-caspase-1 in macrophages.     

Metabolic effects of dietary lactose in adult female rats

As an outgrowth of our interest in the potential toxicity of dietary galactose, we investigated the metabolic effects of high lactose diets in Long-Evans female rats. Seventy-five Long-Evans female rats (25-day-old) were randomized to receive one of 3 diets for 7 months: glucose diet (CON); low lactose diet (10.5%, LLD); or a high lactose diet (41.9%, HLD). Necropsy was performed seven months after randomization. HLD animals had significantly lower body weights than controls (P < 0.01). These animals continued to grow, however at a retarded rate compared to the CON group. The HLD group also had significantly lower triglyceride and non-esterified fatty acid levels than the CON group (P < 0.01 and P < 0.05). Serum glucose concentrations were lower in the HLD group compared to CON animals (P < 0.05), while serum insulin levels were lower than both the LLD and CON animals (P < 0.01 and P < 0.05). Leptin exhibited a similar trend. Thyroid studies revealed no difference in TSH between groups. Free T4 was significantly higher in HLD rats compared to LLD and CON rats while free T3 was lower in the HLD group (P < 0.05). This indicates a possible impairment in T4 to T3 conversion. Our data suggests that a long-term high lactose diet is associated with a decrease in insulin and leptin levels, and an increase in the insulin to glucose ratio. However, these changes are seen in the presence of a decreased body mass. A significant effect on thyroid hormone metabolism is also seen, and may be an adaptive mechanism in lactose-fed rats.     

Quantitative structure-activity relationship studies on 5-phenyl-3-ureido-1,5-benzodiazepine as cholecystokinin-A receptor antagonists

Quantitative structure-activity relationship (QSAR) studies on a series of 5-phenyl-3-ureido-1,5-benzodiazepine-2,4-diones has been carried out using a pool of distance-based topological indices. Step-wise regression analysis indicated that penta-parametric regression expression containing Sz, B, Ip1, Ip2 and Ip3 is the most potent and selective for CCK-A affinity. The predictive potential of the model is discussed on the basis of cross-validation parameters as well as by estimating root mean square (RMSR) of the residuals.     

Drosophila Twins regulates Armadillo levels in response to Wg/Wnt signal

Protein Phosphatase 2A (PP2A) has a heterotrimeric-subunit structure, consisting of a core dimer of approximately 36 kDa catalytic and approximately 65 kDa scaffold subunits complexed to a third variable regulatory subunit. Several studies have implicated PP2A in Wg/Wnt signaling. However, reports on the precise nature of PP2A role in Wg/Wnt pathway in different organisms are conflicting. We show that twins (tws), which codes for the B/PR55 regulatory subunit of PP2A in Drosophila, is a positive regulator of Wg/Wnt signaling. In tws(-) wing discs both short- and long-range targets of Wingless morphogen are downregulated. Analyses of tws(-) mitotic clones suggest that requirement of Tws in Wingless pathway is cell-autonomous. Epistatic genetic studies indicate that Tws functions downstream of Dishevelled and upstream of Sgg and Armadillo. Our results suggest that Tws is required for the stabilization of Armadillo/beta-catenin in response to Wg/Wnt signaling. Interestingly, overexpression of, otherwise normal, Tws protein induce dominant-negative phenotypes. The conflicting reports on the role of PP2A in Wg/Wnt signaling could be due to the dominant-negative effect caused by the overexpression of one of the subunits.     

Complementation of human papillomavirus type 16 E6 and E7 by Jagged1-specific Notch1-phosphatidylinositol 3-kinase signaling involves pleiotropic oncogenic functions independent of CBF1;Su(H);Lag-1 activation

We have analyzed the induction and role of phosphatidylinositol 3-kinase (PI3K) by Notch signaling in human papillomavirus (HPV)-derived cancers. Jagged1, in contrast to Delta1, is preferentially upregulated in human cervical tumors. Jagged1 and not Delta1 expression sustained in vivo tumors by HPV16 oncogenes in HaCaT cells. Further, Jagged1 expression correlates with the rapid induction of PI3K-mediated epithelial-mesenchymal transition in both HaCaT cells and a human cervical tumor-derived cell line, suggestive of Delta1;Serrate/Jagged;Lag2 ligand-specific roles. Microarray analysis and dominant-negatives reveal that Notch-PI3K oncogenic functions can be independent of CBF1;Su(H);Lag-1 activation and instead relies on Deltex1, an alternative Notch effector.     

Cissampelos pareira Linn: Natural Source of Potent Antiviral Activity against All Four Dengue Virus Serotypes

Background

Dengue, a mosquito-borne viral disease, poses a significant global public health risk. In tropical countries such as India where periodic dengue outbreaks can be correlated to the high prevalence of the mosquito vector, circulation of all four dengue viruses (DENVs) and the high population density, a drug for dengue is being increasingly recognized as an unmet public health need.

Methodology/Principal findings

Using the knowledge of traditional Indian medicine, Ayurveda, we developed a systematic bioassay-guided screening approach to explore the indigenous herbal bio-resource to identify plants with pan-DENV inhibitory activity. Our results show that the alcoholic extract of Cissampelos pariera Linn (Cipa extract) was a potent inhibitor of all four DENVs in cell-based assays, assessed in terms of viral NS1 antigen secretion using ELISA, as well as viral replication, based on plaque assays. Virus yield reduction assays showed that Cipa extract could decrease viral titers by an order of magnitude. The extract conferred statistically significant protection against DENV infection using the AG129 mouse model. A preliminary evaluation of the clinical relevance of Cipa extract showed that it had no adverse effects on platelet counts and RBC viability. In addition to inherent antipyretic activity in Wistar rats, it possessed the ability to down-regulate the production of TNF-α, a cytokine implicated in severe dengue disease. Importantly, it showed no evidence of toxicity in Wistar rats, when administered at doses as high as 2g/Kg body weight for up to 1 week.

Conclusions/Significance

Our findings above, taken in the context of the human safety of Cipa, based on its use in Indian traditional medicine, warrant further work to explore Cipa as a source for the development of an inexpensive herbal formulation for dengue therapy. This may be of practical relevance to a dengue-endemic resource-poor country such as India.