Combination of all-<Emphasis Type="Italic">trans</Emphasis> retinoic acid and taxol regressed glioblastoma T98G xenografts in nude mice

Glioblastoma is the most prevalent and highly malignant brain tumor that continues to defy current treatment strategies. This investigation used all-trans retinoic acid (ATRA) and taxol (TXL) as a combination therapy for controlling the growth of human glioblastoma T98G xenografted in athymic nude mice. Histopathological examination revealed that ATRA induced differentiation and combination of ATRA and TXL caused more apoptosis than either treatment alone. Combination therapy decreased expression of telomerase, nuclear factor kappa B (NFκВ), and inhibitor-of-apoptosis proteins (IAPs) indicating suppression of survival factors while upregulated Smac/Diablo. Combination therapy also changed expression of Bax and Bcl-2 proteins leading to increased Bax:Bcl-2 ratio, mitochondrial release of cytochrome c and apoptosis-inducing factor (AIF), and activation of caspase-9. Increased activities of calpain and caspase-3 degraded 270 kD α-spectrin at the specific sites to generate 145 kD spectrin breakdown product (SBDP) and 120 kD SBDP, respectively. Further, increased activity of caspase-3 cleaved inhibitor-of-caspase-activated DNase (ICAD). In situ double immunofluorescent labelings showed overexpression of calpain, caspase-12, caspase-3, and AIF during apoptosis, suggesting involvement of both caspase-dependent and caspase-independent pathways for apoptosis. Our investigation revealed that treatment of glioblastoma T98G xenografts with the combination of ATRA and TXL induced differentiation and multiple molecular mechanisms for apoptosis.     

Direct Evidence for Calpain Involvement in Apoptotic Death of Neurons in Spinal Cord Injury in Rats and Neuroprotection with Calpain Inhibitor

To demonstrate calpain involvement in neurodegeneration in rat spinal cord injury (SCI), we examined SCI segments for DNA fragmentation, neurons for calpain overexpression, neuronal death, and neuroprotection with calpain inhibitor (E-64-d). After the induction of SCI (40 g cm force) on T12, rats were treated within 15 min with vehicle (DMSO) or E-64-d. Sham animals underwent laminectomy only. Animals were sacrificed at 24 h, and five 1-cm long spinal cord segments were collected: two rostral (S1 and S2), one lesion (S3), and two caudal segments (S4 and S5). Agarose gel electrophoresis of DNA samples isolated from the SCI segments showed both random and internucleosomal DNA fragmentation indicating occurrence of necrosis as well as apoptosis mostly in the lesion, moderately in caudal, and slightly in rostral segments from SCI rats. Treatment of SCI rats with E-64-d (1 mg/kg) reduced DNA fragmentation in all segments. The lesion and adjacent caudal segments (S3 and S4) were further investigated by in situ double-immunofluorescent labelings that showed increase in calpain expression in neurons in SCI rats and decrease in calpain expression in SCI rats treated with E-64-d. In situ combined TUNEL and double-immunofluorescent labelings directly detected co-localization of neuronal death and calpain overexpressin in SCI rats treated with only vehicle while attenuation of neuronal death in SCI rats treated with E-64-d. Previous studies from our laboratory indirectly showed neuroprotective effect of E-64-d in SCI rats. Our current results provide direct in situ evidence for calpain involvement in neuronal death and neuroprotective efficacy of E-64-d in lesion and penumbra in SCI rats. Special issue in honor of Naren Banik.     

Current status of lupus genetics

Over the past 40 years more than 100 genetic risk factors have been defined in systemic lupus erythematosus through a combination of case studies, linkage analyses of multiplex families, and case-control analyses of single genes. Multiple investigators have examined patient cohorts gathered from around the world, and although we doubt that all of the reported associations will be replicated, we have probably already discovered many of the genes that are important in lupus pathogenesis, including those encoding human leukocyte antigen-DR, Fcγ receptor 3A, protein tyrosine phosphatase nonreceptor 22, cytotoxic T lymphocyte associated antigen 4, and mannose-binding lectin. In this review we will present what is known, what is disputed, and what remains to be discovered in the world of lupus genetics.     

Agricultural biotechnology for crop improvement in a variable climate: hope or hype?

Developing crops that are better adapted to abiotic stresses is important for food production in many parts of the world today. Anticipated changes in climate and its variability, particularly extreme temperatures and changes in rainfall, are expected to make crop improvement even more crucial for food production. Here, we review two key biotechnology approaches, molecular breeding and genetic engineering, and their integration with conventional breeding to develop crops that are more tolerant of abiotic stresses. In addition to a multidisciplinary approach, we also examine some constraints that need to be overcome to realize the full potential of agricultural biotechnology for sustainable crop production to meet the demands of a projected world population of nine billion in 2050.     

Microsatellite and minisatellite markers based DNA fingerprinting and genetic diversity of blast and ufra resistant genotypes

A total of 78 alleles and 29 loci were detected from nine microsatellite and three minisatellite markers, respectively across 26 blast and ufra disease resistant genotypes. For blast resistant genotypes, the Polymorphic Information Content (PIC) values ranged from 0.280 to 0.726 and RM21 was considered as the best marker. PIC values ranged from 0.5953 to 0.8296 for ufra resistant genotypes and RM23 was the best marker for characterization of ufra resistant genotypes. The genetic similarity analysis using UPGMA clustering generated nine clusters with coefficient of 0.66 for blast resistant genotypes while five genetic clusters with similarity coefficient of 0.42 for ufra resistant genotypes. In order to develop resistant varieties of two major diseases of rice, hybridisation should be made using the parents, BR29 and NJ70507, BR36 and NJ70507 for blast, while BR11 and Aokazi, BR3 and Aokazi, Rayda and BR3 and Rayda and BR11 for ufra.     

Extracellular matrix from porcine small intestinal submucosa (SIS) as immune adjuvants

Porcine small intestinal submucosa (SIS) of Cook Biotech is licensed and widely used for tissue remodeling in humans. SIS was shown to be highly effective as an adjuvant in model studies with prostate and ovarian cancer vaccines. However, SIS adjuvanticity relative to alum, another important human-licensed adjuvant, has not yet been delineated in terms of activation of innate immunity via inflammasomes and boosting of antibody responses to soluble proteins and hapten-protein conjugates. We used ovalbumin, and a hapten-protein conjugate, phthalate-keyhole limpet hemocyanin. The evaluation of SIS was conducted in BALB/c and C57BL/6 mice using both intraperitoneal and subcutaneous routes. Inflammatory responses were studied by microarray profiling of chemokines and cytokines and by qPCR of inflammasomes-related genes. Results showed that SIS affected cytokine and chemokines microenvironments such as up-regulation of IL-4 and CD30-ligand and activation of chemotactic factors LIX and KC (neutrophil chemotactic factors), MCP-1 (monocytes chemotactic factors), MIP 1-α (macrophage chemotactic factor) and lymphotactin, as well as, growth factors like M-CSF. SIS also promoted gene expression of Nod-like receptors (NLR) and associated downstream effectors. However, in contrast to alum, SIS had no effects on pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) or NLRP3, but it appeared to promote both Th1 and Th2 responses under different conditions. Lastly, it was as effective as alum in engendering a lasting and specific antibody response, primarily of IgG1 type.     

Polymorphisms of complement receptor 1 and interleukin-10 genes and systemic lupus erythematosus: a meta-analysis

A number of studies have tested the association of the complement receptor 1 (CR1) and Interleukin-10 (IL10) polymorphisms with systemic lupus erythematosus (SLE), but reported conflicting results. The aim of the study is to explore whether the CR1 and IL10 genes are associated with SLE susceptibility. We surveyed studies on the CR1 and IL10 polymorphisms and SLE using comprehensive Medline search and review of the references. A meta-analysis was conducted in a fixed effects model or random effects model based on between-study heterogeneity. Eighteen comparisons from 13 studies were included in the CR1 meta-analysis and a total of 16 separate comparisons were used for the IL10 meta-analysis. The CR1 meta-analysis showed no significant association of the CR1 functional polymorphisms with SLE. In contrast, the S structural variant of the CR1 showed a significant association (OR=1.544, 95% CI, 1.217–1.959, P<0.001). Stratification by ethnicity indicated that the CR1 S variant was associated with SLE in Caucasians (OR=1.667, 95% CI, 1.193–2.357, P=0.003). The IL10 meta-analysis showed a significant association between SLE and the G11 allele of IL10.G (OR=1.279, 95% CI; 1.027–1.593, P=0.028) in whole populations, and IL10 promoter −1082G allele was associated with SLE in Asians (OR=1.358, 95% CI; 1.015–1.816, P=0.039). In conclusion, the CR1 meta-analysis revealed the association of the S structural variant of the CR1 with SLE and the IL10 meta-analysis showed the association of IL10.G11 allele and SLE in whole populations and the association between promoter -A1082G polymorphism and SLE in Asians.     

Effect of 30 kDa and above buffalo follicular fluid protein treatment and immunization on ovarian functions in goats (Capra hircus)

Information on the use of buffalo follicular fluid (buFF) in modulation of ovarian functions in farm animals is scanty compared to other species. This is an attempt to investigate the effect of direct administration and active immunization of 30 kDa and above buFF proteins on ovarian functions in goats. Treatment of goats (n = 6) with steroid free 30 kDa and above buFF protein fraction during late-luteal phase for 4 days (days 12 or 13 to days 15 or 16) of the natural cycle, delayed the onset of estrus by 24 h compared to control although the mean duration of estrus was unaffected. A 71% increase (P = 0.06) in mean ovulation number was also observed following treatment. However, the population of large (> or =5 mm diameter) follicle was not affected. The ovarian activity calculated as total of ovulation and large follicles increased (1.6 times) significantly (P = 0.02) in treated animals. Active immunization of goats (n = 5) against these proteins did not affect the onset and duration of estrus. Similarly, the ovulation rate, number of large follicles and the ovarian activity did not differ significantly between immunized and control groups. The study revealed that 30 kDa and above buffalo follicular fluid contains some factor(s) that cause delay in the onset of estrus in goats and increase the ovulation rate. Active immunization against these proteins in goat did not show any effect either on onset, duration of estrus or ovulation rate and large follicle population. Detailed study on these buffalo follicular fluid proteins may help to use them further for modulation of ovarian function in farm animals.     

Shigellosis

Shigellosis is a global human health problem. Four species of Shigella i.e. S. dysenteriae, S. flexneri, S. boydii and S. sonnei are able to cause the disease. These species are subdivided into serotypes on the basis of O-specific polysaccharide of the LPS. Shigella dysenteriae type 1 produces severe disease and may be associated with life-threatening complications. The symptoms of shigellosis include diarrhoea and/or dysentery with frequent mucoid bloody stools, abdominal cramps and tenesmus. Shigella spp. cause dysentery by invading the colonic mucosa. Shigella bacteria multiply within colonic epithelial cells, cause cell death and spread laterally to infect and kill adjacent epithelial cells, causing mucosal ulceration, inflammation and bleeding. Transmission usually occurs via contaminated food and water or through person-to-person contact. Laboratory diagnosis is made by culturing the stool samples using selective/differential agar media. Shigella spp. are highly fragile organism and considerable care must be exercised in collecting faecal specimens, transporting them to the laboratories and in using appropriate media for isolation. Antimicrobial agents are the mainstay of therapy of all cases of shigellosis. Due to the global emergence of drug resistance, the choice of antimicrobial agents for treating shigellosis is limited. Although single dose of norfloxacin and ciprofloxacin has been shown to be effective, they are currently less effective against S. dysenteriae type 1 infection. Newer quinolones, cephalosporin derivatives, and azithromycin are the drug of choice. However, fluoroquinolone-resistant S. dysenteriae type 1 infection have been reported. Currently, no vaccines against Shigella infection exist. Both live and subunit parenteral vaccine candidates are under development. Because immunity to Shigella is serotype-specific, the priority is to develop vaccine against S. dysenteriae type 1 and S. flexneri type 2a. Shigella species are important pathogens responsible for diarrhoeal diseases and dysentery occurring all over the world. The morbidity and mortality due to shigellosis are especially high among children in developing countries. A recent review of literature (Kotloff et al.,1999) concluded that, of the estimated 165 million cases of Shigella diarrhoea that occur annually, 99% occur in developing countries, and in developing countries 69% of episodes occur in children under five years of age. Moreover, of the ca.1.1 million deaths attributed to Shigella infections in developing countries, 60% of deaths occur in the under-five age group. Travellers from developed to developing regions and soldiers serving under field conditions are also at an increased risk to develop shigellosis.     

Virtual screening,selection and development of a benzindolone structural scaffold for inhibition of lumazine synthase

Virtual screening of a library of commercially available compounds versus the structure of Mycobacterium tuberculosis lumazine synthase identified 2-(2-oxo-1,2-dihydrobenzo[cd]indole-6-sulfonamido)acetic acid (9) as a possible lead compound. Compound 9 proved to be an effective inhibitor of M. tuberculosis lumazine synthase with a K_i of 70 μM. Lead optimization through replacement of the carboxymethylsulfonamide sidechain with sulfonamides substituted with alkyl phosphates led to a four-carbon phosphate 38 that displayed a moderate increase in enzyme inhibitory activity (K_i 38 μM). Molecular modeling based on known lumazine synthase/inhibitor crystal structures suggests that the main forces stabilizing the present benzindolone/enzyme complexes involve π–π stacking interactions with Trp27 and hydrogen bonding of the phosphates with Arg128, the backbone nitrogens of Gly85 and Gln86, and the side chain hydroxyl of Thr87.