Enhancement of α-tocopherol content through transgenic and cell suspension culture systems in tobacco

The tocopherols are amphipathic antioxidant synthesized by photosynthetic organisms, which forms the essential component in the human diet. To increase the α-tocopherol content in tobacco, two approaches have been attempted in this study: (1) transgenic approach, by constitutive overexpression of the genes encoding Arabidopsis homogentisate phytyltransferase (HPT) and tocopherol cyclase (TC) through Agrobacterium-mediated genetic transformation; (2) non-transgenic approach, by supplementation of intermediates/precursors of vitamin E biosynthesis like tyrosine, p-hydroxyphenyl pyruvic acid, homogentisic acid (HGA) and phytol in different concentrations and combinations using cell suspension culture system. Molecular analyses by PCR, RT-PCR and Southern hybridization were carried out to confirm the HPT and TC expressing transgenic tobacco lines. The α-tocopherol content in transgenic plants expressing HPT and TC increase by 5.5 and 4.1, respectively, over the wild type. These results indicate that, HPT and TC activities are important in tobacco plants for enhancing the vitamin E content. In the second approach, the supplementation of precursor in cell suspension cultures, i.e., combination of 150 μM HGA + 100 μM phytol, showed the maximum enhancement of α-tocopherol, i.e., 36-fold. These findings clearly imply that enhancement of α-tocopherol levels in tobacco system is possible, if we could modulate the vitamin E metabolic pathway. This is a very useful finding for the large-scale production of natural Vitamin E. Among the two systems tested, cell suspension culture-based system is ideal over the transgenic technology due to its efficiency and no biosafety concerns.     

Diosgenin improves vascular function by increasing aortic eNOS expression, normalize dyslipidemia and ACE activity in chronic renal failure rats

In recent years, the role of endothelial dysfunction (ED) and excessive oxidative stress in the development of cardiovascular diseases has been highlighted. The aim of the present study is to evaluate the effect of diosgenin, an antioxidant on chronic renal failure (CRF) induced vascular dysfunction. CRF was induced by feeding the rats with a diet containing 0.75 % adenine and diosgenin was given orally (everyday at the dose of 40 mg/kg). Isometric force measurement was performed on isolated aortic rings in organ baths. Levels of reduced glutathione (GSH), nitric oxide metabolites, and endothelial nitric oxide synthase mRNA in rat aorta were examined. Further, plasma lipid profile, activity of enzymes of lipid metabolism, and aortic angiotensin converting enzyme (ACE) also studied. The overall results have proved that diosgenin attenuates CRF-induced impairment in acetylcholine induced endothelium-dependent and sodium nitroprusside induced endothelium-independent vascular relaxation. Moreover, it elevates the GSH and restores the eNOS mRNA expression level. CRF-induced dyslipidemia and ACE activity was also inhibited by diosgenin treatment. This study indicates that diosgenin have enough potential to protect vasculature against oxidative stress, dyslipidemia which in turn improves the vascular function in CRF milieu.     

Changes in Caenorhabditis elegans exposed to Vibrio parahaemolyticus

Vibrio parahaemolyticus, which owes its origin to the marine environment, is considered as one of the most common causes of infectious diarrhea worldwide. The present study investigated the pathogenicity of V. parahaemolyticus against the model organism, Caenorhabditis elegans. Infection in the host was localized with GFP-tagged V. parahaemolyticus using confocal laser scanning microscopy. The times required for causing infection, bacterial load in intestine, chemotactic response, and alteration in pharyngeal pumping were analyzed in the host system. In addition, the regulation of innate immune-related genes, lys-7, clec- 60, and clec-87, was analyzed using real-time PCR. The role of immune-responsible pmk-1 was studied using mutant strains. The pathogenicity of environmental strain CM2 isolated from the Gulf of Mannar, India was compared with that of a reference strain obtained from ATCC. The pathogen infected animals appeared to ward off infection by upregulating candidate antimicrobial genes for a few hours after the exposure, before succumbing to the pathogen. For the first time, the pathogenicity of V. parahaemolyticus at both the physiological and molecular levels has been studied in detail using the model organism C. elegans.     

Comparative efficacy of live replicating sheeppox vaccine strains in Ovines

In the present study, two sheeppox vaccines made from strains [sheeppox virus-Srinagar (SPPV-Srin) and Ranipet (SPPV-R)] indigenous to India and adapted to Vero cells were compared in terms of their safety, potency, efficacy and antigenic value with the commercial in-use Roumanian Fanar (SPPV-RF) vaccine, a foreign strain adapted in primary lamb testes cells. The safety test indicated that the SPPV (Sri and RF) vaccines were safe while SPPV-R was not completely attenuated and caused excessive adverse reactions at the passage level tested. The immunized animals showed DTH reaction and resisted virulent SPPV challenge, while control animals developed disease. Specific virus could be detected in the controls and animals immunized with lower dilutions of vaccines after challenge but not in any of the sheep immunized with 1 and 100 doses of each vaccine. All vaccines were found potent and the PD₅₀ was highest for SPPV (Srin and R) followed by RF. The immunized animals were seroconverted following vaccination with sustained antibody responses after challenge. In conclusion, indigenous SPPV-Srin vaccine was found to be as efficacious as SPPV-R and SPPV-RF vaccines. Thus, there is potential benefit in replacing the currently used commercial vaccine SPPV-RF with indigenous SPPV-Srin vaccine for use in India.     

In Vivo Suppression of HIV by Antigen Specific T Cells Derived from Engineered Hematopoietic Stem Cells

The HIV-specific cytotoxic T lymphocyte (CTL) response is a critical component in controlling viral replication in vivo, but ultimately fails in its ability to eradicate the virus. Our intent in these studies is to develop ways to enhance and restore the HIV-specific CTL response to allow long-term viral suppression or viral clearance. In our approach, we sought to genetically manipulate human hematopoietic stem cells (HSCs) such that they differentiate into mature CTL that will kill HIV infected cells. To perform this, we molecularly cloned an HIV-specific T cell receptor (TCR) from CD8+ T cells that specifically targets an epitope of the HIV-1 Gag protein. This TCR was then used to genetically transduce HSCs. These HSCs were then introduced into a humanized mouse containing human fetal liver, fetal thymus, and hematopoietic progenitor cells, and were allowed to differentiate into mature human CD8+ CTL. We found human, HIV-specific CTL in multiple tissues in the mouse. Thus, genetic modification of human HSCs with a cloned TCR allows proper differentiation of the cells to occur in vivo, and these cells migrate to multiple anatomic sites, mimicking what is seen in humans. To determine if the presence of the transgenic, HIV-specific TCR has an effect on suppressing HIV replication, we infected with HIV-1 mice expressing the transgenic HIV-specific TCR and, separately, mice expressing a non-specific control TCR. We observed significant suppression of HIV replication in multiple organs in the mice expressing the HIV-specific TCR as compared to control, indicating that the presence of genetically modified HIV-specific CTL can form a functional antiviral response in vivo. These results strongly suggest that stem cell based gene therapy may be a feasible approach in the treatment of chronic viral infections and provide a foundation towards the development of this type of strategy.     

Cytokines expression profile and kinetics of Peste des petits ruminants virus antigen and antibody in infected and vaccinated goats

The present study deals with the co-ordination of cytokine (IL-4 and IFN-γ) expression and kinetics of peste des petits ruminants (PPR) virus antigen and antibody in PPRV infected and vaccinated goats. The infected animals exhibited mixed cytokine (both T_H1 and T_H2) responses in the initial phase of the disease. The infected and dead goats had increased IFN-γ response before their death; while IL-4 remained at the base level. The cytokine expression in recovered animals was almost similar to that of vaccinated ones, where a unique biphasic response of IL-4 expression was observed with an up-regulation of IFN-γ on 7^th days post vaccination (dpv). Analysis of PPR virus antigen and antibody kinetics in different components of blood from infected and vaccinated animals revealed that the PPR virus antigen load was highest in plasma followed by serum and blood of the infected animals, whereas vaccinated animals showed only marginal positivity on 9^th dpv. The antibody titer was high in serum followed by plasma and blood in both vaccinated and infected animals. Therefore, it is inferred that the presence of antigen and antibody were significant with the expression of cytokine, and that a decreased response of IL-4 was noticed during intermediate phase of the disease i.e., 7 to 12^th days post infection (dpi). This indicates the ability to mount a functional T_H2 response after 14^th dpi could be a critical determinant in deciding the survival of the PPR infected animal.     

Study on passive immunity: Time of vaccination in kids born to goats vaccinated against Peste des petits ruminants

In this study, the decay of maternal peste des petits ruminants virus (PPRV) antibodies in kids born to goats vaccinated with Asian lineage IV PPR vaccine and the efficacy of passive immunity against PPRV was assessed to determine the appropriate period for vaccination in kids. Serum samples collected from kids born to vaccinated, unvaccinated and infected goats at different time intervals were tested by PPR competitive ELISA and serum neutralization test (SNT). Maternal antibodies in kids were detectable up to 6 months with a decline trend from the third month onwards and receded below the protective level by the fourth month. The kid with an SN titre of 1:8 at the time of immunization showed significant PPRV specific antibody response (percentage inhibition of 76; SN titers >1:16), when tested on 21 day post-vaccination and was completely protected from infection upon virulent PPRV challenge. Similarly, the kid with 1:8 SN titers was completely protected from PPR infection on active challenge. Therefore, PPR vaccination is recommended in kids, aged 4 months and born to immunized or exposed goats. This could be a suitable period to avoid window of susceptibility in kids to PPRV and the effort to eliminate PPR infection from susceptible populations.