Modulation of Lactate Dehydrogenase Isozymes by Modified Base Queuine

The modified base queuine is a nutrient factor for lower and higher eukaryotes except yeast. It is synthesized in eubacteria and inserted into the wobble position of specific tRNAs (tRNA_GUN) in exchange of guanine at position 34. The tRNAs of Q family are completely modified in terminally differentiated somatic cells. However, mainly free queuine is present in embryonic and fast proliferating cells, tRNA remains Q deficient. Lactate dehydrogenase (LDH) A mRNA and LDH A protein is known to increase when cells are grown in hypoxic conditions. In the present study, the level of LDH isozymes is analyzed in different tissues of normal and cancerous (DLA) mice and the effect of queuine treatment on LDH isozyme is observed. LDH A isozyme is shown to increase in serum and liver of DLA mice. The level and activity of LDH A decreases on queuine treatment. In skeletal muscle and heart, LDH A isozyme decreases while LDH B increases in DLA mice. Queuine administration leads to change back towards normal. In case of brain, LDH A increases but LDH B decreases in DLA mice. Queuine treatment leads to decrease in A4 anaerobic isozymes of LDH. The results suggest that queuine suppresses anaerobic glycolytic pathway, which leads to tumor suppression of DLA mice.     

RNA interference of dual oxidase in the plant nematode Meloidogyne incognita

RNA interference (RNAi) is a powerful tool for the analysis of gene function in model organisms such as the nematode Caenorhabditis elegans. Recent demonstrations of RNAi in plant parasitic nematodes provide a stimulus to explore the potential of using RNAi to investigate disruption of gene function in Meloidogyne incognita, one of the most important nematode pests of global agriculture. We have used RNAi to examine the importance of dual oxidases (peroxidase and NADPH oxidase), a class of enzyme associated with extracellular matrix cross-linking in C. elegans. RNAi uptake by M. incognita juveniles is highly efficient. In planta infection data show that a single 4-h preinfection treatment with double-stranded RNA derived from the peroxidase region of a dual oxidase gene has effects on gene expression that are phenotypically observable 35 days postinfection. This RNAi effect results in a reduction in egg numbers at 35 days of up to 70%. The in vitro feeding strategy provides a powerful tool for identifying functionally important genes, including those that are potential targets for the development of new agrochemicals or transgenic resistance strategies.     

Identification of sites in adenovirus hexon for foreign peptide incorporation

Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His6) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His6 epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His6 epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His6 epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His6 epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His6 epitopes did not appear to mediate His6-dependent viral infection, as assessed in two His6 artificial receptor systems. Our study provided valuable information for studies involving hexon modification.     

5-Aminosalicylic acid reverses endosulfan-induced testicular toxicity in male rats

Pre-treatment with 5-aminosalicylic acid (5-ASA) significantly reduced sperm-shape abnormalities in endosulfan-treated rats. The number of abnormal sperm in the epididymis was markedly increased by endosulfan treatment but pre-treatment with 5-ASA kept these values close to normal. Treatment with 5-ASA at a dose of 25 mg/kg bw was more effective in reducing sperm-shape abnormality and sperm count than at a dose of 50 mg/kg bw. Endosulfan significantly increased the level of lactate dehydrogenase (LDH) in rats but a marked decrease was observed upon pre-treatment with 25 mg/kg bw 5-ASA. Changes in plasma testosterone levels were not significantly correlated with 5-ASA pre-treatment but histopathological analysis of seminiferous tubules and Leydig cells showed significant protection from endosulfan-induced tissue damage such as necrosis. The population of Sertoli cells increased and the lumen of the seminiferous tubules contained a greater number of spermatids. There was a corresponding increase in the number of Leydig cells. A curative study with 5-ASA showed a similar protection from endosulfan-induced toxicity and cellular damage, but the extent of protection was significantly lower.     

Chitin-supplemented formulations improve biocontrol and plant growth promoting efficiency of Bacillus subtilis AF 1

Formulations of a chitinolytic biocontrol and a plant growth promoting Bacillus subtilis AF 1 were prepared in peat, in peat supplemented with either 0.5% chitin or Aspergillus niger mycelium, or in spent compost obtained from Agaricus bisporus cultivation and were evaluated for biocontrol of two fungal pathogens and plant growth promoting activities on pigeon pea and groundnut. A steady increase in cell numbers of introduced B. subtilis AF 1 was observed in all the formulations at 30 degrees C. The increase in cell numbers was about 5.0 log units. Peat or spent compost inoculated with physiologically active and dormant states of B. subtilis AF 1 showed different time period requirements to attain maximum cell numbers. The presence of chitin or A. niger (in peat) or A. bisporus (in spent compost) as supplement in the carrier material improved the multiplication of B. subtilis AF 1. When used as seed treatments, formulations of AF 1 in peat supplemented with chitin or chitin-containing materials showed better control of A. niger (causing crown rot of groundnut) and Fusarium udum (causing wilt of pigeon pea) than AF 1 culture alone, in both groundnut and pigeon pea. Bacillus subtilis AF 1 formulations promoted seed germination and biomass of both groundnut and pigeon pea even under pathogen pressure. Survival of AF 1 on fresh culture-treated and formulation product-treated plants was similar in pathogen-infested soil.     

Bromodomain and extra-terminal (BET) family proteins: New therapeutic targets in major diseases

The bromodomains and extra-terminal domain (BET) family proteins recognize acetylated chromatin through their bromodomains (BDs) and help in regulating gene expression. BDs are chromatin ‘readers’: by interacting with acetylated lysines on the histone tails, they recruit chromatin-regulating proteins on the promoter region to regulate gene expression and repression. Extensive efforts have been employed by scientific communities worldwide to identify and develop potential inhibitors of BET family BDs to regulate protein expression by inhibiting acetylated histone (H3/H4) interactions. Several small molecule inhibitors have been reported, which not only have high affinity but also have high specificity to BET BDs. These developments make BET family proteins an important therapeutic targets for major diseases such as cancer, neurological disorders, obesity and inflammation. Here, we review and discuss the structural biology of BET family BDs and their applications in major diseases.     

α-Tocopherol prevents lymphoma by improving antioxidant defence system of mice

Increased level of ROS causes oxidative stress and leads to various pathological conditions including cancer. Therefore antioxidants should contribute to cancer prevention by improving antioxidant defense system and thereby protecting the cell from oxidative damage. In the present study we have validated the hypothesis by evaluating the antioxidant action of α-tocopherol. The effect of α-tocopherol is analyzed on oxidative stress as well as its regulation on antioxidant defense system. Oxidative stress is measured in terms of reduced glutathione and protein carbonylation. To evaluate the role of α-tocopherol on antioxidant defense system, the activities and expressions of antioxidant enzymes like glutathione peroxidase, catalase and superoxide dismutase are analyzed by activity gel assay and by RT-PCR respectively. These enzyme activities and/or expressions are found to be improved by α-tocopherol in lymphoma bearing mice which brings down the oxidative stress as reflected by increased level of reduced glutathione as well as decreased protein carbonylation. The effect of α-tocopherol is further analyzed on general characteristics of lymphoma growth like body weight, longevity, accumulation of ascites fluid, angiogenesis in peritoneum, morphology of liver and abundance of lymphocytes. The antioxidant α-tocopherol is found to check lymphoma growth. The results suggest that α-tocopherol contributes to lymphoma prevention by improving antioxidant defence system of mice.     

A Temperature-Monitoring Vaginal Ring for Measuring Adherence

BackgroundProduct adherence is a pivotal issue in the development of effective vaginal microbicides to reduce sexual transmission of HIV. To date, the six Phase III studies of vaginal gel products have relied primarily on self-reporting of adherence. Accurate and reliable methods for monitoring user adherence to microbicide-releasing vaginal rings have yet to be established.MethodsA silicone elastomer vaginal ring prototype containing an embedded, miniature temperature logger has been developed and tested in vitro and in cynomolgus macaques for its potential to continuously monitor environmental temperature and accurately determine episodes of ring insertion and removal.ResultsIn vitro studies demonstrated that DST nano-T temperature loggers encapsulated in medical grade silicone elastomer were able to accurately and continuously measure environmental temperature. The devices responded quickly to temperature changes despite being embedded in different thickness of silicone elastomer. Prototype vaginal rings measured higher temperatures compared with a subcutaneously implanted device, showed high sensitivity to diurnal fluctuations in vaginal temperature, and accurately detected periods of ring removal when tested in macaques.ConclusionsVaginal rings containing embedded temperature loggers may be useful in the assessment of product adherence in late-stage clinical trials.     

Host‐induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce

Host‐induced gene silencing (HIGS) is an RNA interference‐based approach in which small interfering RNAs (siRNAs) are produced in the host plant and subsequently move into the pathogen to silence pathogen genes. As a proof‐of‐concept, we generated stable transgenic lettuce plants expressing siRNAs targeting potentially vital genes of Bremia lactucae, a biotrophic oomycete that causes downy mildew, the most important disease of lettuce worldwide. Transgenic plants, expressing inverted repeats of fragments of either the Highly Abundant Message #34 (HAM34) or Cellulose Synthase (CES1) genes of B. lactucae, specifically suppressed expression of these genes, resulting in greatly reduced growth and inhibition of sporulation of B. lactucae. This demonstrates that HIGS can provide effective control of B. lactucae in lettuce; such control does not rely on ephemeral resistance conferred by major resistance genes and therefore offers new opportunities for durable control of diverse diseases in numerous crops.     

Mitochondrial Control Region Alterations and Breast Cancer Risk: A Study in South Indian Population

Background

Mitochondrial displacement loop (D-loop) is the hot spot for mitochondrial DNA (mtDNA) alterations which influence the generation of cellular reactive oxygen species (ROS). Association of D-loop alterations with breast cancer has been reported in few ethnic groups; however none of the reports were documented from Indian subcontinent.

Methodology

We screened the entire mitochondrial D-loop region (1124 bp) of breast cancer patients (n = 213) and controls (n = 207) of south Indian origin by PCR-sequencing analysis. Haplotype frequencies for significant loci, the standardized disequilibrium coefficient (D′) for pair-wise linkage disequilibrium (LD) were assessed by Haploview Software.

Principal Findings

We identified 7 novel mutations and 170 reported polymorphisms in the D-loop region of patients and/or controls. Polymorphisms were predominantly located in hypervariable region I (60%) than in II (30%) of D-loop region. The frequencies of 310‘C’ insertion (P = 0.018), T16189C (P = 0.0019) variants and 310‘C’ins/16189C (P = 0.00019) haplotype were significantly higher in cases than in controls. Furthermore, strong LD was observed between nucleotide position 310 and 16189 in controls (D′ = 0.49) as compared to patients (D′ = 0.14).

Conclusions

Mitochondrial D-loop alterations may constitute inherent risk factors for breast cancer development. The analysis of genetic alterations in the D-loop region might help to identify patients at high risk for bad progression, thereby helping to refine therapeutic decisions in breast cancer.