Detailed characterization of the mRNA mapping in the HindIII fragment K region of the herpes simplex virus type 1 genome.

We have isolated as recombinant DNA clones, in the plasmid pBR322, regions of the herpesvirus type 1 genome spanning the region between 0.53 and 0.6 on the prototypical arrangement. This 11,000-base-pair region corresponds to 10% of the large unique region and encodes five major and several minor mRNA species abundant at different times after infection, which range in length from 7 to 1 kilobase. In this report, we have used RNA transfer blots and S1 nuclease digestion of hybrids between viral DNA and polyribosomal RNA to precisely localize (+/- 0.1 kilobase) these mRNA's. Comparison of neutral and alkaline gels of S1 nuclease-digested hybrids indicates no internal introns in the coding sequences of these mRNA's, although noncontiguous leader sequences near (ca. 0.1 kilobase) the 5' ends of any or all mRNA's could not be excluded. The 5' ends of several late mRNA's that are encoded opposite DNA strands map very close to one another, and the 3' ends of a major late and a major early mRNA, which are partially colinear, terminate in the same region. In vitro translation of the viral mRNA's isolated by hybridization with DNA bound to cellulose and fractionation of mRNA species on denaturing agarose gels allowed us to assign specific polypeptide products to each of the mRNA's characterized. Among other results, it was demonstrated unequivocally that two major late mRNA's, which partially overlap, encode the same polypeptide.     

Survival ofPseudomonas solanacearum in soil

Summary Studies on the survival ofPseudomonas solanacearum, the causal agent of bacterial wilt of tomato, under laboratory conditions showed that soil amendments had little effect on the population of the pathogen. When the host plant was grown in amended soil there was a positive influence on growth of the pathogen. The population level of the pathogen incorporated into the soil was reduced to one-half within a period of 46 days. The pathogen survived in the rhizosphere of non-host plants belonging to the families Acanthaceae and Leguminosae even in the absence of the natural hosts, but its incidence in the rhizosphere of plants belonging to Graminae and Cyperaceae was comparatively low indicating possibilities of reducing the inoculum potential of the pathogen in tomato fields by allowing such plants to grow during fallow periods. Part of the thesis of the Senior Author presented to the Kerala Agricultural University, Trichur, Kerala State, India.     

Microbial metabolism of phenolic amines: degradation of dl-synephrine by an unidentified arthrobacter.

Microorganisms capable of degrading dl-synephrine were isolated from soil of Citrus gardens by enrichment culture, with dl-synephrine as the sole source of carbon and nitrogen. An organism which appears to be an arthrobacter, but which cannot be identified with any of the presently recognized species was predominant in these isolates. It was found to metabolize synephrine by a pathway involving p-hydroxyphenylacetaldehyde, p-hydroxyphenylacetic acid, and 3,4-dihydroxyphenylacetic acid as intermediates. Some of the enzymes of this pathway were demonstrated in cell-free extracts. An aromatic oxygenase, which could also be readily obtained in a cell-free system, was found to degrade 3,4-dihydroxyphenylacetic acid by meta cleavage.     

Chemical Investigation of Marine-Derived Fungus Aspergillus flavipes for Potential Anti-Inflammatory Agents

The marine fungus, Aspergillus flavipes (MTCC 5220), was isolated from the pneumatophore of a mangrove plant Acanthus ilicifolius found in Goa, India. The crude extract of A. flavipes was found to show anti-inflammatory activity. It blocked interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) production in lipopolysaccharide (LPS)-activated THP-1 cells with IC₅₀ of 2.69±0.5 μM and 6.64±0.4 μM, respectively. The chemical investigation led to the isolation of optically inactive 4β-[(1E)-propen-1-yl]cyclopentane-1β,2β-diol ( 1 ) along with a new optically active diastereoisomeric compound, 4β-[(1E)-propen-1-yl]cyclopentane-1β,2α-diol ( 2 ). In addition, the fungus also produced known compounds (+)-terrein ( 3 ), butyrolactone I ( 4 ) and butyrolactone II ( 5 ) in high yields. Among these, (+)-terrein ( 3 ) exhibited IL-6 and TNF-α inhibition activity with IC₅₀ of 8.5±0.68 μM and 15.76±0.18 μM, respectively, while butyrolactone I ( 4 ) exhibited IC₅₀ of 12.03±0.85 μM (IL-6) and 43.29±0.76 μM (TNF-α) inhibition activity with low toxicity to host cells in LPS stimulated THP-1 cells. This is the first report of the isolation and characterization of 4β-[(1E)-propen-1-yl]cyclopentane-1β,2α-diol ( 2 ). The structures of all the isolated compounds were elucidated on the basis of extensive detailed NMR spectroscopic data. Anti-inflammatory activity of the fungi A. flavipes is presented here for the first time, which was due to (+)-terrein and butyrolactone I, as the major constituents and they can be further explored in the therapeutic area.     

Effect of doxorubicin on heart mitochondrial enzymes in rats: a protective role for alpha-tocopherol.

Effect of doxorubicin on heart mitochondrial enzymes was studied in rats with or without the administration of alpha-tocopherol. Rats were treated with doxorubicin 2.5 mg/kg, ip body wt once a week for 8 weeks. Alpha-tocopherol was co-administered orally for 2 months (400 mg/kg body wt daily). TCA cycle enzyme, NADH-dehydrogenase, cytochrome-C-oxidase and Na+,K(+)-ATPase activities were found to be decreased in doxorubicin treatment. A significant decrease in protease activity was observed with a concomitant increase in mitochondrial protein level. Mitochondrial lipid peroxide level was found to be increased with a decrease in thiol content. Alpha-tocopherol co-administration was found to maintain the mitochondrial enzyme activities as well as the thiol content. The results are discussed with reference to the antioxidant nature of alpha-tocopherol.     

Chiral Imidazolidin-4-one with catalytic amount of Dicationic ionic liquid act as a recoverable and reusable Organocatalyst for asymmetric Diels-Alder reaction

Imidazolidin-4-one is used as a recoverable organocatalyst for the asymmetric Diels-Alder reaction in the presence of catalytic amount of dicationic ionic liquid and trifluoroacetic acid as a co-catalyst. The Diels-Alder reaction between model substrate cyclopentadiene and crotonaldehyde gave the product in 95% conversion and 87% ee of the endo-product. The catalyst was shown better reusability when the 20 mol% of dicationic ionic liquid was used and catalyst was reused upto 5 cycles, conversion remains upto 3 recycles but ee of endo- 9 was slightly droped.     

Regulation of Opioid Receptors by Their Endogenous Opioid Peptides

Cellular and Molecular Neurobiology - Activation of μ, δ, and κ opioid receptors by endogenous opioid peptides leads to the regulation of many emotional and physiological responses....     

Analytical validation of quantitative immunohistochemical assays of tumor infiltrating lymphocyte biomarkers

Tumor infiltrating lymphocytes (TIL), especially T-cells, have both prognostic and therapeutic applications. The presence of CD8+ effector T-cells and the ratio of CD8+ cells to FOXP3+ regulatory T-cells have been used as biomarkers of disease prognosis to predict response to various immunotherapies. Blocking the interaction between inhibitory receptors on T-cells and their ligands with therapeutic antibodies including atezolizumab, nivolumab, pembrolizumab and tremelimumab increases the immune response against cancer cells and has shown significant improvement in clinical benefits and survival in several different tumor types. The improved clinical outcome is presumed to be associated with a higher tumor infiltration; therefore, it is thought that more accurate methods for measuring the amount of TIL could assist prognosis and predict treatment response. We have developed and validated quantitative immunohistochemistry (IHC) assays for CD3, CD8 and FOXP3 for immunophenotyping T-lymphocytes in tumor tissue. Various types of formalin fixed, paraffin embedded (FFPE) tumor tissues were immunolabeled with anti-CD3, anti-CD8 and anti-FOXP3 antibodies using an IHC autostainer. The tumor area of stained tissues, including the invasive margin of the tumor, was scored by a pathologist (visual scoring) and by computer-based quantitative image analysis. Two image analysis scores were obtained for the staining of each biomarker: the percent positive cells in the tumor area and positive cells/mm² tumor area. Comparison of visual vs. image analysis scoring methods using regression analysis showed high correlation and indicated that quantitative image analysis can be used to score the number of positive cells in IHC stained slides. To demonstrate that the IHC assays produce consistent results in normal daily testing, we evaluated the specificity, sensitivity and reproducibility of the IHC assays using both visual and image analysis scoring methods. We found that CD3, CD8 and FOXP3 IHC assays met the fit-for-purpose analytical acceptance validation criteria and that they can be used to support clinical studies.