Establishment and optimization of epithelial cell cultures from human ectocervix,transformation zone,and endocervix optimization of epithelial cell cultures

Cervical cancer is a major public health problem and research using cell culture models has improved understanding of this disease. The human cervix contains three anatomic regions; ectocervix with stratified squamous epithelium, endocervix with secretory epithelium, and transformation zone (TZ) with metaplastic cells. Most cervical cancers originate within the TZ. However, little is known about the biology of TZ cells or why they are highly susceptible to carcinogenesis. The goal of this study was to develop and optimize methods to compare growth and differentiation of cells cultured from ectocervix, TZ or endocervix. We examined the effects of different serum-free media on cell attachment, cell growth and differentiation, and cell population doublings in monolayer culture. We also optimized conditions for organotypic culture of cervical epithelial cells using collagen rafts with human cervical stromal cells. Finally, we present a step-by-step protocol for culturing cells from each region of human cervix.     

Lecithin retinol acyltransferase contains cysteine residues essential for catalysis

Lecithin retinol acyltransferase (LRAT) is an essential enzyme in vitamin A metabolism and mobilization. The membrane-bound enzyme catalyzes the transfer of an acyl group from the sn-1 position of lecithin to vitamin A to generate retinyl esters. The sequence of LRAT is novel and hence does not suggest a mechanistic class to which the enzyme belongs. However, the activity of the enzyme is exceedingly sensitive to affinity labeling and group-specific reagents directed toward thiol groups. LRAT from human retinal pigment epithelium has cysteine residues at positions 161, 168, 182, and 208. Site-specific mutagenic studies show that C182 and C208 can be converted to alanines with little affect on activity. The activities of the C161A and C168A mutants are virtually nil. Moreover, while C168S is substantially active, C161S possesses only a few percent of the activity of wild-type (WT) LRAT. Also, pH-rate profiles show that C168S has virtually the same profile as WT LRAT, while C161S shows an aberrant profile quite unlike that of WT LRAT. Therefore, LRAT is a thiol acyltransferase and C161 may be the essential nucleophilic residue critical for catalysis.     

Phosphate Glass Fibre Composites for Bone Repair

We investigate high-modulus degradable materials intended to replace metals in biomedical applications.These are typicallycomposites comprising a polylactide(PLA)matrix reinforced with phosphate glass fibres,which provide reinforcementsimilar to E-glass but are entirely degradable in water to produce,principally,calcium phosphate.We have made compositesusing a variety of fibre architectures,from non-woven random mats to unidirectional fibre tapes.Flexural properties in theregion of 30 GPa modulus and 350 MPa strength have been achieved-directly comparable to quoted values for human corticalbone.In collaboration with other groups we have begun to consider the development of foamed systems with structures mimickingcancellous bone and this has shown significant promise.The fibres in these foamed structures provide improved creepresistance and reinforcement of the pore walls.To date the materials have exhibited excellent cellular responses in vitro andfurther studies are due to include consideration of the surface character of the materials and the influence of this on cell interaction,both with the composites and the glass fibres themselves,which show promise as a standalone porous scaffold.     

Chemo-enzymatic synthesis of the exocyclic olefin isomer of thymidine monophosphate

Exocyclic olefin variants of thymidylate (dTMP) recently have been proposed as reaction intermediates for the thymidyl biosynthesis enzymes found in many pathogenic organisms, yet synthetic reports on these materials are lacking. Here we report two strategies to prepare the exocyclic olefin isomer of dTMP, which is a putative reaction intermediate in pathogenic thymidylate biosynthesis and a novel nucleotide analog. Our most effective strategy involves preserving the existing glyosidic bond of thymidine and manipulating the base to generate the exocyclic methylene moiety. We also report a successful enzymatic deoxyribosylation of a non-aromatic nucleobase isomer of thymine, which provides an additional strategy to access nucleotide analogs with disrupted ring conjugation or with reduced heterocyclic bases. The strategies reported here are straightforward and extendable towards the synthesis of various pyrimidine nucleotide analogs, which could lead to compounds of value in studies of enzyme reaction mechanisms or serve as templates for rational drug design.     

Stress-induced effects, which inhibit host defenses, alter leukocyte trafficking

Acute cold restraint stress (ACRS) has been reported to suppress host defenses against Listeria monocytogenes, and this suppression was mediated by beta1-adrenoceptors (β1-ARs). Although ACRS appears to inhibit mainly early innate immune defenses, interference with leukocyte chemotaxis and the involvement of β1-AR (or β2-AR) signaling had not been assessed. Thus, the link between sympathetic nerve stimulation, release of neurotransmitters, and changes in blood leukocyte profiles, including oxidative changes, following ACRS was evaluated. The numbers of leukocyte subsets in the blood were differentially affected by β1-ARs and β2-ARs following ACRS; CD3⁺ (CD4 and CD8) T-cells were shown to be decreased following ACRS, and the T cell lymphopenia was mediated mainly through a β2-AR mechanism, while the decrease in CD19⁺ B-cells was influenced through both β1- and β2-ARs, as assessed by pharmacological and genetic manipulations. In contrast to the ACRS-induced loss of circulating lymphocytes, the number of circulating neutrophils was increased (i.e., neutrophilia), and this neutrophilia was mediated through β1-ARs. The increase in circulating neutrophils was not due to an increase in serum chemokines promoting neutrophil emigration from the bone marrow; rather it was due to neutrophil release from the bone marrow through activation of a β1-AR pathway. There was no loss of glutathione in any of the leukocyte subsets suggesting that there was minimal oxidative stress; however, there was early production of nitric oxide and generation of some protein radicals. Premature egress of neutrophils from bone marrow is suggested to be due to norepinephrine induction of nitric oxide, which affects the early release of neutrophils from bone marrow and lessens host defenses.     

Factors influencing the distribution of leopard in a semiarid landscape of Western India

Previously, factors governing distribution of leopard (Panthera pardus fusca) in forest habitats of the Indian subcontinent were unknown. The present study assessed the influence of different ecogeographic variables determining the distribution of leopards in and around Sariska Tiger Reserve through MaxEnt habitat suitability model based on camera trapping method. Camera trapping was used to collect presence/absence information in the study area from December 2008 to June 2010. Information of 11 macrohabitat characteristics and variables (habitat types, prey species, Normalized Difference Vegetation Index (NDVI), elevation, livestock, village, water source, etc.) were collected along with leopard presence data. The probability of presence of leopards increased with decreasing distance to water and increasing encounter rate of peafowl (Pavo cristatus), chital (Axis axis), sambar (Rusa unicolor), and wild pig (Sus scrofa). It was found that the probability of presence of leopards increased with increasing area of Zizyphus mixed forest patches and NDVI. Results of this study showed that the probability of presence of leopards was higher in habitat types with intermediate cover, high wild prey base, and water sources. They also indicated that leopards are not always ‘generalists’ showing some degree of specialization, at least in their choice of habitat, and this information is useful for conserving leopard in human-dominated landscapes.     

GxcDD,a putative RacGEF,is involved in <Emphasis Type="Italic">Dictyostelium</Emphasis> development

Background  

Rho subfamily GTPases are implicated in a large number of actin-related processes. They shuttle from an inactive GDP-bound form to an active GTP-bound form. This reaction is catalysed by Guanine nucleotide exchange factor (GEFs). GTPase activating proteins (GAPs) help the GTPase return to the inactive GDP-bound form. The social amoeba Dictyostelium discoideum lacks a Rho or Cdc42 ortholog but has several Rac related GTPases. Compared to our understanding of the downstream effects of Racs our understanding of upstream mechanisms that activate Rac GTPases is relatively poor.     

Gender discrimination in undernutrition with mediating factors among Bengalee school children from Eastern India

This study was undertaken to determine age and sex variations in the prevalence of underweight and stunting, and to assess the impact of some socio-economic variables on undernutrition among 6–16 year old school children of Bengalee ethnicity in Chapra, West Bengal, India. The subjects were selected randomly from various schools and madrassas of the Chapra Block. A total of 725 children (342 boys and 383 girls) aged 6–16 years were measured and data on their socio-economic status were collected. Age and sex combined rates of underweight and stunting were 44.40% and 37.20%, respectively. Weight-for-age Z-score (WAZ) showed significant association with per-capita income (PCI) among boys (F = 5.45) and girls (F = 8.14). Height-for-age Z-score (HAZ) has also shown the association with per-capita income among boys (F = 4.43) and girls (F = 9.69). The WAZ was significantly associated with fathers’ educational status (FOS) (t = ?2.95) and the number of living rooms (NLR) (t = ?2.91) among girls. The HAZ showed significant association with number of siblings (NS) among girls (F = 4.25). Linear regression analyses revealed that NLR (t = 2.04) and NS (t = 1.95) had a significant impact on HAZ among boys. Among girls, PCI (t = 3.38), FOS (t = 2.87) and NLR (t = 2.81) had a significant impact on WAZ and also PCI (t = 3.28) and FOS (t = 2.90) had a significant impact on HAZ. NLR had significant associations with underweight (χ² = 3.59) and stunting (χ² = 4.20) among boys. Among girls, PCI had significant associations with underweight (χ² = 11.15) and stunting (χ² = 11.64). FOS also showed significant associations with underweight (χ² = 8.10) as well as stunting (χ² = 8.28) among girls. NLR showed a significant association with underweight (χ² = 7.75). Logistics regression analyses revealed that FOS (Wald = 8.00) and NLR (Wald = 4.09) were significant predictors of stunting among boys. Among girls, PCI was a significant predictor of underweight (Wald = 10.95) as well as stunting (Wald = 10.45). FOS, NLR and NS were also significant predictors of stunting (Wald = 8.16), underweight (Wald = 7.68) and stunting (Wald = 6.97) respectively. The present study revealed that the nutritional status of the children was unsatisfactory and it is of paramount importance not only to increase the amount of food supplementation given but also to promote gender equality.