Association between tumour necrosis-α gene polymorphisms and acne vulgaris in a Pakistani population.

BACKGROUND: The cytokine tumour necrosis factor (TNF)-α is a well-studied potent candidate mediator that is systemically involved in a variety of inflammatory diseases. Several single nucleotide polymorphisms (SNPs) of the TNF-α gene have been studied with regard the pathogenesis of acne vulgaris, but the results have been inconclusive. AIM: This case-control study investigated the association of the TNF -308 G>A and -238 G>A SNPs with acne vulgaris in a high-risk Pakistani population. METHODS: In total, 160 healthy controls and 140 patients with acne were enrolled in this study. Polymorphisms were determined by PCR and restriction fragment length polymorphism analysis. RESULTS: Our data showed that the TNF -308 G>A and TNF -238 G>A SNPs were present at a significantly higher rate in cases than in controls (P < 0.01 and P < 0.02; respectively). There was a significant difference between the G and A alleles from patients with acne and controls for -308 G>A (OR = 1.5, 95% CI = 1.07-2.19, P < 0.02) and -238 G>A (OR=1.6, 95% CI = 1.06-2.44, P = 0.02) genotype. Moreover, the severity of acne was significantly associated with TNF genotype (TNF -308 G>A: χ² = 34.6, P < 0.001; TNF -238 G>AL χ² = 12.9, P < 0.01). CONCLUSION: Our data suggest that the TNF -308 G>A and TNF -238 G>A SNPs may contribute to the pathogenesis of acne in the study population. Furthermore, patients with severe acne showed an increased frequency of mutant TNF genotypes at -308 and -238 compared with patients with less severe acne.

Insulin over expression induces heart abnormalities via reactive oxygen species regulation, might be step towards cardiac hypertrophy.

Insulin is known to regulate blood—glucose level and promote its utilization as an energy source in cardiac tissues under normal physiological conditions as well as stimulates signaling pathways that involved cell growth and proliferation. Although recently insulin generated free radicals via NAD(P)H has been documented but the molecular mechanism is still under investigation. The aim of present study is to elucidate the reactive oxygen species (ROS) dependent possible role of insulin in cardiac abnormalities, including hypertrophy by regulation of antioxidants enzyme (SOD) activity. In the current study, 60 cardiac patients and 50 healthy individuals as well as the rat model with insulin administration were under investigation. Oxidant, anti—oxidant biochemical assays, hypertrophic marker expression via immunobloting and histopathology were performed. We observed statistically significant elevation of the reactive oxygen species level in the serum of patients as well as in the insulin administrated rat model, a mild expression of cardiac marker in experimental models along with abnormal histopathology of hearts. However, super oxide dismutase free radical scavenger activity was down regulated upon insulin treatment compared to control rats. Conclusively, the present study showed that over expression of insulin might stimulate cardiac hypertrophic signal via up regulation of free radicals and down regulation of antioxidants enzymes including SOD activity.

Diameter-controlled synthesis of α-Mn2O3 nanorods and nanowires with enhanced surface morphology and optical properties.

Single crystalline α-Mn(2)O(3) nanorods, and nanowires with and without nanoparticles on them have been successfully synthesized by a template-free hydrothermal route. The variation in hydrothermal temperature has not only affected the diameter of the nanostructure but also noticeably affected the morphology and optical properties of the α-Mn(2)O(3) nanostructure. The influence of temperature on the diameter, crystallinity, surface morphology and optical properties of the α-Mn(2)O(3) nanostructure have been characterized by x-ray diffraction, scanning electron microscopy, energy dispersive x-ray analysis, transmission electron microscopy, high resolution transmission electron microscopy, Raman spectroscopy and UV-visible spectroscopy and photoluminescent (PL) spectroscopy. The results showed in our experimental conditions that single crystalline nanorods of the α-Mn(2)O(3) were obtained at a temperature of 180 °C, while single crystalline nanowires were obtained by increasing the temperature to 240 and 300 °C. Nanowires with nanoparticles on them were obtained by increasing the temperature to 240 °C and nanowires without nanoparticles on them were obtained by increasing the temperature to 300 °C. The nanorods and nanowires obtained had a well-defined morphology. The nanowires synthesized at 300 °C exhibited an intense orange band PL spectrum.

Galectin 1 is involved in vascular smooth muscle cell proliferation.

OBJECTIVE: Smooth muscle cell (SMC) migration and proliferation are the key steps in the development of atherosclerosis and restenosis. Matricellular proteins have been implicated in cell adhesion, migration and proliferation. Here we investigated the role of the matricellular protein galectin-1 (Gal-1), a beta-galactoside-binding lectin, in SMC proliferation in atheroma and DNA synthesis in cell culture. METHODS: Protein expression was visualised by tissue section immunostaining. RNA expression was analysed using Northern blot analysis. DNA synthesis of human vascular SMCs was determined by 3H-thymidine incorporation. Recombinant glutathione S-transferase-galectin-1 fusion protein (Gal FP) binding to extracellular matrix (ECM) proteins was measured by ELISA. Gal-1 binding to cells and ECM was estimated using 125I-labelled Gal FP. RESULTS: Prominent Gal-1 staining coincided with SMC proliferation in human coronary endarterectomy samples in organoid culture. In cell culture, Gal-1 mRNA was upregulated in growing SMCs. Gal FP increased serum-induced DNA synthesis of human SMCs on plastic or endogenous ECM, but not of a rat PAC1 SM cell line. Also, Gal FP slightly increased SMC adhesion to ECM. SMCs exhibited a complex pattern of receptor-ligand interactions with Gal FP. The Gal-1 binding to SMCs was much stronger than to ECM, produced by these SMCs. We identified new ECM proteins: thrombospondin, vitronectin and osteopontin, which bound to Gal FP in a dose- and beta-galactoside-dependent manner in ELISA. CONCLUSIONS: Gal-1 binding to SMCs was stronger than to ECM, although ECM of atherosclerotic blood vessels contained additional ECM proteins which bound to Gal-1. Gal-1 was upregulated during SMC growth and Gal FP enhanced serum-induced DNA synthesis in SMCs. Overall, Gal-1 upregulation is likely to provide a reinforcement of serum-induced events during vascular injury.

The interleukin-6 and interleukin-1A gene promoter polymorphism is associated with the pathogenesis of acne vulgaris.

Acne vulgaris is a skin disorder with a complex pathogenesis. Better treatment strategies require comprehensive knowledge of molecular factors contributing to the acne pathophysiology. Recent studies are focused on investigating the influence of inflammatory cytokines on the disease. This case-control study investigated the association of IL-6-572 G/C and IL-1A-889 C/T gene polymorphisms with acne in a Pakistani population. Pakistani subjects (380 healthy controls and 430 acne patients) were enrolled in this study. Polymorphism in the promoter region of IL-6-572 and IL-1A-889 was determined by polymerase chain reaction and restriction fragment length polymorphism. The IL-6-572 and IL-1A-889 variant genotypes were significantly associated with the acne pathogenesis. The IL-6-572C and the IL-1A-889T alleles were significantly high in the patient vs. control group (p < 0.0001 for both loci). The IL-6-572 G/C and IL-1A-889 C/T variant allele haplotypes showed significantly high prevalence in patients with acne; G-T (P = 0.0014), C-C (P < 0.0001), and C-T (P < 0.0001). This is the first report on the association between the IL-6-572 G/C polymorphism and acne among any population. The IL-1A-889 C/T polymorphism is also significantly linked with acne in the study population; the -889 C/T association with acne has been reported in one ethnic group previously. Our findings suggest that the IL-6-572C and IL-1A-889T alleles may contribute to the pathogenesis of acne in a Pakistani population. Further studies are required to verify these findings in other populations.

Ultrasonographic renal size in individuals without known renal disease.

OBJECTIVE: In order to establish some preliminary data of our population, we determined the ultrasonographic kidney dimensions in individuals without known renal disease. We assessed whether age, sex, side, body mass index (BMI) and presence or absence of diabetes mellitus and hypertension affect the renal size. METHODS: Ultrasonographic kidney measurements were performed on 194 adult patients without known kidney lesions. Measurements included length, width, cortical thickness and estimation of renal size which was obtained by multiplying the first three variables. The effect of age, gender, side, height, weight, BMI, hypertension and diabetes mellitus was statistically analyzed. RESULTS: The mean kidney length was 10.4 +/- 0.8 cm, mean with 4.5 +/- 0.6 cm and mean cortical thickness 1.6 +/- 0.2 cm. The estimated mean renal size was 76 +/- 22 cm3. Kidney length did not significantly differ between right and left, however, kidney width, cortical thickness and size did (p < 0.05). Right kidneys were smaller than the left ones. In univariate analysis, the mean renal size correlated with age, sex, side, BMI and absence or presence of hypertension and diabetes mellitus. In a multivariate analysis, however, the only significant factors affecting renal size were sex and BMI. CONCLUSION: We conclude that renal size is related to age, side, sex and the individual's height and weight. Population-based studies are needed to establish the normal values for the Pakistani population.

Biogenesis of structural intercellular junctions during cleavage in the mouse embryo.

The preimplantation embryo differentiates the trophectoderm epithelium which, from the 32-cell stage, generates the blastocoel of the blastocyst and, after implantation, gives rise to most extraembryonic lineages of the conceptus. Trophectoderm differentiation begins at compaction (8-cell stage) when cell-cell adhesion, mediated by uvomorulin, and epithelial cell polarisation first occur. Here, we review our work on the biogenesis of tight junctions and desmosomes during epithelial differentiation. Tight junction construction begins at compaction and appears to be a gradual process, both at morphological and molecular levels. This maturation pattern may be due in part to sequential expression of tight junction constituents from the embryonic genome. Tight junction formation is dependent upon uvomorulin adhesion but can be inhibited by different means without apparently disturbing cell adhesion or polarisation. Cell interactions appear to regulate tight junction tissue specificity, in part by controlling the level of synthesis of constituents. Desmosome formation begins at the 32-cell stage, particularly as the embryo initiates blastocoel accumulation, and, in contrast with tight junction formation, does not appear to be a gradual process. Thus, nascent desmosomes appear mature in terms of their molecular composition. Desmosomal proteins are synthesised well in advance of desmosome formation but the synthesis of the principal glycoprotein components begins at the blastocyst stage and may regulate the timing of junction assembly. Implications of these differing patterns of biogenesis for the embryo are discussed.

Localisation of tight junction protein cingulin is temporally and spatially regulated during early mouse development.

The molecular maturation of the tight junction in the mouse early embryo has been investigated by monitoring the distribution of cingulin, a 140 x 10(3) M(r) peripheral (cytoplasmic) membrane constituent of the junction, at different stages of development and in different experimental situations. Although tight junction formation does not begin until compaction at the 8-cell stage, cingulin is detectable in oocytes and all stages of cleavage, a factor consistent with our biochemical analysis of cingulin expression (Javed et al., 1992, Development 117, 1145-1151). Using synchronised egg and embryo stages and isolated cell clusters, we have identified three sites where cingulin is localised, the cytocortex, punctate cytoplasmic foci and tight junctions themselves. Cytocortical cingulin is present at the cumulus-oocyte contact site (both cell types), in unfertilised and fertilised eggs and in cleavage stages up to 16-cell morulae, particularly at microvillous domains on the embryo outer surface (eg. apical poles at compaction). Embryo manipulation experiments indicate that cortical cingulin is labile and dependent upon cell interactions and therefore is not merely an inheritance from the egg. Cingulin cytoplasmic foci are evident only in outer cells (prospective trophectoderm) from the 32-cell stage, just prior to cavitation, and decline from approx. 8 hours after cavitation has initiated. The appearance of these foci is insensitive to cycloheximide treatment and they colocalise with apically derived endocytic vesicles visualised by FITC-dextran, indicating that the foci represent the degradation of cytocortical cingulin by endocytic turnover. Cingulin is detectable at the tight junction site between blastomeres usually from the 16-cell stage, although earlier assembly occurs in a minority (up to 20%) of specimens. Cingulin assembly at the tight junction is sensitive to cycloheximide and is identifiable approx. 10 hours after cell adhesion is initiated and ZO-1 protein assembles. Collectively, our results indicate that (i) cingulin from nonjunctional sites does not contribute to tight junction assembly and (ii) the molecular maturation of the junction appears to occur progressively over at least two cell cycles.

Tight junction protein cingulin is expressed by maternal and embryonic genomes during early mouse development.

The expression of the tight junction peripheral membrane protein, cingulin (140 x 10(3) M(r), was investigated in mouse eggs and staged preimplantation embryos by immunoblotting and immunoprecipitation. Polyclonal antibody to chicken brush cingulin detected a single 140 x 10(3) M(r) protein in immunoblots of unfertilised eggs and all preimplantation stages. Relative protein levels were high in eggs and early cleavage stages, declined during later cleavage and increased again in expanding blastocysts. Quantitative immunoprecipitation of metabolically labelled eggs and staged embryos also revealed a biphasic pattern for cingulin synthesis with relative net levels being high in unfertilised eggs, minimal during early cleavage, rising 2.3-fold specifically at the onset of compaction (8-cell stage, when tight junction formation begins), and increasing further at a linear rate during morula and blastocyst stages. Cingulin synthesis in eggs is not influenced by fertilisation (or aging, if unfertilised), but this level declines sharply after first cleavage. These results indicate that cingulin is expressed by both maternal and embryonic genomes. The turnover of maternal cingulin (unfertilised eggs) and embryonic cingulin at a stage before tight junction formation begins (4-cell stage) is higher (t1/2 approximately 4 hours) than cingulin synthesised after tight junction formation (blastocysts; t1/2 approximately 10 hours). This increase in cingulin stability is reversed in the absence of extracellular calcium. Cingulin synthesis is also tissue-specific in blastocysts, being up-regulated in trophectoderm and down-regulated in the inner cell mass. Taken together, the results suggest that (i) cingulin may have a role during oogenesis and (ii) cell-cell contact patterns regulate cingulin biosynthesis during early morphogenesis, contributing to lineage-specific epithelial maturation.

Epithelial differentiation and intercellular junction formation in the mouse early embryo.

Trophectoderm differentiation during blastocyst formation provides a model for investigating how an epithelium develops in vivo. This paper briefly reviews our current understanding of the stages of differentiation and possible control mechanisms. The maturation of structural intercellular junctions is considered in more detail. Tight junction formation, essential for blastocoele cavitation and vectorial transport activity, begins at compaction (8-cell stage) and appears complete before fluid accumulation begins a day later (approx 32-cell stage). During this period, initial focal junction sites gradually extend laterally to become zonular and acquire the peripheral tight junction proteins ZO-1 and cingulin. Our studies indicate that junction components assemble in a temporal sequence with ZO-1 assembly preceding that of cingulin, suggesting that the junction forms progressively and in the 'membrane to cytoplasm' direction. The protein expression characteristics of ZO-1 and cingulin support this model. In contrast to ZO-1, cingulin expression is also detectable during oogenesis where the protein is localised in the cytocortex and in adjacent cumulus cells. However, maternal cingulin is metabolically unstable and does not appear to contribute to later tight junction formation in trophectoderm. Cell-cell interactions are important regulators of the level of synthesis and state of assembly of tight junction proteins, and also control the tissue-specificity of expression. In contrast to the progressive nature of tight junction formation, nascent desmosomes (formed from cavitation) appear mature in terms of their substructure and composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative immunohistochemical detection of oxidized low density lipoprotein in the rabbit arterial wall.

Quantitative immunohistochemical techniques were developed for mapping low density lipoprotein (LDL) oxidation within arterial tissue. Antibodies were raised by immunizing rabbits with Cu(2+)-oxidized rabbit LDL. ELISAs showed that they reacted strongly with oxidized rabbit LDL, weakly with other oxidized lipoproteins, and not at all with native LDL. Using optimized histological procedures, the antibodies were applied to sections of calibration gels containing LDL at various concentrations and levels of oxidation, and to sections of aortas from normal and heritable hyperlipidemic rabbits. Binding was measured with a rhodamine-labeled secondary antibody and carefully calibrated techniques of digital imaging fluorescence microscopy. Values obtained using a nonspecific primary antibody were subtracted. Specific binding to calibration sections increased linearly with respect to the concentration of oxidized LDL and the duration of its exposure to Cu2+, approximately linearly with respect to its modified lysine content, and nonlinearly with respect to its relative electrophoretic mobility. Specific staining was detected in sections of aortas from heritable hyperlipidemic but not normal rabbits. In the former, it was higher in the intima than in the media and was greater downstream than upstream of intercostal branch ostia; the average level was lower in those branches with the least intimal thickening but the difference between upstream and downstream regions was larger. These results correlate with the known pattern of lipid deposition in hyperlipidemic rabbit aortas. A small but significant amount of specific staining was observed in sections which were devoid of intimal thickening, which is consistent with LDL oxidation occurring prior to disease or during its earliest stages.

Glutamate dehydrogenase (NADP-dependent) mRNA in relation to enzyme synthesis in Euglena gracilis. Evidence for post-transcriptional control.

Cells of Euglena gracilis Klebs strain z Pringsheim had high NADP-dependent glutamate dehydrogenase activity when grown on glutamate as nitrogen source but activity was completely repressed in cells grown on ammonium (NH4+). A 120-fold purification of NADPH-glutamate dehydrogenase (subunit Mr = 45 000) was achieved from glutamate-grown cells by affinity chromatography on blue Sepharose CL-6B. Antisera raised against the homogeneously pure protein were used to demonstrate that increase in NADPH-glutamate dehydrogenase activity on transfer from NH4+ to glutamate medium resulted from an increase in the amount of protein. Glutamate NH4+-grown cells were labelled with L-[35S]methionine and anti-(NADPH-glutamate dehydrogenase) used to immunoprecipitate the dehydrogenase from cell extracts. NADPH-glutamate dehydrogenase protein was detected in glutamate-grown but not NH4+-grown cells. Anti-(NADPH-glutamate dehydrogenase) was used to detect NADPH-glutamate dehydrogenase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. NADPH-glutamate dehydrogenase mRNA was present in glutamate NH4+-grown cells, there being no apparent difference in mRNA abundance between cells showing a tenfold difference in NADPH-glutamate dehydrogenase specific activity. These results indicate that the synthesis of this dehydrogenase is regulated primarily at the post-transcriptional level.

Plasminogen activator and plasminogen activator inhibitor gene expression in human saphenous vein organ culture.

We describe the expression of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) in saphenous vein culture. Smooth muscle cells (SMC) are quiescent in fresh tissue, whereas these cells acquire a proliferating phenotype when venous segments are cultured in the presence of serum. t-PA and PAI-1 were localized immunohistochemically and quantified using biochemical techniques. t-PA and PAI-1 mRNA was quantified by reverse transcription polymerase chain reaction (RT--PCR) assay. Immunostaining showed an increase in the positivity of proliferating cell nuclear antigen (PCNA) from 10-day tissue culture. Tissue sections from fresh vein showed minimal t-PA and maximal PAI-1 immunostaining. In contrast, 10-day cultures showed an increase in t-PA and a decline in PAI-1 staining. Biochemical analysis revealed a 118% increase in t-PA and a 50% decrease in PAI-1 antigen levels from 10-day tissue cultures. RT--PCR demonstrated that the mRNA encoding t-PA increased, while PAI-1 decreased after 10 days of culture. In conclusion, venous culture showed an up-regulation of t-PA and a repression of PAI-1 gene expression during SMC proliferation in the vessel wall. The PAI-1 repression observed in venous culture is in contrast to the situation observed in human atheroma. A shift in the t-PA/PAI-1 balance may have a role in vascular remodeling.

Molecular maturation of cell adhesion systems during mouse early development.

During cleavage, the mouse embryo expresses a variety of cell adhesion systems on its cell surfaces. We have reviewed biogenetic and assembly criteria for the formation of the uvomorulin/catenin, tight junction and desmosome adhesion systems as the trophectoderm differentiates. Each system reveals different mechanisms regulating molecular maturation. Adhesion processes contribute to the generation of distinct tissues in the blastocyst by modifying the expression pattern of blastomeres entering the non-epithelial inner cell mass lineage. Cell adhesion also influences the spatial organisation, but rarely the timing of expression, of proteins involved in trophectoderm differentiation.