Pressure-induced leukocyte margination in lung postcapillary venules

Although pressure elevation in lung postcapillary venules increases endothelial P-selectin expression, the extent to which P-selectin causes lung leukocyte margination remains controversial. To address this issue, we optically viewed postcapillary venules of the isolated blood-perfused rat lung by real-time fluorescence imaging. To determine leukocyte margination in single postcapillary venules, we quantified the fluorescence of leukocytes labeled in situ with rhodamine 6G (R6G). Although baseline fluorescence was sparse, a 10-min pressure elevation by 10 cmH(2)O markedly increased R6G fluorescence. Both stopping blood flow during pressure elevation and eliminating leukocytes from the perfusion blocked the fluorescence increase, affirming that these fluorescence responses were attributable to pressure-induced leukocyte margination. A P-selectin-blocking MAb and the L- and P-selectin blocker fucoidin each inhibited the fluorescence increase, indicating that P-selectin was critical for inducing margination. Time-dependent imaging of blood-borne fluorescent beads revealed reduction of plasma velocity during pressure elevation. After pressure returned to baseline, a similar reduction of plasma velocity, established by manually decreasing the perfusion rate, prolonged margination. Our findings show that in lung postcapillary venules, the decrease in plasma velocity critically determines pressure-induced leukocyte margination.     

Cytochrome P4502D6(193-212): a new immunodominant epitope and target of virus/self cross-reactivity in liver kidney microsomal autoantibody type 1-positive liver disease

Cytochrome P4502D6 (CYP2D6), target of liver kidney microsomal autoantibody type 1 (LKM1), characterizes autoimmune hepatitis type 2 (AIH2) but is also found in patients with chronic hepatitis C virus (HCV) infection. To provide a complete linear epitope B cell map of CYP2D6, we tested peptides spanning the entire sequence of CYP2D6. In addition to confirming previously described antigenic sites, we identified four new epitopes (193-212, 238-257, 268-287, and 478-497). CYP2D6(193-212) is immunodominant and was the target of 12 of 13 (93%) patients with AIH2 and 5 of 10 (50%) HCV/LKM1-positive patients. Because LKM1 is present in both AIH2 and a viral infection, we tested whether Abs to CYP2D6(193-212) arise through cross-reactive immunity between virus and self. We identified a hexameric sequence "RLLDLA" sharing 5 of 6 aa with "RLLDLS" of HCV(2985-2990) and all 6 aa with CMV(130-135). Of 17 CYP2D6(193-212)-reactive sera, 11 (7 AIH and 4 HCV) reacted by ELISA with the HCV homologue, 8 (5 AIH and 3 HCV) with the CMV homologue, and 8 (5 AIH and 3 HCV) showed double reactivity. Autoantibody binding to CYP2D6(193-212) was inhibited by preincubation with HCV(2977-2996) or CMV(121-140). Recombinant HCV-nonstructural protein 5 and CMV-UL98 proteins also inhibited Ab binding to CYP2D6(193-212). Affinity-purified CYP2D6(193-212)-specific Ab inhibited the metabolic activity of CYP2D6. The demonstrated similarity and cross-reactivity between CYP2D6(193-212) and two unrelated viruses suggests that multiple exposure to viruses mimicking self may represent an important pathway to the development of autoimmunity.     

Why is trehalose an exceptional protein stabilizer? An analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose

Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 m trehalose was observed to raise the transition temperature, Tm of RNase A by as much as 18 degrees C and Gibbs free energy by 4.8 kcal mol-1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (DeltaCp) in trehalose solutions for all the studied proteins. An increase in the DeltaG and a decrease in the DeltaCp values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (DeltaG versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 m concentration 4-7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.     

Comparison of the prevention of aflatoxin b(1)-induced genotoxicity by quercetin and quercetin pentaacetate

Earlier work carried out in our laboratory highlighted the mode of action of acetoxy 4-methylcoumarins in preventing the genotoxicity of aflatoxin B(1) (AFB(1)). We have in this report extended the observations to quercetin pentaacetate (QPA), which unlike quercetin (Q) has demonstrated time-dependent inhibition of liver microsome catalysed AFB(1) epoxidation as measured by AFB(1) binding to DNA. The action of QPA is similar to that of the acetoxy 4-methylcoumarins in that they are acted upon by microsomal transacetylase leading to modulation of catalytic activities of certain enzymes (such as P-450 enzymes, NADPH cytochrome C reductase and glutathione S-transferase) possibly by way of protein acetylation. In the present work, we have documented the transacetylase-mediated action of QPA in preventing genotoxicity due to AFB(1).     

Subcellular localization of LGN during mitosis: evidence for its cortical localization in mitotic cell culture systems and its requirement for normal cell cycle progression

Mammalian LGN/AGS3 proteins and their Drosophila Pins orthologue are cytoplasmic regulators of G-protein signaling. In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in their asymmetric division. Using overexpression studies in different cell line systems, we demonstrate here that, like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used. We find that in WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis. Overexpression of truncated protein domains further identified the Galpha-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture. In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Galpha subunits of heterotrimeric G-proteins. The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression.     

Involvement of reactive oxygen species in gastric ulceration: protection by melatonin

Uncontrolled hydrochloric acid secretion and ulceration in the stomach due to various factors are serious global problems today. Although the mechanism of acid secretion from the parietal cell is now fairly known, the mechanism of gastric ulceration is still not clear today. Among various causes of gastric ulceration, lesions caused by stress, alcohol consumption, Helicobacter pylori infection and use of nonsteroidal antiinflammatory drugs have been shown to be mediated largely through the generation of reactive oxygen species especially hydroxyl radical (*OH). A number of excellent drugs have been proved useful in controlling hyperacidity and ulceration but their long term uses are not devoid of disturbing side-effects. Hence, the search is still on to find out a compound possessing antisecretory, antiulcer and antioxidant properties which will serve as a powerful therapeutic agent to cure gastric hyperacidity and ulcer. This article describes the role of reactive oxygen species in gastric ulceration, drugs controlling them with their merits and demerits and, the role of melatonin, a pineal hormone in protecting the gastric lesions with a final commentary on how melatonin research with respect to gastric pathophysiology can be taken forward with a view to projecting this indole as a promising therapeutic agent to control gastric ulceration in humans.     

Age trends in anthropometric characteristics among 6-9 years old Bengalee Hindu school girls of Kolkata, India

A cross-sectional study of 431 6-9 years old urban Bengalee Hindu schoolgirls of Kolkata, India, was undertaken to study age trends in anthropometric characteristics including regional and subcutaneous adiposity. The anthropometric variables measured included height, weight, sitting height (SH), waist (WC), hip (HPC), thigh (TC), mid-upper arm (MUAC) and medial calf (MC) circumferences as well as triceps (TSF), biceps (BSF), subscapular SUBSF), suprailliac (SUPSF) and medial calf (MCASF) skinfolds. The results revealed, that there was a significant increasing age trend for all the anthropometric variables including the two derived variables: body mass index (BMI) and subischial leg length (SLL). For all variables, the lowest and the highest means were observed at the age of 6 and 9 years, respectively. The maximum increase in weight, BMI, all linear measurements, WC and HPC were observed during the period 6-7 years of age. In general, all skinfolds recorded similar yearly increments. More importantly, this study clearly indicated that among Bengalee girls aged 6-9 years, the highest amount of linear growth (height, SH and SLL) was observed at 6 years of age. The overall adiposity (BMI) also recorded the maximum increment during this period. The unique data presented here can be used as reference values for urban Bengalee Hindu girls aged 6-9 years.     

Physical exercise, body mass index, subcutaneous adiposity and body composition among Bengalee boys aged 10-17 years of Kolkata, India

A comparative study of 215 sedentary (no regular physical exercise undertaken) and 313 physically active (regular physical exercise undertaken) Bengalee boys aged 10-17 years was undertaken to investigate the differences in overall adiposity (body mass index), subcutaneous adiposity (skinfolds) and body composition (percent body fat, fat mass and fat mass index). Both groups had a similar age. The results revealed that boys who did not undertake regular physical exercise (NPE) had a significantly greater mean body mass index (BMI) compared with those who undertook regular physical exercise (PE); p < 0.001. The means for all the skinfolds as well as percent body fat (PBF), fat mass (FM) and fat mass index (FMI) were significantly higher among the NPE group. The percentile distributions of all these variables and indices were consistently higher among the NPE group. The results of ANOVA of physical exercise (PE = yes, NPE = no) and PBF, FM and FMI, with age as covariate, revealed that PE had a significant negative effect on all these measures of body composition even after controlling for the impact of age. The means in each case were greater among the NPE group. In conclusion, this study provided evidence that Bengalee boys, who undertook regular physical exercise, had significantly less adiposity compared with those who did not undertake regular physical exercise.