Hydroxyl radical formation in chondrocytes and cartilage as detected by electron paramagnetic resonance spectroscopy using spin trapping reagents

Chondrocytes have been shown to produce superoxide and hydrogen peroxide, suggesting possible formation of hydroxyl radical in these cells. In this study, we used electron spin resonance/spin trapping technique to detect hydroxyl radicals in chondrocytes. We found that hydroxyl radicals could be detected as α-hydroxyethyl spin trapped adduct of 4-pyridyl 1-oxide N-tert-butylnitrone (4-POBN) in chondrocytes stimulated with phorbol 12-myristate 13-acetate in the presence of ferrous ion. The formation of hydroxyl radical appears to be mediated by the transition metal-catalyzed Haber-Weiss reaction since no hydroxyl radical was detected in the absence of exogenous iron. The hydroxyl radical formation was inhibited by catalase but not by superoxide dismutase, suggesting that the hydrogen peroxide is the precursor. Cytokines, IL-1 and TNF enhanced the hydroxyl radical formation in phorbol 12-myristate 13-acetate treated chondrocytes. Interestingly, hydroxyl radical could be detected in unstimulated fresh human and rabbit cartilage tissue pieces in the presence of iron. These results suggest that the formation of hydroxyl radical in cartilage could play a role in cartilage matrix degradation.     

Cupincin: A Unique Protease Purified from Rice (Oryza sativa L.) Bran Is a New Member of the Cupin Superfamily

Cupin superfamily is one of the most diverse super families. This study reports the purification and characterization of a novel cupin domain containing protease from rice bran for the first time. Hypothetical protein OsI_13867 was identified and named as cupincin. Cupincin was purified to 4.4 folds with a recovery of 4.9%. Cupincin had an optimum pH and temperature of pH 4.0 and 60°C respectively. Cupincin was found to be a homotrimer, consisting of three distinct subunits with apparent molecular masses of 33.45 kDa, 22.35 kDa and 16.67 kDa as determined by MALDI-TOF, whereas it eluted as a single unit with an apparent molecular mass of 135.33 ± 3.52 kDa in analytical gel filtration and migrated as a single band in native page, suggesting its homogeneity. Sequence identity of cupincin was deduced by determining the amino-terminal sequence of the polypeptide chains and by and de novo sequencing. For understanding the hydrolysing mechanism of cupincin, its three-dimensional model was developed. Structural analysis indicated that cupincin contains His313, His326 and Glu318 with zinc ion as the putative active site residues, inhibition of enzyme activity by 1,10-phenanthroline and atomic absorption spectroscopy confirmed the presence of zinc ion. The cleavage specificity of cupincin towards oxidized B-chain of insulin was highly specific; cleaving at the Leu₁₅-Tyr₁₆ position, the specificity was also determined using neurotensin as a substrate, where it cleaved only at the Glu₁-Tyr₂ position. Limited proteolysis of the protease suggests a specific function for cupincin. These results demonstrated cupincin as a completely new protease.     

Chemical composition and fungitoxic activity of essential oil of Thuja orientalis L. grown in the north-western Himalaya

The essential oil from fresh leaves of Thuja orientalis L. grown in the north-western Himalaya was isolated by means of hydrodistillation and analyzed by GC and GC/MS. Twenty-two compounds representing 94.0% of the total oil were identified. The leaf oil contained alpha-pinene (29.2%), Delta-3-carene (20.1%), alpha-cedrol (9.8%), caryophyllene (7.5%), alpha-humulene (5.6%), limonene (5.4%), alpha-terpinolene (3.8%) and alpha-terpinyl acetate (3.5%) as major constituents. The essential oil showed antifungal activity against Alternaria alternata in a direct bioautography assay. Two main bioactive compounds named as b1 (Rf = 0.54) and b2 (Rf = 0.80) were observed and tested for antifungal activity; they produced an inhibition zone of 5 and 10 mm in diameter, respectively. The components b1 and b2 were further purified by preparative thin layer chromatography and their antifungal efficacy was re-tested. The minimum inhibitory amount (MIA) of b1 and b2 against A. alternata was determined as 30.5 and 4.5 microg, respectively, using a bioautography assay. The bioactive constituent corresponding to b1 was determined as alpha-cedrol by using GC/MS analysis. The potential of essential oils as a source of natural biocides is discussed.     

Comparison of the Abiotic Preferences of Macroinvertebrates in Tropical River Basins

We assessed and compared abiotic preferences of aquatic macroinvertebrates in three river basins located in Ecuador, Ethiopia and Vietnam. Upon using logistic regression models we analyzed the relationship between the probability of occurrence of five macroinvertebrate families, ranging from pollution tolerant to pollution sensitive, (Chironomidae, Baetidae, Hydroptilidae, Libellulidae and Leptophlebiidae) and physical-chemical water quality conditions. Within the investigated physical-chemical ranges, nine out of twenty-five interaction effects were significant. Our analyses suggested river basin dependent associations between the macroinvertebrate families and the corresponding physical-chemical conditions. It was found that pollution tolerant families showed no clear abiotic preference and occurred at most sampling locations, i.e. Chironomidae were present in 91%, 84% and 93% of the samples taken in Ecuador, Ethiopia and Vietnam. Pollution sensitive families were strongly associated with dissolved oxygen and stream velocity, e.g. Leptophlebiidae were only present in 48%, 2% and 18% of the samples in Ecuador, Ethiopia and Vietnam. Despite some limitations in the study design, we concluded that associations between macroinvertebrates and abiotic conditions can be river basin-specific and hence are not automatically transferable across river basins in the tropics.     

Immunobiological function of normal rabbit synovial cells

The ability of enzyme-dissociated primary cultures of synovial cells (Sy) to present antigen was investigated. Adult rabbits were immunized in foot pads with bovine serum albumin (BSA) in complete Freund's adjuvant (CFA) or with CFA alone. Four to six weeks later draining popliteal lymph node cells (LNC) and synovial cells were obtained. Synovial cells were cultured overnight with or without antigen. About 20% of these synovial cells had Fc receptors and 15% C3 receptors. As a positive control splenic adherent cells (SAC) were similarly treated. Next day, autologous lymph node cells were added to the extensively washed and irradiated synovial cells or splenic adherent cells. Lymphocyte proliferation was measured by [3H]thymidine uptake. Synovial cells as well as splenic adherent cells induced mixed lymphocyte reaction (MLR) and autologous mixed lymphocyte reaction (AMLR) and effectively presented antigen for specific immune response to the priming antigen. Thus, the synovium contains macrophage-like cells that can effectively interact with lymphocytes and participate in the immune phenomena in the joints of patients with rheumatoid arthritis.     

Testing Linear Contrasts of Means in Experimental Design Without Assuming Normality and Homogeneity of Variances

We investigate the efficiencies of TIKU'S (1967, 1980) modified maximum likelihood (MML) estimators of location and scale parameters of symmetric distributions and show that they are remarkably efficient (jointly). We develop test statistics (based on MML estimators), analogous to the classical tests based on sample means and variances, for testing the equality of two means (the population variances not necessarily equal). We show that these tests are remarkably robust to distributional assumptions and generally more powerful than the well-known nonparametric tests (WILCOXON , normal-score, KOLMOGOROV -SMIRNOV ). We generalize the results to testing linear contrasts of means in experimental design (the error variances not necessarily equal). We show that the analogous tests based on ‘adaptive’ robust estimators (wave, bisquare, HAMPEL ,) etc., GROSS (1976, and other ‘adaptive’ robust estimators) give misleading Type I errors.     

Salinity tolerance within the grass speciesAgrostis stolonifera L

Summary Eight populations ofAgrostis stolonifera, collected from maritime and inland habitats, were grown in solution culture and in sand culture at various concentrations of sodium chloride from 0 ppm to 5,000 ppm Na. NaCl had less effect upon the dry weight yield of populations from maritime habitats, with soils of high Na content, than upon populations from inland or maritime habitats with soils of low Na content. The correlation between per cent reduction in dry weight yield and the Na content of the native soil of each population was r=−0.73. Populations from inland or maritime habitats, with soils of low Na content, contained more Na in their shoot materials, and higher Na/K ratios, than did populations from high Na soils, at all Na concentrations. The effect of NaCl upon the root elongation of each population was not correlated with the effect upon dry weight yield, nor with the Na content of their native soil. The effect of Na Cl upon root elongation was highly dependent upon the composition of the culture solution used. It is concluded that the effect of NaCl on root elongation is not an adequate measure of salinity tolerance in this species.     

Establishment ofEncarsia(=Prospaltella) perniciosi,a specific parasite of San Jose scale,on apple in Kashmir

Attempts were made to reduce populations of San Jose scaleQuadraspidiotus perniciosus (Comstock) by releasing populations of the parasiteEncarsia (=Prospaltella) perniciosi Tower. Several releases were made, between May and November in both 1985 and 1986. TheEncarsia were released into 2 apple orchards infested with scale. Increases in the numbers ofEncarsia caught on sticky traps in these orchards, coupled with an increase from 2.3% to 9.1% in the overall level of parasitism of the scales, suggested that the released parasites had become established. Other natural enemies found associated with San Jose scale on apple in Kashmir included 3 parasites (Marietta carnesi Howard,Azotus kashmirensis Narayanan andAphytis paramaculicornis De Bach) and 5 predators, 4 of which were coccinellids (Chilocorus bijugus Muls,Pharoscymus flexibilis, Exochomus sp. andCoccinella septempunctata) and the remaining one was a chrysopid (Chrysoperla carnea).