Influence of triacontanol on somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L. and <Emphasis Type="Italic">Coffea canephora</Emphasis> P. ex Fr.

Summary A highly reproducible method for regeneration of Coffea arabica and C. canephora plants via direct somatic embryogenesis from cultured leaf and stem segments of regenerated plants was developed. Embryogenesis was influenced by the presence of triacontanol (TRIA) in the medium. TRIA incorporated at 4.55 and 11.38 μM in half-strength MS basal medium containing 1.1 μM 6-benzyladenine (BA) and 2.28 μM indole-3-acetic acid (IAA) induced direct somatic embryogenesis in both species. A maximum of 260±31.8 and 59.2±12.8 somatic embryos per culture were induced from in vitro leaf explants of C. arabica and C. canephora, respectively. TRIA also induced embryo formation from in vitro stem segment callus tissues along with multiplication of primary embryos into secondary embryos. By using TRIA, it was possible to obtain somatic embryogenesis in C. arabica and C. canephora.     

Recognition of PSA-derived peptide antigens by T cells from prostate cancer patients without any prior stimulation

Prostate-specific antigen (PSA) is a valuable marker antigen for prostate cancer. Lately considerable interest has been generated in the prospect of developing a vaccine for prostate cancer with PSA-derived peptide epitopes to induce cytotoxic T-cell (CTL) response. We report here that T cells capable of exhibiting PSA epitope-specific effector function-in their native state, i.e, without having to be further stimulated, in vitro-are detectable in more than half of the prostate cancer patients we studied. Ex vivo cultured autologous dendritic cells (DC) were used to present four HLA-A2-binding PSA peptide epitopes to freshly isolated peripheral blood lymphocytes (PBL) from patients and healthy volunteers. Ten out of 14 patients' PBL recognized at least one of the four peptides and 6 out of 10 patients' PBL recognized more than one peptide antigen as measured by IFN-gamma secretion upon stimulation of the PBL with the peptide antigen. Intracytoplasmic cytokine analysis for IFN-gamma in purified CD8(+) cells after stimulation with peptide antigens was tested in 6 patients and this technique demonstrated a similar response. Freshly isolated and purified CD8(+) cells when tested, also recognized the epitopes, as measured by IFN assay, when presented by transporter associated with antigen-processing (TAP) deficient T2 cells in an MHC-I restricted fashion. PBL from 9 normal donors when tested in identical fashion did not show any IFN-gamma production in recognition to the peptide antigens. Interestingly, neither of these CD8(+) T cells having IFN-gamma-producing ability did show any cytolytic activity in their native state against peptide loaded target cells or tumor cells when tested in cytotoxicity assay. In long term cocultures stimulation of purified CD8(+) T cells with matured DC pulsed with PSA peptides generated a PSA-specific CTL response in 4 of 6 patients studied and in 2 of 9 normal donors. While our observations of CTL generation are consistent with the prior reports that have demonstrated that specific CD8(+) CTL could be generated which recognize PSA-derived epitopes by in vitro stimulation by one means or another, this observation that IFN-gamma-producing CD8(+) T cells are present in patients which are antigen experienced, and do not require in vitro stimulation, is novel and has major implications for prostate cancer vaccine preparation.     

Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8

Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.     

Tumor necrosis factor induces apoptosis in hepatoma cells by increasing Ca(2+) release from the endoplasmic reticulum and suppressing Bcl-2 expression

Tumor necrosis factor (TNF) plays an import role in the control of apoptosis. The most well known apoptotic pathway regulated by TNF involves the TNFR1-associated death domain protein, Fas-associated death domain protein, and caspase-8. This study examines the mechanism of TNF-induced apoptosis in FaO rat hepatoma cells. TNF treatment significantly increased the percentage of apoptotic cells. TNF did not activate caspase-8 but activated caspase-3, -10, and -12. The effect of TNF on the expression of different members of the Bcl-2 family in these cells was studied. We observed no detectable changes in the steady-state levels of Bcl-X(L), Bax, and Bid, although TNF suppresses Bcl-2 expression. Dantrolene suppressed the inhibitory effect of TNF on Bcl-2 expression. TNF induced release of Ca(2+) from the endoplasmic reticulum (ER) that was blocked by dantrolene. Importantly, the expression of Bcl-2 blocked TNF-induced apoptosis and decreased TNF-induced Ca(2+) release. These results suggest that TNF induces apoptosis by a mechanism that involves increasing Ca(2+) release from the ER and suppression of Bcl-2 expression.     

Influence of Antioxidants on the Peroxidative Swelling of Mitochondria in vitro

To clarify the role of prooxidative processes during in vitro swelling of freshly isolated rat liver mitochondria, the influence of different antioxidants and free-radical scavengers was tested. Ascorbate below 10 mmol/L without externally added Fe²⁺ acted as a prooxidant and enhanced swelling. Higher concentrations in the presence of Fe²⁺ showed antioxidant properties and a decrease in swelling and lipid peroxidation. Swelling was abolished by -tocopherol and reduced to 50% by butylated hydroxytoluene. Glutathione supplementation decreased both swelling and lipid peroxidation. Oxidized glutathione caused swelling without any effect on peroxidation. Hydrogen peroxide, cumene hydroperoxide and t-butyl hydroperoxide caused progressive decreases in glutathione and reduced niacinamide coenzyme levels, suggesting prooxidative changes. Dithiothreitol was found to abolish this effect. Thus, antioxidants reverse superoxide-induced mito chondrial swelling and lipid peroxidation in vitro.     

Activated charcoal filter effectively reduces p-benzosemiquinone from the mainstream cigarette smoke and prevents emphysema

In this paper, we have made a comparative evaluation of the cytotoxicity and pathophysiological effects of mainstream smoke from cellulose acetate (CA)-filtered cigarettes with that of charcoal-filtered cigarettes developed in our laboratory. Previously, we had demonstrated that the mainstream smoke from an Indian CA-filtered commercial cigarette contains p-benzosemiquinone (p-BSQ), a major, highly toxic, long-lived water-soluble radical. Here, we have examined 16 brands of different CA-filtered cigarettes including Kentucky research cigarettes, and observed that mainstream smoke from all the cigarettes contains substantial amounts of p-BSQ (100–200 μg/cigarette). We also show that when the CA filter is replaced by a charcoal filter, the amount of p-BSQ in the mainstream smoke is reduced by 73–80%, which is accompanied by a reduction of carbonyl formation in bovine serum albumin to the extent of 70–90%. The charcoal filter also prevented cytotoxicity in A549 cells as evidenced by MTT assay, apoptosis as evidenced by FACS analysis, TUNEL assay, overexpression of Bax, activation of p53 and caspase 3, as well as emphysematous lung damage in a guinea pig model as seen by histology and morphometric analysis. The results indicate that the charcoal filter developed in our laboratory may protect smokers from cigarette smoke-induced cytotoxity, protein modification, apoptosis and emphysema.     

Isolation and characterization of alkalotolerant bacteria and optimization of process parameters for decolorization and detoxification of pulp and paper mill effluent by Taguchi approach

Four different bacterial strains were isolated from pulp and paper mill sludge in which one alkalotolerant isolate (LP1) having higher capability to remove color and lignin, was identified as Bacillus sp. by 16S RNA sequencing. Optimization of process parameters for decolorization was initially performed to select growth factors which were further substantiated by Taguchi approach in which seven factors, % carbon, % black liquor, duration, pH, temperature, stirring and inoculum size, at two levels, applying L-8 orthogonal array were taken. Maximum color was removed at pH 8, temperature 35°C, stirring 200 rpm, sucrose (2.5%), 48 h, 5% (w/v) inoculum size and 10% black liquor. After optimization 2-fold increase in color and lignin removal from 25–69% and 28–53%, respectively, indicated significance of Taguchi approach in decolorization and delignification of lignin in pulp and paper mill effluent. Enzymes involved in the process of decolorization of effluent were found to be xylanase (54 U/ml) and manganese peroxidase (28 U/ml). Treated effluent was also evaluated for toxicity by Comet assay using Saccharomyces cerevisiae MTCC 36 as model organism, which indicated 58% reduction after treatment by bacterium.     

Changes in carbonic anhydrase may be the initial step of altered metabolism in hypertension

Carbonic anhydrase (CA) is a well characterized pH regulatory enzyme in most of the tissues in the body. Changes in activities of CA have been associated with altered metabolism, especially in diabetes mellitus. Insulin resistance and hyperinsulinemia are common in hypertension. To investigate the possible role of CA, we measured the CA activity spectrophotometrically using p-nitrophenyl acetate as a substrate and acetazolamide, the specific inhibitor, in erythrocytes from normotensive and essential hypertensive subjects. Further, to evaluate the insulin action on CA, we used two different hemolysates; (i) insulin applied into hemolysate and (ii) hemolysate from insulin treated erythrocytes in vitro before the determination of CA activity. Two different levels of CA activities were obtained in these patients. CA activities were much lower (mean +/- SD, 0.88 +/- 0.19 U/min/mL) and higher (mean +/- SD, 1.77 +/- 0.23 U/min/mL) in patients than the normotensive controls (mean +/- 1 SD, 1.41 +/- 0.1 U/min/mL). These differences in both the groups were statistically significant (p less than 0.001). Similarly, total esterase activities in patients were (1.41 +/- 0.27 U/min/mL) that was 30% less in low activity group and (2.47 +/- 0.25 U/min/mL) that was 22% more in higher activity group in comparison with those from normotensives (2.02 +/- 0.17 U/min/mL). The relative percent of CA activities of insulin treated erythrocytes from normotensives and hypertensives were 11% and 18% higher than without insulin (p less than 0.05). No difference was observed when insulin was applied in the hemolysate. We conclude that essential hypertensive patients are associated with altered CA activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disruption of Elements Uptake due to Excess Chromium in Indian Medicinal Plants

Chromium and its compounds may cause disturbance in the nutrient level of the plants. Iron, manganese, copper, and zinc are essential nutrient elements and required for balanced growth and development of plants, but chromium uptake sometimes disturbed their concentration in plants. Therefore, in the present paper, an effort has been made to observe the effect of different levels of Cr on nutrient uptake of Phyllanthus amarus and Solanum nigrum, the medicinally important plants of indigenous systems of medicine having hepatoprotective and diuretic properties. The study revealed that Cr causes significant changes in nutrient uptake as compared to control plants. Besides, Cr-treated plants showed growth depression and decrease in fresh and dry weight too. With the increase in Cr supply, accumulation of Cr in roots was increased significantly. Concentration of manganese and zinc was also increased. However, copper concentration in both the plants seemed less affected by Cr.