Phytoremediation potential of chromium-containing tannery effluent-contaminated soil by native Indian timber-yielding tree species

Twenty-six native Indian tree species that are used for the enhanced tree cover program of the forest department (Government of Tamilnadu, India) were screened for phytoremediation of tannery effluent-contaminated soil containing high chromium content. Out of 26 tree species tested, 10 timber-yielding tree species were selected for further phytoremediation monitoring. After a series of treatments with tannery effluent sludge, the chromium content was measured in the plant parts. The saplings of Acacia auriculiformis, Azadirachta indica, Albizzia lebbeck, Dalbergia sisso, and Thespesia populnea were identified as efficient bioaccumulators of chromium from Cr-contaminated soil. Acacia auriculiformis accumulates higher amounts of Cr in both the root and stem. Dalbergia sisso and T. populnea were found to accumulate higher quantity of Cr in the roots, whereas A. indica, A. richardiana, and A. lebbeck accumulate Cr in their stem. The stress response of the plant species was assessed by quantifying the antioxidative enzymes such as catalase, superoxide dismutase, glutathione reductase, and DHAR. Activity of all the enzymes was observed to gradually increase following treatment with tannery effluent sludge.     

Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization

Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they share features of both PTKs and STKs in the core. Finally, our studies provide an evolutionary framework for identifying and characterizing disease and drug resistance mutations in the kinase core.     

The Carboxyl-terminal Domain of Atypical Protein Kinase C�� Binds to Ceramide and Regulates Junction Formation in Epithelial Cells

Atypical protein kinase Cs (PKCs) (aPKCζ and λ/ι) have emerged as important binding partners for ceramide, a membrane-resident cell signaling lipid that is involved in the regulation of apoptosis as well as cell polarity. Using ceramide overlay assays with proteolytic fragments of PKCζ and vesicle binding assays with ectopically expressed protein, we show that a protein fragment comprising the carboxyl-terminal 20-kDa sequence of PKCζ (C20ζ, amino acids 405–592) bound to C16:0 ceramide. This sequence is not identical to the C1 domain (amino acids 131–180), which has been suggested to serve as a potential ceramide binding domain. Using immunocytochemistry, we found that a C20ζ protein fragment ectopically expressed in two epithelial cell types (neural progenitors and Madin-Darby canine kidney cells) co-distributed with ceramide. Stable expression of C20ζ-EGFP in Madin-Darby canine kidney cells disrupted the formation of adherens and tight junctions and impaired the epithelium integrity by reducing transepithelial electrical resistance. Disruption of cell adhesion and loss of transepithelial electrical resistance was prevented by incubation with C16:0 ceramide. Our results show, for the first time, that there is a novel ceramide binding domain (C20ζ) in the carboxyl terminus of aPKC. Our results also show that the interaction of ceramide with this binding domain is essential for cell-to-cell contacts in epithelia. Therefore, ceramide interaction with the C20ζ binding domain is a potential mechanism by which ceramide and aPKC regulate the formation of junctional complexes in epithelial cells.Epithelial cells play essential roles in multicellular organisms by forming physiological and mechanical barriers and controlling tissue architecture, because they acquire apicobasal and cell-to-cell (planar) polarity (1, 2). Adherens junctions (AJs)² and tight junctions (TJs) are major structures responsible for cell-to-cell adhesion in epithelial cells (3). The regulation of junction formation requires endocytosis, redistribution, and recycling of junctional proteins, such as E-cadherin (4), and ZO-1. Many factors, including EGF, EGFR, Src kinase, Rho family GTPases Cdc42 and Rac1, and atypical PKC (aPKC), have been found to regulate junction formation (5–9). In Madin-Darby canine kidney (MDCK) cells, Cdc42 modulates AJs by regulating E-cadherin ubiquitination and degradation (9), whereas aPKC directly localized at TJs is required for the asymmetric differentiation of the premature junction complex during epithelial cell polarization (1, 10).The protein kinase C (PKC) family comprises serine/threonine kinases, which consist of a carboxyl-terminal catalytic domain and an amino-terminal regulatory domain (Fig. 1A). The regulatory domain includes an inhibitory pseudosubstrate domain and allosteric sites for activation by phosphatidylserine and, depending on the isoform, calcium (C2 domain) and/or diacylglycerol (C1 domain). aPKC is a subfamily of PKC, which consists of the isoforms ζ and λ/ι. The aPKC isoforms contain only half of the C1 domain, and hence, their activity is not affected by calcium or diacylglycerol/phorbol esters (see Fig. 1A and Refs. 11–13).Open in a separate windowFIGURE 1.Binding of ceramide to the COOH terminus of PKCζ. A, primary structure of aPKC, the caspase 3 proteolytic fragment ζCasp II, and the NH₂-terminal deletion mutant C20ζ-EGFP. B, 2 μg of recombinant His-tagged PKCζ was proteolytically digested by 20 ng of recombinant caspase 3. Proteolysis by caspase 3 occurred first after amino acid 239 (4-h incubation) and then after amino acid 459 (10-h incubation, ζCasp II). C, binding to ceramide spotted on nitrocellulose (overlay assay). FL PKCζ and the COOH-terminal proteolytic fragment ζCasp II bound to C16 ceramide. D, C16 ceramide vesicle binding assay (LIMAC). Ectopically expressed C20ζ-EGFP prepared from a cell lysate was bound to ceramide vesicles; EGFP was not. Protein was detected using anti-aPKC and anti-GFP antibodies. Lanes 1–3, loading input for ceramide vesicles; lanes 4–6, eluate of vesicle binding columns (output). Lanes 7 (input) and 8 (output) show that PKCζ-EGFP did not bind to vesicles prepared with sphingomyelin (SM) instead of ceramide. E, subcellular fractionation of cells expressing FL PKCζ-EGFP or C20ζ-EGFP.Apart from its function in apoptosis (13–15) and cell growth (16), aPKC has been found to play a pivotal role in cell polarity, both in neuroepithelial cells (17–20) or other epithelial cell types (1, 10). Consistently, the gene knock-out of aPKC shows loss of cell junction formation and detachment of neural progenitor cells from the neuroepithelium (8, 21–23). We and others have found that the sphingolipid ceramide activates aPKC, recruits it to structured microdomains, and regulates cell polarity and motility (24–28). Using lipid vesicle-mediated affinity chromatography (LIMAC) assays, we showed for the first time that ceramide directly binds to aPKC (25). Yet which domain of aPKC binds to ceramide is not known.Using lipid overlay and LIMAC assays, we show here that a COOH-terminal 20-kDa domain of PKCζ (C20ζ) binds to ceramide. Similar to its full-length counterpart, the C20ζ protein fragment resides in cellular membranes, where it co-distributes with ceramide in both C17.2 (neural progenitor) and MDCK cells. To study the function of this ceramide binding domain, we established a stably transfected MDCK cell line expressing C20ζ-EGFP. In these cells, the protein level of E-cadherin is reduced, and the cellular distribution of E-cadherin, ZO-1, and β-catenin is disrupted when compared with EGFP-transfected cell lines. Further, transepithelial electrical resistance (TER) assays show that the C20ζ-EGFP cell line has reduced impedance when compared with the control cell line expressing EGFP. This finding suggests that the C20ζ protein fragment is a dominant negative mutant of PKCζ. The effects of this dominant negative mutant can be, at least partially, rescued by incubation with C16:0 ceramide, suggesting that ceramide regulates aPKC and aPKC-dependent cell junction formation by interaction with the COOH-terminal domain.     

T-h-2 immunity and CD3+CD45RBlow-activated T cells in mice immunized with recombinant bacillus Calmette-Guérin expressing HIV-1 principal neutralizing determinant epitope

The genetic engineering of Mycobacterium bovis-bacillus Calmette-Guérin to express foreign epitopes is an attractive strategy in the field of epitope vaccines. We constructed an 'epitope-trap vector' with Mycobacterium tuberculosis chaperonin-10 as a carrier antigen and used it to express the HIV-1 principal neutralizing determinant epitope. We also identified a new chaperonin-10 promoter that was hyperexpressive compared with the heat shock protein-65 promoter. Splenocytes from recombinant bacillus Calmette-Guérin-immunized mice showed enhanced lymphocyte proliferation and interleukin-4 (but not interferon-gamma) secretion. The recombinant bacillus Calmette-Guérin-immunized group also exhibited mild delayed-type hypersensitivity reaction and a high frequency of CD3+CD45RBlow-activated T cells, together with high titer of antiprincipal neutralizing determinant immunoglobulin G antibodies. Thus, this epitope delivery system induced strong epitope-specific T-h-2 polarization.     

Two generation reproduction study of ethylbenzene by inhalation in Crl-CD rats

BACKGROUND: This study was conducted to evaluate the potential adverse effects of ethylbenzene (EB) on reproductive capability from whole-body inhalation exposure of F0 and F1 parental animals. METHODS: Four groups of Crl:CD(SD)IGS BR rats (30/sex/group for F0 and 25/sex/group for F1) were exposed to 0, 25, 100, and 500 ppm EB for 6 hr/day for at least 70 consecutive days before mating. Inhalation exposure for the F0 and F1 females continued throughout mating, gestation through gestation day (GD) 20, and lactation days (LD) 5-21. On LD 1-4, females received EB in corn oil via oral gavage at dose levels of 26, 90, and 342 mg/kg/day (divided into three equal doses, approximately 2 hr apart), as calculated from a physiologically-based pharmacokinetic (PBPK) model to provide similar maternal blood area-under-concentration (AUC) as provided by inhalation. Pups were weaned on postnatal day (PND) 21 and exposure of the F1 generation started on PND 22. Estimates of internal exposure were determined by measuring EB concentrations in blood collected from F1 dams (4/group) and their culled pups 1 hr after the last gavage dose on PND 4. On PND 22, blood was collected from these same F1 dams and their weanlings for EB analysis 1 hr after a 6-hr inhalation exposure. The remainder of the F2 generation was not directly exposed. RESULTS: EB exposure did not affect survival or clinical observations. Male rats in the 500 ppm group in both generations gained weight more slowly than the controls. There were no indications of adverse effects on reproductive performance in either generation. Male and female mating and fertility indices, pre-coital intervals, spermatogenic endpoints, ovarian follicle counts, reproductive organ weights, lengths of estrous cycle and gestation, live litter size, pup weights, developmental landmarks, and postnatal survival were unaffected. No adverse exposure-related macroscopic pathology was noted at any level. CONCLUSIONS: Increased liver weights were found in the animals exposed to 500 ppm. F1 maternal whole blood EB concentrations of 0.49, 3.51, or 18.28 mg/L were found 1 hr after administration of a composite oral dose of 26, 90, or 342 mg/kg/day, respectively, but no detectable EB was found in blood samples of their F2 PND 4 culled pups. F1 maternal mean whole blood EB levels 1 hr after a 6-hr inhalation exposure on postpartum day (PPD) 22 was 0.11 mg/L (25 ppm), 0.56 mg/L (100 ppm), and 11 mg/L (500 ppm). For the offspring exposed with their dams on PND 22, F2 pup blood EB concentrations ranged from 0.017-0.039 mg/L (25 ppm), 0.165-0.465 mg/L (100 ppm), and 8.82-15.74 mg/L (500 ppm). Because decreased weight gain in the 500 ppm males was transient and no histopathological changes were associated with the increased liver weights in the 500 ppm male and female groups, these changes were not considered adverse. Therefore, for parental systemic toxicity, 100 ppm was considered a NOEL and 500 ppm a NOAEL in this study. The 500 ppm exposure concentration was considered a NOAEL for F0 and F1 reproductive toxicity and offspring developmental endpoints.     

Diversity of microorganisms in solar salterns of Tamil Nadu,India

In this study, the diversity of prokaryotes inhabiting crystallizer ponds of three solar salterns, located along Bengal Bay in Tamil Nadu, India was examined. Unlike other salterns studied the Tamil Nadu salterns are fed by hypersaline spring water mixed with seawater and led to the ponds from bore wells. In addition, prokaryotic community development is restricted as salterns operate only during the arid part of the year. Both culture-based and culture-independent polymerase chain reaction 16S rRNA molecular phylogenetic approaches were employed. Representatives of the family Halobacteriaceae dominated in cultivable portion of diversity encountered with members of genera Haloferax, Halorubrum, Haloarcula, Halobacterium and Halogeometricum recovered in pure culture. In contrast, members of Bacteria were recovered from only one sampling site and were represented by members of genera Salinibacter, Cytophaga and Marinococcus. Based on culture-independent sampling, the predominant members of the haloarchaeal crystallizer community belonged to the genus Natrinema.     

Improved method of in vitro regeneration in Leucaena leucocephala — a leguminous pulpwood tree species

Leucaena leucocephala is a fast growing multipurpose legume tree used for forage, leaf manure, paper and pulp. Lignin in Leucaena pulp adversely influences the quality of paper produced. Developing transgenic Leucaena with altered lignin by genetic engineering demands an optimized regeneration system. The present study deals with optimization of regeneration system for L. leucocephala cv. K636. Multiple shoot induction from the cotyledonary nodes of L. leucocephala was studied in response to cytokinins, thidiazuron (TDZ) and N⁶-benzyladenine (BA) supplemented in half strength MS (½-MS) medium and also their effect on in vitro rooting of the regenerated shoots. Multiple shoots were induced from cotyledonary nodes at varied frequencies depending on the type and concentration of cytokinin used in the medium. TDZ was found to induce more number of shoots per explant than BA, with a maximum of 7 shoots at an optimum concentration of 0.23 µM. Further increase in TDZ concentration resulted in reduced shoot length and fasciation of the shoots. Liquid pulse treatment of the explants with TDZ did not improve the shoot production further but improved the subsequent rooting of the shoots that regenerated. Regenerated shoots successfully rooted on ½-MS medium supplemented with 0.54 µM α-naphthaleneacetic acid (NAA). Rooted shoots of Leucaena were transferred to coco-peat and hardened plantlets showed ≥ 90 % establishment in the green house.Key words: Cotyledonary nodes, Multiple shoot induction, Pulse treatment, TDZ     

<Emphasis Type="Italic">BRCA1</Emphasis> and <Emphasis Type="Italic">BRCA2</Emphasis> germline mutation analysis among Indian women from south India: identification of four novel mutations and high-frequency occurrence of 185delAG mutation

Mutations in the BRCA1 and BRCA2 genes profoundly increase the risk of developing breast and/or ovarian cancer among women. To explore the contribution of BRCA1 and BRCA2 mutations in the development of hereditary breast cancer among Indian women, we carried out mutation analysis of the BRCA1 and BRCA2 genes in 61 breast or ovarian cancer patients from south India with a positive family history of breast and/or ovarian cancer. Mutation analysis was carried out using conformation-sensitive gel electrophoresis (CSGE) followed by sequencing. Mutations were identified in 17 patients (28.0%); 15 (24.6%) had BRCA1 mutations and two (3.28%) had BRCA2 mutations. While no specific association between BRCA1 or BRCA2 mutations with cancer type was seen, mutations were more often seen in families with ovarian cancer. While 40% (4/10) and 30.8% (4/12) of families with ovarian or breast and ovarian cancer had mutations, only 23.1% (9/39) of families with breast cancer carried mutations in the BRCA1 and BRCA2 genes. In addition, while BRCA1 mutations were found in all age groups, BRCA2 mutations were found only in the age group of ≤40 years. Of the BRCA1 mutations, there were three novel mutations (295delCA; 4213T→A; 5267T→G) and three mutations that have been reported earlier. Interestingly, 185delAG, a BRCA1 mutation which occurs at a very high frequency in Ashkenazi Jews, was found at a frequency of 16.4% (10/61). There was one novel mutation (4866insT) and one reported mutation in BRCA2. Thus, our study emphasizes the importance of mutation screening in familial breast and/or ovarian cancers, and the potential implications of these findings in genetic counselling and preventive therapy.     

Is gender difference in postnatal thyroid growth associated with specific expression patterns of androgen and estrogen receptors?

Variations in sex steroids bioavailability were linked to the gender difference in the growth of thyroid glands of neonatal rats. In the present study we tested androgen receptor (AR) and estrogen receptor (ER) concentrations by ligand binding assay, and expression of their genes by RT-PCR and Western blot in the thyroid glands of neonatal rats. AR concentration remained elevated from postnatal day (PND) 10 onwards in males, whereas it decreased by PND 20 in females. AR mRNA and protein expressions were higher in males than females, which increased by PND 10, decreased after PND 15 and reached the nadir by PND 20. ER concentration increased by PND 10 and decreased thereafter in both sex. ERα mRNA expression diminished by PND 15 in both sex; while ERβ mRNA decreased by PND 15 to reach the nadir by PND 20 in males, it was augmented by PND 10 in females to reach the peak by PND 15 and diminished by PND 20. ERα protein expression increased by PND 10 and remained elevated till PND 20 in both sex. ERβ protein expression in males increased by PND 10 and decreased by PND 20, while it remained static up to PND 15 and decreased in females. Testosterone stimulated [³H]-thymidine uptake and the expression of IGF-1 and NIS genes in thyrocytes of both sex in vitro, while estradiol stimulated them in females but not in males. We conclude that androgens influence the growth and differentiation of thyrocytes through augmented expression of AR, IGF-1 and NIS in either sex, whereas estrogen imparts the gender difference, which may be at a level beyond the expression of ERs.