Geochemical characteristics of the Rajmahal coals in Dhulia North Block, Eastern India: implication to their utilization and environment.

The borehole coal samples of Dhulia North Block from the Rajmahal Basin, Eastern India, were systematically analyzed based on the chemical composition and concentration of major and trace elements (including rare earth elements, REEs) to assess the distribution of REEs and their environmental implications with utilization potential. The Dhulia North Block coals are characterized by the predominant major oxides of SiO2, Al2O3, and Fe2O3, accounting for 94% of the total ash composition, indicating the presence of quartz, clay-rich minerals, and pyrite. Compared with the average world coal ash, the total REE content in the analyzed samples ranged from 341.0 to 810.4 ppm, which is substantially higher. Hot humid climate conditions with intermediate igneous source rocks of the basin were demonstrated by the major oxide ratios (Al2O3/TiO2 < 20) and plots of TiO2 with Al2O3 and Zr. The redox-sensitive elements such as V, Ni, Cr, and Co found in the Dhulia North Block coal indicate that an oxic sedimentary environment existed in the basin when coal was formed. The low sulfur content (1% in most samples) indicates freshwater conditions in the basin at the time of organic matter deposition. The outlook coefficient (Coutl) varies between 0.7 and 1.6, indicating that the Dhulia North Block coals are a prospective source of REEs. The Dhulia North Block coals are characterized by low H/C and O/C atomic ratios ranging from 0.56 to 0.90 and 0.10 to 0.22, respectively, and contain type-III kerogens, indicating gas-prone source rock. Further, the basic-to-acid oxide ratio suggested that Dhulia North Block coals were suitable for utilization during combustion processes.

Mobility of metals and metalloids from SHOS coal ash and slag deposit: mineralogical and geochemical constraints.

Deposits remained after coal combustion are a well-known occurrence in the world; unfortunately, only a small percentage of such deposits are adequately regulated and, consequently, pose a serious threat to the local environment. Attenuation of negative consequences presupposes knowledge of a number of features, both of the deposit and the local environment as well the interaction with local biota. In this study, unregulated waste generated from decades of coal mining and combustion of superhigh-organic-sulfur Rasa coal, enriched in Se-U-Mo-V and located in a vulnerable karst region, was investigated. To assess the impact of landfill on the environment, in addition to its general geochemical and mineralogical features, the human health risk was assessed and the leaching of elements from the landfill, local soil, and the coal itself was investigated. For the latter, three extraction procedures, ASTM, EP, and TCLP (pH 4.93 and 2.9), were employed, mimicking different environmental conditions, including the sporadic occurrence of acid rains in the region. The soil around the landfill displayed enrichment in the majority of elements compared to expected values, with exception of Se, Mo, U, V, Sr, and Cu found at the highest levels in landfill samples. Mobility of elements was found to be controlled by both pH and mineralogy (carbonates and sulfates), whereby the overall highest relative mobility was observed in landfill samples for elements prevalently bound to sulfate phases. Calculated Hazard Quotient describes this landfill as a risk to the environment and human health through different pathways.

Production of monoclonal antibodies against recombinant human zona pellucida glycoproteins: utility in immunolocalization of respective zona proteins in ovarian follicles.

The zona pellucida (ZP) glycoproteins play an important role in oocyte development and gamete biology. To analyze their expression in follicles during various developmental stages, murine monoclonal antibodies (MAbs) were generated against the baculovirus-expressed recombinant human ZP2, ZP3 and ZP4. A panel of MAbs specific for the respective zona protein in ELISA and Western blot, and devoid of cross-reaction with other zona proteins was selected. Immunohistochemistry has shown that ZP2 MAb, MA-1620, did not react with oocytes in resting primordial follicles but showed reactivity with degenerating oocytes in primordial follicles undergoing atresia, and with oocytes in growing and antral follicles. Three MAbs against ZP3 did not react with oocytes in primordial follicles, but reacted only with oocytes in growing and antral follicles. Out of four MAbs against ZP4, three MAbs reacted with oocytes in primordial, growing and antral follicles. No reactivity of these MAbs with other ovarian cell types and other tissues studied (endometrium, uterine cervix, fallopian tubes and kidney) was detected except for a strong reactivity of ZP2 MA-1620 with epithelial cells of the uterine ectocervix or endometrium in some samples investigated. Altogether, these studies document generation of MAbs exhibiting high specificity for human zona proteins, which will be useful reagents to study their immunobiology.

Immunocontraceptive approaches in females.

In the female reproductive system, there are multiple points, where immunointerception is feasible. Among these, the most promising are those that interfere immunologically at the level of sperm-egg interaction or neutralize human chorionic gonadotropin (hCG), which is required for the establishment and maintenance of pregnancy. Immunization studies in various animal models including non-human primates with zona pellucida (ZP) glycoproteins have demonstrated curtailment of fertility. To circumvent the ovarian pathology often observed following immunization with ZP glycoproteins, synthetic peptides devoid of 'oophoritogenic' T cell epitopes as immunogens have been proposed. Though of utility for controlling wildlife populations, its application in humans will have to wait till the safety of ZP glycoprotein vaccine is established beyond doubt. Clinical trials in women have established that immunocontraceptive vaccine based on Beta-subunit of hCG inhibits fertility. To make it a practical proposition, novel strategies have to be adopted to increase its immunogenicity so that 'contraceptive' antibody titers are achieved in near to 100% recipients.

Role of proteasomal activity in the induction of acrosomal exocytosis in human spermatozoa.

Sperm-associated proteasomes have been suggested to play an important role during fertilization in animals. To delineate the role of these proteasomes during fertilization in humans, the present study reports proteasomal proteolytic activity both in noncapacitated and capacitated human spermatozoa, which is not altered in the presence of baculovirus-expressed recombinant human zona pellucida glycoprotein-3 (ZP3) and zona pellucida glycoprotein-4 (ZP4). However, inhibition of proteasomal proteolytic activity by clasto-lactacystin beta-lactone (CLBL) and Z-Leu-Leu-Leu-CHO (MG132), which are specific inhibitors of the 20S proteasomal core proteases, led to a significant (P < 0.05) inhibition of induction of acrosome reaction mediated by both recombinant human ZP3 and ZP4. Both inhibitors, however, failed to inhibit the induction of acrosomal exocytosis mediated by pharmacological agonist, calcium ionophore (A23187). The binding of recombinant human ZP3 and ZP4, labelled with fluorescein isothiocyanate, to the capacitated spermatozoa was not affected in the presence of proteasomal inhibitors. These observations suggest a role of the sperm proteasome in the induction of ZP3- and ZP4-mediated acrosomal exocytosis upstream of calcium signalling in humans.

Structural and functional attributes of zona pellucida glycoproteins.

A translucent matrix termed the zona pellucida (ZP) surrounds the mammalian oocyte. It plays a critical role in fertilization by acting as a "docking site" for binding of spermatozoa followed by induction of the acrosome reaction in the zona bound sperm. Recent analyses of the genes of the human oocyte revealed that the ZP matrix is composed of four glycoproteins, designated as ZP1, ZP2, ZP3 and ZP4, instead of 3 found in the mouse ZP. Comparison of the deduced amino acid (aa) sequences of the human ZP glycoproteins with those from various species, revealed that these are evolutionarily conserved. Phylogenetic analysis revealed that ZP1 and ZP4 may be related as these have the highest sequence identity at the aa level within a given species. Each zona protein has a signal sequence driving these proteins to the endoplasmic reticulum, a aproximately 260 aa long 'ZP domain' comprising of 8-10 conserved cysteine residues, a C-terminal, hydrophobic transmembrane-like region and a short cytoplasmic tail. In order to understand the structure-function relationship of human ZP glycoproteins, our lab has cloned and expressed ZP2, ZP3 and ZP4 proteins both in E. coli as well as baculovirus expression systems. Simultaneously, our group has been able to amplify the cDNA encoding human ZP1. Employing baculovirus-expressed recombinant ZP glycoproteins; our group has provided evidence for the first time that in human, in addition to ZP3, ZP4 is also able to induce acrosomal exocytosis in the capacitated spermatozoa. ZP3 mediated induction of the acrosome reaction can be inhibited by pertussis toxin suggesting the involvement of G, protein in downstream signaling in contrast to ZP4, which follows a G, protein independent pathway. Hence, elucidation of the role of individual ZP glycoproteins in humans will provide a better insight into the gamete interaction culminating in fertilization.

Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C.

To facilitate our understanding of the role of zona pellucida glycoproteins during fertilization in humans, recombinant human zona pellucida glycoprotein-A (hZPA), -B (hZPB) and -C (hZPC) were obtained by using Escherichia coli and baculovirus expression systems. Analysis by SDS-PAGE and Western blot of the Ni-NTA affinity purified recombinant proteins revealed that the baculovirus-expressed hZPA, hZPB and hZPC have an apparent molecular weight of approximately 110, approximately 70-75 and approximately 65 kDa, respectively, as compared to approximately 80, approximately 65 and approximately 50 kDa of the respective E. coli-expressed proteins. Lectin binding studies revealed that the baculovirus-expressed recombinant zona proteins were glycosylated. Major oligosaccharides were represented by strong reactivity with Concanavalin A (mannose alpha 1-3 or mannose alpha 1-6 residues) and Jacalin (alpha-O glycosides of Gal or GalNAc moieties). A significant increase in acrosomal exocytosis was observed when capacitated human sperm were incubated in vitro with baculovirus-expressed hZPB (P=0.0005) and hZPC (P=0.0005) The E. coli-expressed hZPB, hZPC and baculovirus-expressed hZPA failed to induce any significant increase (P>0.05) in acrosome reaction. In contrast to hZPC, the acrosome reaction induced by recombinant hZPB was not inhibited by pertussis toxin. These studies, for the first time, have demonstrated that in humans, ZPB also induces acrosomal exocytosis through a Gi independent pathway.

Spectrum of beta-thalassemia mutations and their association with allelic sequence polymorphisms at the beta-globin gene cluster in an Eastern Indian population.

In this report, the spectrum of beta-thalassemia mutations and genotype-to-phenotype correlations were defined in large number of patients (beta-thalassemia carriers and major) with varying disease severity in an Eastern Indian population mainly from the state of West Bengal. The five most common beta-thalassemia mutations were detected, which included IVS1-5 (G-->C), codon 15 (G-->A), codon 26 (G-->A), codon 30 (G-->C), and codon 41/42 (-TCTT). These accounted for 85% in 80 beta-thalassemic alleles deciphered from 56 patients, including beta-thalassemia major and carriers, and 15% of alleles remained uncharacterized in these patients. Expression of the human beta-globin gene is regulated by an array of cis-acting DNA elements, including five DNase I hypersensitive sites (HSs) in the locus control region (LCR), promoters that incorporate certain silencer elements, and enhancers at 3' of the beta-globin gene. For detailed studies and to understand the molecular basis of beta-thalassemia, we studied two groups of subjects: a group of 12 patients from four families having beta-thalassemia major and carrier phenotype and a control group of 26 healthy individuals. In these two groups, we examined portions of the beta-globin gene locus control region HSs 1, 2, 3, and 4, which included the (CA)(x)(TA)(y) repeat motif, the (AT)(x)N(y)(AT)(z) repeat motif, the inverted repeat sequence TGGGGACCCCA, the promoter region of the (G)gamma-globin gene, an (AT)(x)(T)(y) repeat 5' of the silencer region, and the beta-globin gene and its 3' flanking region. We investigated the allelic sequence polymorphisms in these regions and their association with the beta-thalassemia mutations to know the possible genotype-phenotype relationship in beta-thalassemia patients. An analysis of cis-acting regulatory regions showed varied sequence haplotypes associated with some frequent beta-thalassemia mutations in this Eastern Indian population.