Population genetic structure of Indian shad, Tenualosa ilisha inferred from variation in mitochondrial DNA sequences.

Indian shad, Tenualosa ilisha, is a commercially important anadromous fish representing major catch in Indo-pacific region. The present study evaluated partial Cytochrome b (Cyt b) gene sequence of mtDNA in T. ilisha for determining genetic variation from Bay of Bengal and Arabian Sea origins. The genomic DNA extracted from T. ilisha samples representing two distant rivers in the Indian subcontinent, the Bhagirathi (lower stretch of Ganges) and the Tapi was analyzed. Sequencing of 307 bp mtDNA Cytochrome b gene fragment revealed the presence of 5 haplotypes, with high haplotype diversity (Hd) of 0.9048 with variance 0.103 and low nucleotide diversity (π) of 0.14301. Three population specific haplotypes were observed in river Ganga and two haplotypes in river Tapi. Neighbour-joining tree based on Cytochrome b gene sequences of T. ilisha showed that population from Bay of Bengal and Arabian Sea origins belonged to two distinct clusters.

Modification of photosynthesis and growth responses to elevated CO2 by ozone in two cultivars of winter wheat with different years of release.

The beneficial effects of elevated CO2 on plants are expected to be compromised by the negative effects posed by other global changes. However, little is known about ozone (O3)-induced modulation of elevated CO2 response in plants with differential sensitivity to O3. An old (Triticum aestivum cv. Beijing 6, O3 tolerant) and a modern (T. aestivum cv. Zhongmai 9, O3 sensitive) winter wheat cultivar were exposed to elevated CO2 (714 ppm) and/or O3 (72 ppb, for 7h d(-1)) in open-topped chambers for 21 d. Plant responses to treatments were assessed by visible leaf symptoms, simultaneous measurements of gas exchange and chlorophyll a fluorescence, in vivo biochemical properties, and growth. It was found that elevated CO2 resulted in higher growth stimulation in the modern cultivar attributed to a higher energy capture and electron transport rate compared with the old cultivar. Exposure to O3 caused a greater growth reduction in the modern cultivar due to higher O3 uptake and a greater loss of photosystem II efficiency (mature leaf) and mesophyll cell activity (young leaf) than in the old cultivar. Elevated CO2 completely protected both cultivars against the deleterious effects of O3 under elevated CO2 and O3. The modern cultivar showed a greater relative loss of elevated CO2-induced growth stimulation due to higher O3 uptake and greater O3-induced photoinhibition than the old cultivar at elevated CO2 and O3. Our findings suggest that the elevated CO2-induced growth stimulation in the modern cultivar attributed to higher energy capture and electron transport rate can be compromised by its higher O3 uptake and greater O3-induced photoinhibition under elevated CO2 and O3 exposure.

Differential drought-induced modulation of ozone tolerance in winter wheat species.

Recent reports challenge the widely accepted idea that drought may offer protection against ozone (O(3)) damage in plants. However, little is known about the impact of drought on the magnitude of O(3) tolerance in winter wheat species. Two winter wheat species with contrasting sensitivity to O(3) (O(3) tolerant, primitive wheat, T. turgidum ssp. durum; O(3) sensitive, modern wheat, T. aestivum L. cv. Xiaoyan 22) were exposed to O(3) (83ppb O(3), 7h d(-1)) and/or drought (42% soil water capacity) from flowering to grain maturity to assess drought-induced modulation of O(3) tolerance. Plant responses to stress treatments were assessed by determining in vivo biochemical parameters, gas exchange, chlorophyll a fluorescence, and grain yield. The primitive wheat demonstrated higher O(3) tolerance than the modern species, with the latter exhibiting higher drought tolerance than the former. This suggested that there was no cross-tolerance of the two stresses when applied separately in these species/cultivars of winter wheat. The primitive wheat lost O(3) tolerance, while the modern species showed improved tolerance to O(3) under combined drought and O(3) exposure. This indicated the existence of differential behaviour of the two wheat species between a single stress and the combination of the two stresses. The observed O(3) tolerance in the two wheat species was related to their magnitude of drought tolerance under a combination of drought and O(3) exposure. The results clearly demonstrate that O(3) tolerance of a drought-sensitive winter wheat species can be completely lost under combined drought and O(3) exposure.

Mechanism of benzo(a)pyrene induction of alpha-human chorionic gonadotropin gene expression in human lung tumor cells.

Human lung cells (ChaGo) derived from a bronchogenic carcinoma synthesize and secrete in the culture medium the alpha subunit of the glycoprotein hormone, human chorionic gonadotropin (alpha-hCG). The synthesis of alpha-hCG by ChaGo cells could be further stimulated by treatment with sublethal concentrations of the polycyclic aromatic hydrocarbons (PAHs), benzo(a)pyrene (BaP), or dimethylbenzanthracene. The production of alpha-hCG could be correlated to the levels of alpha-hCG-specific mRNA sequences in control and PAH-treated cells. Further analysis of the RNA species (Northern blot) revealed that the level of the mature (approximately 1.0 kb) and the high molecular weight alpha-hCG specific nuclear RNA sequences (approximately 2.2 and 5 kb) were all greater in PAH-treated cells. Addition of [3H]BaP (0.25 microgram/ml) in the culture medium of ChaGo cells led to immediate uptake of the radioactive compound apparently by simple diffusion. SDS PAGE and subsequent fluorography revealed that the radioactive compound interacted and formed covalent complexes with cytoplasmic and nuclear proteins. This covalent interaction of the [3H]BaP molecule with cellular proteins could be significantly inhibited by either inhibiting the activity of the enzyme aryl hydrocarbon hydroxylase with 7,8-benzoflavone or by reducing the cellular concentration of the enzyme by simultaneous incubation with cycloheximide. These results suggested that in ChaGo cells, the observed covalent complexes were formed by the interaction of the BaP metabolites with cellular proteins. The concentrations at which 7,8-benzoflavone or cycloheximide inhibited formation of metabolites from [3H]BaP and their covalent interaction with cell protein did not affect the BaP-induced stimulation of alpha-hCG gene expression. However, the cytotoxic effects of BaP in ChaGo cells seemed to be exerted by the metabolism of the compounds. Results presented in this report suggest that BaP metabolism and the interaction of the metabolites with cell proteins were not essential for the BaP-induced modulation of alpha-hCG gene expression.

Regulated expression of the prolactin gene in rat pituitary tumor cells.

Prolactin (PRL) gene expression in three strains of GH cells (rat pituitary tumor cells) has been quantitated by measurement of: (a) intracellular and extracellular PRL, (b) cytoplasmic translatable PRL-specific mRNA (mRNAPRL), and (c) molecular hybridization of cytoplasmic poly(A) RNA to cDNAPRL (DNA complementary to mRNAPRL). Three GH cell lines utilized in this investigation were a PRL-producing (PRL+) strain, GH4C1, a PRL nonproducing 5-bromo-deoxyuridine resistnat (PRL- BrdUrdr) strain, F1BGH12C1, and a new strain, 928-9b, derived by fusion of PRL+ cells with a nuclear monolayer of the PRL-, BrdUrdr GH cell strain. PRL production is a characteristic of 928-9b cells, but the level of PRL production (2-4 micrograms/mg protein/24 h) is much lower than that of the PRL+ strain, GH4C1 (15-25 micrograms/mg protein/24 h). Levels of cytoplasmic translatable mRNAPRL and cytoplasmic PRL-RNA sequences quantitated with a cDNAPRL probe were also much lower in 928-9b as compared to the PRL+ parent. PRL-RNA sequences could not be detected in the PRL- strain. Thyrotopin-releasing hormone (TRH) stimulates PRL synthesis about threefold and inhibit a growth hormone (GH) synthesis 72% in the PRL+ strain. TRH has no effect on the synthesis of either PRL or GH in the 928-9b strain, although TRH receptors could be detected in these cells. Stimulation of PRL synthesis in the PRL+ strain by TRH could be correlated with increases in levels of cytoplasmic translatable mRNAPRL and increases in cytoplasmic PRL-RNA sequences. These results demonstrate that the graded expression of the PRL gene at the basal level, and in response to TRH, is caused by the regulated production of specific mRNA, i.e., mRNAPRL in these three GH cell strains.

On the mechanism of 5-bromodeoxyuridine induction of prolactin synthesis in rat pituitary tumor cells.

GH12C1, a clonal strain of rat pituitary tumor cells in culture (GH cells), does not produce detectable amounts of prolactin. 5-Bromodeoxyuridine (BrdUrd), the thymidine analogue, at sublethal concentrations (3-5 microgram/ml) induces prolactin synthesis in these cells. BrdUrd also induces prolactin synthesis in F1BGH12C1 cells, a BrdUrd resistant (BrdUrdr) substrain isolated from GH12C1 cells. The F1BGH12C1 strain is not drug dependent, but its resistance to BrdUrd is a stable phenotype. The significant features of the induction of prolactin synthesis in the BrdUrdr strain are the increased net synthesis of prolactin and the shortening of the lag period of prolactin induction. As BrdUrd concentration in the growth medium is increased, the rise in prolactin synthesis parallels the increased incorporation of BrdUrd into DNA. Prolactin synthesis is first detected when BrdUrd replaces 20-25% of the thymidine in DNA. BrdUrd can replace up to 75-80% of the thymidine within 2 d of treatment. Partial starvation of these cells under specified growth conditions does not affect the general growth pattern of the cells, general protein synthesis, and thymidine uptake, but does affect DNA synthesis. When cells are cultured under conditions in which DNA synthesis is preferentially inhibited, BrdUrd does not induce prolactin synthesis, suggestive of a DNA-mediated mechanism of action for the drug.

Loss of tumorigenic potential by human lung tumor cells in the presence of antisense RNA specific to the ectopically synthesized alpha subunit of human chorionic gonadotropin.

A clonal strain of human lung tumor cells in culture (ChaGo), derived from a bronchogenic carcinoma, synthesizes and secretes large amounts of alpha (alpha) and a comparatively lower level of beta (beta) subunit of the glycoprotein hormone, human chorionic gonadotropin (HCG). ChaGo cells lost their characteristic anchorage-independent growth phenotype in the presence of anti-alpha-HCG antibody. The effect of the antibody was partially reversed by addition of alpha-HCG to the culture medium. ChaGo cells were transfected with an expression vector (pRSV-anti-alpha-HCG), that directs synthesis of RNA complementary to alpha-HCG mRNA. The transfectants produced alpha-HCG antisense RNA which was associated with the reduced level of alpha-HCG. Transfectants also displayed several altered phenotypic properties, including altered morphology, less mitosis, reduced growth rate, loss of anchorage-independent growth, and loss of tumorigenicity in nude mice. Treatment of transfectants with 8,bromo-cAMP resulted in increased accumulation of alpha-HCG mRNA, no change in the level of alpha-HCG antisense RNA, release of the inhibition of [3H]thymidine incorporation, and restoration of anchorage-independent growth phenotype. The overexpression of c-myc, observed in ChaGo cells, was unaffected by the reduced level of alpha-HCG. These results suggest that ectopic synthesis of the alpha subunit of HCG plays a functional role in the transformation of these human lung cells.

Identification of DNA sequence responsible for 5-bromodeoxyuridine-induced gene amplification.

Bromodeoxyuridine (BrdUrd) treatment of the prolactin nonproducing subclone of GH cells (rat pituitary tumor cells) induces amplification of a 20-kilobase DNA fragment including all of the prolactin gene coding sequences. This amplified DNA segment, which is flanked by two unamplified regions, thus designates a unit of BrdUrd-induced amplified sequence. Cloned DNA segments, 10.3 kilobases long, from the 5' end of the rat prolactin gene of BrdUrd-responsive and -nonresponsive cells, were ligated to the thymidine kinase gene of herpes simplex virus type 1 (HSV1TK), and the hybrid DNA was transferred to thymidine kinase-deficient mouse fibroblast cells by transfection. The HSV1TK gene and the rat prolactin gene were amplified together in drug-treated transfectants carrying the hybrid DNA HSV1TK gene and rat prolactin gene of BrdUrd-responsive GH cells. These results suggest that the 10.3-kilobase DNA segment at the 5' end of the rat prolactin gene of BrdUrd-responsive GH cells carries the information for drug-induced gene amplification (amplicon) and that another gene, such as the HSV1TK gene, is also amplified when the latter is placed adjacent to this segment.

Assessing the genetic relatedness of higher ozone sensitivity of modern wheat to its wild and cultivated progenitors/relatives.

Modern wheat (Triticum aestivum L.) is one of the most ozone (O(3))-sensitive crops. However, little is known about its genetic background of O(3) sensitivity, which is fundamental for breeding O(3)-resistant cultivars. Wild and cultivated species of winter wheat including donors of the A, B and D genomes of T. aestivum were exposed to 100 ppb O(3) or charcoal-filtered air in open top chambers for 21 d. Responses to O(3) were assessed by visible O(3) injury, gas exchange, chlorophyll fluorescence, relative growth rate, and biomass accumulation. Ozone significantly decreased light-saturated net photosynthetic rate (-37%) and instantaneous transpiration efficiency (-42%), but increased stomatal conductance (+11%) and intercellular CO(2) concentration (+11%). Elevated O(3) depressed ground fluorescence (-8%), maximum fluorescence (-26%), variable fluorescence (-31%), and maximum photochemical efficiency (-7%). Ozone also decreased relative growth rate and the allometric coefficient, which finally reduced total biomass accumulation (-54%), but to a greater extent in roots (-77%) than in the shoot (-44%). Winter wheat exhibited significant interspecies variation in the impacts of elevated O(3) on photosynthesis and growth. Primitive cultivated wheat demonstrated the highest relative O(3) tolerance followed by modern wheat and wild wheat showed the lowest. Among the genome donors of modern wheat, Aegilops tauschii (DD) behaved as the most O(3)-sensitive followed by T. monococcum (AA) and Triticum turgidum ssp. durum (AABB) appeared to be the most O(3)-tolerant. It was concluded that the higher O(3) sensitivity of modern wheat was attributed to the increased O(3) sensitivity of Aegilops tauschii (DD), but not to Triticum turgidum ssp. durum (AABB) during speciation.

Necrosis and inhibition of growth of human lung tumor by anti-alpha-human chorionic gonadotropin antibody.

Human lung tumor cells (ChaGo) established in culture from a bronchogenic squamous cell carcinoma synthesize and secrete large amounts of human chorionic gonadotropin (HCG), predominantly the alpha subunit of the glycoprotein hormone. ChaGo cells lose their transformation phenotypes following treatment with anti-alpha-HCG antibody or following inhibition of intracellular synthesis of alpha-HCG by the anti-sense RNA technique. We report that tumors induced by ChaGo cells in female athymic mice undergo necrotic degeneration following local or intraperitoneal administration of alpha-HCG-specific-antibody. The alpha-HCG antibody did not affect the growth of tumor induced by alpha-HCG nonproducing human tumor cells. Histopathological examinations of the anti-alpha-HCG antibody-treated tumor tissues showed active necrosis. When the antibody treatment was discontinued, the tumorigenesis process resumed. When anti-alpha-HCG antibody was administered simultaneously with ChaGo cells, a concentration-dependent inhibition of tumor growth in athymic mice was completely prevented at higher concentrations of the specific antibody.

Expression of c-erbB proto-oncogene during dimethylbenzanthracene-induced tumorigenesis in hamster cheek pouch.

Repeated local applications of dimethylbenzanthracene (DMBA) induce epidermoid carcinomas in 14 weeks in the cheek pouch of Syrian hamsters. Characteristic histopathological changes similar to the ones observed in human oral cancers can be detected during the development of these tumors in the hamster cheek pouch. Results presented in this report demonstrate that the cellular proto-oncogene c-erbB is amplified and expressed in an epidermoid carcinoma cell line (HCPC-1), derived from one such cheek pouch tumor. The expression of the gene cannot be detected in the untreated cheek pouch tissue whereas the level of expression of the gene is significantly high in the tumor cell line. The expression of c-erbB gene can also be detected in cheek pouch tissue at an early stage (8-9 weeks) of tumor development and in all tumor-bearing tissue of subsequent stages. The stage of expression of c-erbB gene does not coincide with the stage in which extensive hyperplasia of the epithelial cells (5 weeks) is observed. Rather, this coincides with the onset of the early invasion of the connective tissue by the dysplastic epithelium. The expression of c-erbB gene increases in parallel with the increased tumor burden. These results implicate the possible role of c-erbB gene in the genesis of DMBA-induced oral epithelial carcinomas.

c-Ha-ras gene mutation and activation precede pathological changes in DMBA-induced in vivo carcinogenesis.

We have previously reported a stage-specific and sequential overexpression of the c-Ha-ras and c-erbB genes in 7, 12-dimethylbenzanthracene (DMBA)-induced in vivo carcinogenesis in hamster buccal pouch epithelium (HBPE). In this investigation, the immunoreactive protein product of the c-Ha-ras gene (p21 protein) was identified in HBPE cells, specifically in treated tissues and cultured cells established after 3 weeks of DMBA treatment. Microscopic examination did not show any histopathological changes in these tissues. The p21 protein was detected in a few selective cells, which were dispersed away from the more densely populated basal layer. The overexpression of the c-Ha-ras gene was accompanied by a point mutation of A----T in codon 61 (CAA), inducing an amino acid substitution from the wild-type glutamine to leucine in the peptide. The concurrent molecular modifications preceded any detectable histopathological changes. The cellular morphology and orientation in treated HBPE at this early stage was indistinguishable from the control tissue. Yet the genetic alterations, such as the point mutation and overexpression of the gene, were evident at the predysplastic stage. Amplification and overexpression of the second proto-oncogene, c-erbB, and its product, epidermal growth factor receptor (EGFR), were detected in HBPE cells at the later stages of extensive cell proliferation and invasion. By using double antibodies and two immunoreporter systems, we demonstrated overexpression of both c-Ha-ras and c-erbB genes in the same HBPE cells during this chemically induced in vivo carcinogenesis.

Inhibitors of HIV-1 transcription.

No curative drug against HIV has yet been found, despite enormous efforts aimed at reverse transcriptase and a variety of other targets. The long terminal repeat (LTR) of HIV-1 has recently become a promising site for antiviral action. This article briefly summarizes information on the nature of this target and potential anti-LTR expression drugs.

Defective distal regulatory element at the 5' upstream of rat prolactin gene of steroid-nonresponsive GH-subclone.

The prolactin-nonproducing (PRL-) GH cell strains (rat pituitary tumor cells in culture). GH12C1 and F1BGH12C1, do not respond to steroid hormones estradiol or hydrocortisone (HC). However, the stimulatory effect of estradiol and the inhibitory effect of hydrocortisone on prolactin synthesis can be demonstrated in the prolactin-producing GH cell strain, GH4C1. In this investigation we have examined the 5' end flanking region of rat prolactin (rat PRL) gene of steroid-responsive, GH4C1 cells to identify the positive and negative regulatory elements and to verify the status of these elements in steroid-nonresponsive F1BGH12C1 cells. Results presented in this report demonstrate that the basel level expression of the co-transferred Neo gene (neomycin phosphoribosyl transferase) is modulated by the distal upstream regulatory elements of rat PRL gene in response to steroid hormones. The expression of adjacent Neo gene is inhibited by dexamethasone and is stimulated by estradiol in transfectants carrying distal regulatory elements (SRE) of steroid-responsive cells. These responses are not observed in transfectants with the rat PRL upstream sequences derived from steroid-nonresponsive cells. The basal level expression of the host cell alpha-2 tubulin gene is not affected by dexamethasone. We report here the identification of the distal steroid regulatory element (SRE) located between 3.8 and 7.8 kb upstream of the transcription initiation site of rat PRL gene. Both the positive and the negative effects of steroid hormones can be identified within this upstream sequence. This distal SRE appears to be nonfunctional in steroid-nonresponsive cells. Though the proximal SRE is functional, the defect in the distal SRE makes the GH substrain nonresponsive to steroid hormones. These results suggest that both the proximal and the distal SREs are essential for the mediation of action of steroid hormones in GH cells.

Pentoxifylline inhibits HIV-1 LTR-driven gene expression by blocking NF-kappa B action.

The drug pentoxifylline (PTX) (1-(5'-oxohexyl) 3,7-dimethylxanthine) down-regulates human immunodeficiency virus (HIV-1) long terminal repeat (LTR)-directed gene expression in the human monocytic cell line U38. This effect of PTX has been tested in clinical trials. In this investigation, a similar inhibitory effect of PTX on HIV-1 LTR-driven gene expression was observed (a) in a stable transfectant of the human embryo kidney cell line 293-27-2 carrying an expression plasmid construct with the HIV-1 LTR fused to the bacterial lac Z gene, and also (b) by transient transfection of human embryo kidney cells 293 S with fusion plasmid constructs with wild-type [pHIV-LTR-chloramphenicol acetyltransferase (CAT)] or mutated (pHIV-LTR-mut-CAT) nuclear factor kappa B (NF-kappa B) motifs. In both stable and transient transfection studies, 4-beta-phorbol-12-beta-myristate-acetate (PMA)-induced or tumor necrosis factor-alpha (TNF-alpha)-induced activation of the HIV-1 LTR was correlated with a concomitant elevated level of NF-kappa B interaction with its motifs. The inducibility of HIV-1 LTR-driven gene expression by PMA or TNF-alpha in transiently transfected cells was completely eliminated by point mutations in the NF-kappa B motifs, suggesting that NF-kappa B plays a major role in the activation of the HIV-1 LTR by these agents in this cell system.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of the level of estrogen receptor and functional variants in human breast cancers by novel assays.

The level of estrogen receptor (ER) is a key determinant for the management of ER-positive [ER(+)] breast cancer patients. Growth of many human breast cancers is regulated by estrogen (E2) and progesterone (Pr). Generally, the ER in ER(+) breast cancer patients is targeted for therapy with antihormones. However 40% of ER(+) patients do not respond to antihormone therapy. Thus, the identification of antihormone resistant ER(+) breast cancers is essential for therapeutic predictions. Although 3H-E2 binding and immunodetection can identify ER, these procedures do not assess the functional state of the receptor molecule. In this study we describe a novel and rapid assay for the detection of ER and its functional state on the basis of the downstream interaction with its response element (ERE) based on the preferential binding of DNA-protein complex (ERE-ER) to a nitrocellulose membrane (NMBA). This method permits measurement of both the total and the functional fraction of ER. The ER status was examined in breast cancer cell lines and in breast cancer biopsy specimens by (i) 3H-E2 binding assay, (ii) immunodetection assays and (iii) by its interaction with 32P-ERE. The sensitive NMBA assay was validated with well-characterized ER(+) breast cancer cell lines and also identified functional variants of ER among breast tumor biopsy specimens.

cHa-ras proto-oncogene. Amplification and overexpression in UV-B-induced mouse skin papillomas and carcinomas.

The role of cellular proto-oncogene activation in shortwave UV light in the B range (UV-B)--induced skin carcinogenesis was investigated. Epidermal papillomas and carcinomas were induced on the depilated skin surface of Sencar mice with single-dose UV-B irradiation (7 x 10(4) J/m2). The tumors thus initiated were present in 18.8% of treated animals and were primarily benign papillomas, while a few (6 of 17) progressed to form squamous cell carcinomas. A 5- to 10-fold stimulation of cHa-ras gene expression in both papillomas and carcinomas was observed. Other cellular proto-oncogenes such as cKi-ras, c-myc, or c-fos specific messenger RNAs were not detected in these UV-B--induced skin tumors. Subsequent Southern blot analysis revealed a threefold to fivefold amplification of cHa-ras gene in skin papillomas and carcinomas. However, only the carcinoma and not the papilloma DNA induced foci in the classic NIH-3T3 transformation assay, suggesting that activation of cHa-ras gene alone is not sufficient to exhibit this phenotypic expression of transformed cells. The NIH-3T3 transformants exhibited (1) anchorage independent growth on soft agar, (2) tumor induction in athymic mice, and (3) overexpression and amplification of the cHa-ras gene. We propose that overexpression of a ras gene by gene amplification plays a role in the UV-B--induced skin carcinogenesis process.

Environmental auditing programme in India.

The concept of environmental auditing in industrial units in India was formally introduced in March 1992 with the overall objective minimising consumption of resources and promoting use of clean technologies in industrial production to minimise generation of wastes. A series of discussions were held among the concerned regulatory agencies before and after making environmental auditing and submission of Annual Environmental Statements, a mandatory requirement for the industries. This followed organisation of a number of workshops, seminars and training programmers all over the country by different agencies dealing with environment protection, conducting of audit studies in 125 selected polluting industries in the country by the Central Pollution Control Board (CPCB), Delhi, and a number of other activities. The present paper gives the details of the activities before and after the introduction of this requirement in the country. The aspects covered include (i) the evolution of the concept, (ii) the history of the introduction of the scheme in the country and the organisational structure, (iii) the mandatory requirement and its applicability, (iv) the implementing agencies and their responsibilities, (v) summary of the training programmes/workshops organised in the country, (vi) case studies conducted by CPCB in selected polluting industries and the major findings, (vii) development of guidelines for conducting environmental audit in different categories of polluting industries, and (viii) the current status of the scheme and plans for its effective implementation.