Genesis of arsenic-rich groundwater and the search for alternative safe aquifers in the Gangetic Plain, India.

Distribution and mobilization of groundwater arsenic from a 1580-km(2) area in the Gangetic Plain was studied. A two-tier aquifer system made up of Quaternary sand layers exists within 300 m below ground. Arsenic concentration exceeding >50 microg/L is confined within the active floodplain of the Ganga River, affecting the top aquitard and upper 5- to 20-m slice of the underlying shallow aquifer. The genesis of arsenic was investigated by principal component analyses involving total dissolved solids, Ca(+2), Mg(+2), Na(+), K(+), HCO3-, Cl(-1), SO4(-2), NO3-, Fetotal, and Astotal and analyzed for 57 groundwater samples, hydrochemical facies analyses, aquifer-aquitard configuration, and water-level behaviour. A 20- to 25-m thick deeper aquifer, appearing at 190 to 205 m below ground and separated from the shallow aquifer by a thick clay sequence, was low in arsenic load (<2 microg/L). Hydrostratigraphy and pumping tests revealed that the deeper aquifer can be used for community drinking in contaminated areas.

Cell cycle-dependent stimulation of the HIV-1 promoter by Tat-associated CAK activator.

Activation of the HIV-1 promoter by the virally encoded Tat protein is characterized by efficient processive transcription, mediated by host cell factors that are tethered to the promoter with the Tat-TAR RNA complex. Importantly, viral gene activation has been shown to be stimulated in mitogenically induced cells, although the link between cell cycle regulation and viral gene activation is unclear. We reported a Tat-associated CAK/CTD kinase from mitogenically induced primary human T-cells (TTK) (S. Nekhai et al., 1997, J. Virol. 71, 7436-7441). Here, biological activity of the kinase has been studied by direct microinjection at the individual-cell level. The TTK-dependent Tat response is maximal during G1 phase as shown by co-injection with Tat protein in cells synchronized at the various stages of the cell cycle. The cell cycle dependence of the Tat response was confirmed by inhibiting G0 --> G1 progression with the expression of dominant negative mutant Ras(Asn17) or the cyclin-dependent kinase CDK4. The results support a mechanism whereby transactivation of the HIV promoter is regulated by cell growth signal transduction pathways that target the Tat cofactor.

A human primary T-lymphocyte-derived human immunodeficiency virus type 1 Tat-associated kinase phosphorylates the C-terminal domain of RNA polymerase II and induces CAK activity.

Tat protein mediates transactivation of human immunodeficiency virus type 1 (HIV-1), which results in more-efficient transcript elongation. Since phosphorylation of C-terminal domain (CTD) of RNA polymerase II correlates with its enhanced processivity, we studied the properties of a Tat-associated CTD kinase derived from mitogenically stimulated human primary T lymphocytes (TTK). TTK binds to full-length Tat and specifically phosphorylates CTD and CDK2. This dual kinase activity is characteristic of CDK-activating kinase (CAK). The CTD kinase activity is induced upon mitogenic stimulation of primary T lymphocytes. Fractionation of T-cell lysate demonstrates that Tat-associated CTD kinase activity elutes in two peaks. About 60% of Tat-associated CTD kinase copurifies with CDK2 kinase activity and contains the CAK components CDK7 and cyclin H. The rest of Tat-associated kinase is free of CDK2 kinase activity and the CAK components and thus may represent a novel CTD kinase. The kinase activities of TTK are blocked by the adenosine analog 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) as well as by the kinase inhibitor H8 at concentrations known to block transcript elongation. Importantly, the Tat-associated kinase markedly induced CAK. We suggest that the mechanism of Tat-mediated processive transcription of the HIV-1 promoter includes a Tat-associated CAK activator.

Human chromosome 6- and 11-encoded factors support human immunodeficiency virus type 1 Rev function in A9 cells.

The precise mechanism of Rev-mediated expression of human immunodeficiency virus (HIV-1) late genes is not well characterized. We recently proposed a requirement for HIV-1 Rev responsive element (RRE) RNA binding host nuclear proteins in Rev function. In this report, using a transient transfection assay of Rev function, we further demonstrate the role of host cell factors in HIV-1 Rev function. Murine A9 cells, which are inefficient in forming RRE-host protein ribonucleoprotein complexes, are also inefficient in supporting Rev function. We also show that host cell factor(s) encoded by human chromosomes 6 and 11 can support HIV-1 Rev-mediated expression of unspliced viral mRNAs in murine A9 cells.

Inhibition of HIV-1 replication in viral mutants with altered TAR RNA stem structures.

Human immunodeficiency virus type 1 (HIV-1) Tat-mediated trans-activation requires the structural integrity of TAR RNA and the cooperative interaction of human host cell proteins. The TAR domain, minimally required for tat response, includes the Tat binding pyrimidine bulge, the TAR RNA upper stem, and the loop sequences. However, little is known about the significance of the 5'-stem structure of TAR in the regulation of viral growth. We designed viral mutations, specifically in the TAR RNA lower stem structure, and studied their effects on the kinetics of viral growth in T-lymphocyte cell lines and in activated human peripheral blood mononuclear cells. Mutations that destabilized the lower TAR stem structure inhibited viral growth to various degrees in different CD4+ T-cells. These results suggest that the structural integrity of the lower stem structure of TAR plays an important role in viral growth, presumably by binding to specific host cell proteins that stabilize Tat-TAR interactions.

Interaction of nuclear protein p140 with human immunodeficiency virus type 1 TAR RNA in mitogen-activated primary human T lymphocytes.

Several lines of evidence suggest that cellular proteins play a role during human immunodeficiency virus type 1 (HIV-1) Tat-mediated trans activation. A recent report from this laboratory has shown that a 140-kDa HeLa nuclear protein (p140) binds specifically to the lower stem region of the Tat response element, TAR RNA. Since HIV-1 trans activation is most efficient in proliferating T cells, we investigated the binding of p140 to TAR RNA in unstimulated and mitogen-activated, G1-phase primary T lymphocytes. TAR RNA/protein-binding activity was low in resting cells but increased significantly within 2 h of activation and remained elevated for at least 48 h. Corresponding increases in p140 protein levels were observed with most but not all donors, suggesting that an additional nuclear factor(s) may be required for efficient binding of this protein to TAR RNA in activated T cells.

Human immunodeficiency virus type 1 Rev-responsive element RNA binds to host cell-specific proteins.

RNase protection-gel retention studies show human host cell-specific ribonucleoprotein complexes with human immunodeficiency virus type 1 Rev-responsive element (RRE) RNA. Nuclear proteins from rodent or murine cells appear to lack the ability to form these complexes. Human-mouse somatic cell hybrids retaining a single human chromosome, either 6 or 12, form the RRE-nuclear-protein complexes. One of the complexes requires the entire RRE RNA, while the other needs RRE RNA stem-loops 1 and 2 only. Two major proteins with molecular masses of 120 and 62 kDa specifically bind to RRE RNA. Rodent cells (CHO) either lack or contain small amounts of these RRE-binding proteins.

Transforming growth factor beta increases the expression of HIV-1 gene in transfected human mesangial cells.

Human immunodeficiency virus type 1 (HIV) infection is often complicated by focal glomerulosclerosis (FGS) and other renal lesions collectively termed HIV associated nephropathy (HIVAN). FGS is characterized by glomerular mesangial expansion and increased synthesis of matrix components. The molecular pathogenic mechanisms associated with the development of HIV associated nephropathy are unknown. Experimental animal models suggest a role for cytokines and growth factors, particularly transforming growth factor beta (TGF-beta), in the pathogenesis of glomerulosclerosis. Patients with AIDS have elevated plasma and tissue levels of TGF-beta. We carried out experiments to determine whether primary human mesangial cells (HMC) in culture can be transfected with HIV-1 genes. HMC were transfected with a chloramphenicol acetyl transferase (CAT) reporter construct containing HIV-1 acetyl transferase (CAT) reporter construct containing HIV-1 LTR sequences. Our results show successful transfection of HMC with HIV-1 LTR gene. HMC transfected with LTR gene are responsive to the HIV-1 regulatory gene product Tat. To study whether TGF-beta can modulate the expression of HIV-1 LTR gene in HMC, HMC transfected with an HIV-1 LTR CAT plasmid were treated with TGF-beta and other growth factors two hours before harvest. TGF-beta specifically increased the expression of the HIV-1 gene in HMC in a dose dependent manner. We further studied whether up-regulation of HIV-1 LTR expression in HMC was mediated by the effect of TGF-beta on the interaction of transcription factors to their binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of human immunodeficiency virus type I Rev protein with nuclear scaffold nucleoside triphosphatase activity.

Human immunodeficiency virus type I encodes a regulatory protein, termed Rev, which is associated with the appearance of unspliced and partially spliced viral RNAs in the cytoplasm. Rev is believed to function via interaction with a sequence element in the env region of the viral RNA, termed the Rev-responsive element (RRE). In this study, we use a stably transfected, Rev-producing mouse cell line to show that low, functional levels of Rev are associated with the nuclear scaffold (NS). Immunohistochemical studies localize Rev to the NS. Furthermore, immunoblot analyses demonstrate the presence of Rev in NS preparations isolated from Rev-producing cells and document binding of purified Rev protein to isolated NS or to cloned lamin C in vitro. Results with an in vitro RNA transport assay suggest that Rev is associated with a significant defect in transport of RNAs which lack RRE, whereas transport of RRE-containing transcripts proceeds efficiently. This Rev-induced transport defect appears to be mediated via direct inhibition of NS nucleoside triphosphatase, an enzyme thought to be involved in the nucleocytoplasmic transport process. NS preparations isolated from Rev-producing cells show a significantly lower nucleoside triphosphatase activity than those from control preparations. Addition of Rev protein to isolated NS produces a significant inhibition of NS nucleoside triphosphatase activity, which is specifically reversed by addition of RRE transcripts. These data suggest that a major aspect of Rev function may involve selective modulation of host cell nucleocytoplasmic transport mechanisms via interaction with the NS.

Inactivation of splicing factors in HeLa cells subjected to heat shock.

The nuclear extracts from HeLa cells subjected to heat shock at 43 or 46 degrees C for 2 h were unable to splice pre-mRNA in vitro. Analysis of snRNPs in the extracts revealed that the U4.U5.U6 small nuclear ribonucleoprotein particle (snRNP) complex was disrupted at both temperatures while U1 and U2 snRNPs remained unaffected at 43 degrees C but were disrupted to certain extent during heat shock at 46 degrees C. During splicing reaction, the extract from cells heat shocked at 43 degrees C formed intermediate splicing complexes alpha and beta but was unable to form a functional spliceosome, complex gamma. Addition of fractions from a normal nuclear extract restored splicing activity only in the extract from cells subjected to heat shock at 43 degrees C. Using this complementation assay, we have partially purified the factor(s) inactivated at this temperature. The purified factor(s) was essentially devoid of snRNAs and snRNPs and resistant to micrococcal nuclease, indicating that the factor(s) inactivated by in vivo heat shock at 43 degrees C is a protein. We have also subjected the nuclear extracts from normal HeLa cells to in vitro heat treatment at 43 or 46 degrees C. The results indicate that during in vitro heat treatment of the extracts the damage to splicing machinery is more extensive than that during in vivo heat shock. These experiments also suggest that the factor(s) inactivated by heat shock at 43 degrees C is different from previously identified thermolabile splicing factors.

Placental transfer of metals of coal fly ash into various fetal organs of rat.

Fly ash (100 mg/kg body weight) was administered intratracheally to 14-day pregnant rats for 6 consecutive days. On day 20 of gestation the translocation of metals present in the fly ash to various maternal and fetal organs was studied. Fly ash administration to pregnant mothers retarded the growth of fetal heart and kidney as determined by their weights. Fly ash instillation increased organ levels of nearly all the metals studied in both mother and fetus. Most of the metals present in coal fly ash were transferred in significant amounts through placenta to several fetal organs. However, the pattern of their distribution into various fetal organs was different for different metals.

Inhibition of pre-mRNA splicing by 5-fluoro-, 5-chloro-, and 5-bromouridine.

Pre-mRNA transcripts of the human beta-globin gene containing 5-fluoro-, 5-chloro-, and 5-bromouridine were tested for splicing in vitro. Pre-mRNA containing 5-fluorouridine was spliced accurately and efficiently in the nuclear extract from HeLa cells, whereas 5-chloro-, and 5-bromouridine containing transcripts were not spliced. Analysis of the splicing reactions by electrophoresis on nondenaturing polyacrylamide gels showed that the latter two transcripts were unable to form active splicing complexes. Treatment of HeLa cell cultures with 5-fluorouridine decreased the splicing activity of the nuclear extracts in a dose- and time-dependent fashion. The decrease in splicing activity of these extracts appears to be due in part to a decreased level of U-2 small nuclear RNA and the corresponding ribonucleoprotein particle, U2-snRNP.

In vitro studies of the inhibition of protein kinase C from rat brain by di-(2-ethylhexyl)phthalate.

The environmental contaminant di(2-ethylhexyl)phthalate (DEHP) has been shown to inhibit the phosphorylation of histone by purified protein kinase C (PK-C) from rat brain in a concentration-dependent manner. The inhibition does not involve making the substrate unavailable, although DEHP does bind to some extent to histone. DEHP displaces phorbol dibutyrate from PK-C, indicating that DEHP binds to the regulatory domain of the enzyme. Since DEHP does not affect the PK-C dependent phosphorylation of protamine, DEHP probably does not bind at the catalytic site. DEHP non-competitively blocked activation of PK-C by either phosphatidyl serine or calcium ion. Inhibition of histone phosphorylation by DEHP was enhanced if diglyceride was present, and the enhancement was stereoselective for the isomeric form of the diglyceride. The mechanism of the inhibition is thought to involve interference with the interaction between calcium ion and the regulatory domain of PK-C, and would have significance only for those PK-C substrates that require calcium activation of the enzyme. Thus the presence of DEHP in the high nanomolar concentration range alters the effective substrate specificity of PK-C.

In vitro secretion and uptake of radioactive vitamin A in rats fed different dietary proteins.

Generally, protein energy malnutrition is accompanied with vitamin A deficiency. The present study was undertaken to evaluate the importance of the quality and the quantity of dietary proteins on the in vitro secretion and uptake of vitamin A in growing rats. The results show that the in vitro release of radioactive vitamin A was greater in rats fed Bengal gram diets as compared to those fed casein diets. However, this relationship between the two groups of rats was reversed for the in vitro uptake of radioactive vitamin A from plasma by various extrahepatic tissues. Thus the vitamin A status of the animal is profoundly influenced by the quality and the quantity of dietary proteins.

In-vitro modulation of protein kinase C activity by environmental chemical pollutants.

A number of environmental chemical pollutants have been reported to cause tumors or help in the propagation of tumors in experimental animals. The in-vitro effects of a few chemical contaminants were studied on the histone phosphorylation and 3H Phorbol dibutyrate (PdBu) binding of partially purified Ca2+/phospholipid dependent protein kinase c (PKC) from the brains of Fischer F344 and B6C3F1 mice. The enzyme was prepared by a modified method which gave approximately 75-fold purification. A differential effect of various compounds was observed on the phosphorylation activity and PdBu binding of PKC from rats and mice. The reported tumor promoting ability and effect on protein kinase C activity appeared to be related in the case of the rat enzyme, although causality cannot be inferred.

Placental transfer and fetal distribution of 3H-retinoic acid in rats.

The placental transfer of 3H-retinoic acid in vitamin A deprived and vitamin A supplemented pregnant female rats was studied on 20th day of gestation and compared with 3H-retinyl acetate. Radiolabelled compounds were administered to pregnant mothers orally in groundnut oil six hours before sacrifice. The distribution of radioactivity of the two compounds was studied in maternal intestine, liver and plasma and fetal brain, heart liver lung and placenta. The transfer of 3H-retinoic acid across placenta was restricted as compared to that of 3H-retinyl acetate which may explain the reason why retinoic acid does not support fetal growth.

Vitamin A and biochemical development. I. Effect of postnatal excess vitamin-A administration on lipogenesis in various organs of the rat.

The effect of the administration of an excessive dose of vitamin A (1,000 IU) to rat pups on the 4th, 6th, 8th and 10th days of age was studied on the contents of saponifiable (total fatty acids) and non-saponifiable (cholesterol) lipids. Lipogenesis and cholesterogenesis was studied with the help of sodium-1-14C-acetate incorporation. The heart, liver and lung showed impaired lipogenesis while in the brain only cholesterogenesis was affected.

Vitamin A and biochemical development. II. Effect of postnatal excess vitamin-A administration on the activity of pyridine nucleotide-linked dehydrogenases.

The effect of excess vitamin A, (1,000 IU) administration to rat pups on the 4th, 6th, 8th and 10th days of age was studied on the activities of combined HMP dehydrogenases, malic enzyme and isocitrate dehydrogenase on the 11th, 17th and 23rd days of age. The developmental pattern and the effect of vitamin-A administration differs from enzyme to enzyme and organ to organ.

Developmental pattern of DNA and proteins in brain, liver, lung and heart of rats given excess vitamin A postnatally.

The effect of an excessive dose of vitamin A (1,000 IU) to rat pups on 4, 6, 8 and 10 days of age was studied on growth and organ weights. The effects on the amounts of DNA and proteins and incorporation of 3H-thymidine into DNA and 3H-leucine into proteins of brain, liver, lung and heart was studied on 11, 17 and 23 days of age. Excess vitamin A administration affected the postnatal development of DNA and protein metabolism of these organs differently.