TRPC and Orai proteins have both been proposed to form Ca 2؉ -selective, store-operated calcium entry (SOCE) channels that are activated by store-depletion with Ca 2؉ chelators or calcium pump inhibitors. In contrast, only TRPC proteins have been proposed to form nonselective receptor-operated calcium entry (ROCE) cation channels that are activated by Gq/Gi-PLC␤ signaling, which is the physiological stimulus for store depletion. We reported previously that a dominant negative Orai1 mutant, R91W, inhibits Ca 2؉ entry through both SOCE and ROCE channels, implicating Orai participation in both channel complexes. However, the argument for Orai participating in ROCE independently of store depletion is tenuous because store depletion is an integral component of the ROCE response, which includes formation of IP3, a store-depleting agent. Here we show that the R91W mutant also blocks diacylglycerol (DAG)-activated Ca 2؉ entry into cells that stably, or transiently, express DAG-responsive TRPC proteins. This strongly suggests that Orai and TRPC proteins form complexes that participate in Ca 2؉ entry with or without activation of store depletion. To integrate these results with recent data linking SOCE with recruitment of Orai and TRPCs to lipid rafts by STIM, we develop the hypothesis that Orai:TRPC complexes recruited to lipid rafts mediate SOCE, whereas the same complexes mediate ROCE when they are outside of lipid rafts. It remains to be determined whether the molecules forming the permeation pathway are the same when Orai:TRPC complexes mediate ROCE or SOCE.diacylglycerol ͉ STIM1 ͉ store operated calcium entry ͉ transient receptor potential

plasma Fraction TIT in 40-50% yield. Average specific activities of these preparations are 10,300 "Iowa" units per mg protein, 69,000 units per mg niftrogen, and 99,400 units per mg tyrosine. The higher activity of human thrombin indicates that it is either a smaller molecule or more active than bovine or equine throm)bin.

Ozone has been proposed for water disinfection because it is more efficient than chlorine for killing microbes and results in much lower levels of carcinogenic trihalomethanes than does chlorination. Ozone leads to formation of hypobromous acid in surface waters with high bromine content and forms brominated organic by-products and bromate. The carcinogenicity and chronic toxicity of potassium bromate (KBrO3) was studied in male B6C3F1 mice and F344/N rats to confirm and extend the results of previous work. Mice were treated with 0, 0.08, 0.4, or 0.8 g/L KBrO3 in the drinking water for up to 100 wk, and rats were provided with 0, 0.02, 0.1, 0.2, or 0.4 g/L KBrO3. Animals were euthanatized, necropsied, and subjected to a complete macroscopic examination. Selected tissues and gross lesions were processed by routine methods for light microscopic examination. The present study showed that KBrO3 is carcinogenic in the rat kidney, thyroid, and mesothelium and is a renal carcinogen in the male mouse, KBrO3 was carcinogenic in rodents at water concentrations as low as 0.02 g/L (20 ppm; 1.5 mg/kg/day). These data can be used to estimate the human health risk that would be associated with changing from chlorination to ozonation for disinfection of drinking water.

In this study, we have characterized the cDNA clone SQ37 that was isolated previously from a rabbit squamous cell library. The gene encodes a 14-kDa protein that appears to function as a component ofthe cross-linked envelope in squamous differentiating cells. The protein, which has been named cornifin, has a high content of proline (31%), glutamine (20%), and cysteine (11%) and contains 13 repeats of an octapeptide (consensus sequence, EPCQPKVP) at its C terminus. SQ37 mRNA and protein are induced during squamous differentiation of rabbit tracheal (RbTE) cells and human epidermal keratinocytes. This induction is repressed by retinoids. Immunohistochemical studies reveal SQ37 immunoreactivity in fragmented cross-linked envelopes from squamousdifferentiated RbTE cells and in the suprabasal layers of the epidermis. In situ hybridization analysis showed that the presence of SQ37 mRNA is restricted to the suprabasal layers.Treatment of RbTE cells with a Ca2W ionophore induces cross-linking of the SQ37 protein into higher molecular weight complexes. This cross-linking reaction appears to be mediated by transglutaminase type I. Our observations suggest that the protein encoded by SQ37 participates in the assembly of the cross-linked envelope.Epithelial cells from many different tissues are able to undergo squamous differentiation. In the tracheobronchial epithelium this differentiation occurs during vitamin A deficiency and after toxic or mechanical injury (1, 2). In other tissues, including skin, squamous differentiation constitutes the normal pathway of differentiation (3). The existence of histologically distinct layers in squamous epithelia is indicative of a multistage process of differentiation (3,4). This view is supported by observations that each of these stages can be defined by the expression of specific biochemical and molecular markers (5-8). The formation of the cross-linked envelope is a characteristic feature during later stages of differentiation (9-11). The highly insoluble cross-linked envelope consists of a layer of covalently linked protein assembled just beneath the plasma membrane. Transglutaminases catalyze the formation of E-(y-glutamyl)lysine isopeptide bonds between cross-linked envelope precursors (11-15). Several proteins have been implicated in the formation of the cross-linked envelope, including involucrin and loricrin, which have been studied in detail (11,(16)(17)(18)(19)(20)(21)(22). The genes for these proteins have been cloned and sequenced (22)(23)(24).Previously, this laboratory has reported (25) the isolation of several cDNA clones, including SQ37, that encode mRNAs abundantly expressed in differentiating squamous RbTE cells. In the present study, the DNA and protein sequences of cDNA clone SQ37 were analyzed. The predicted coding region of SQ37 shows high homology with that of the human gene for the small proline-rich protein 1 (spr-1) (26,27). However, no function has been described for either gene product. Evidence presented in this study is consistent with the hypothe...

Male B6C3F, mice were exposed to dichloroacetic acid (DCA) in the drinking water in order to establish a dose response for the induction of hepatocellular cancer and to examine several modes of action for the carcinogenic process. Groups of animals were exposed to control, 0.05, 0.5, 1, 2, or 3.5 g/L DCA in the drinking water for 90-100 wk. Mean daily doses (MDD) of 8, 84, 168, 315, and 429 mg/kg/d of DCA were calculated. The prevalence (percent of animals) with hepatocellular carcinoma (HC) was significantly increased in the 1-g/L (71%), 2-g/L (95%), and 3.5-g/L (100%) treatment groups when compared to the control (26%). HC multiplicity (tumors/animal) was significantly increased by all DCA treatments-0.05 g/L (0.58), 0.5 g/L (0.68), 1 g/L (1.29), 2 g/L (2.47), and 3.5 g/L (2.90)-compared to the control group (0.28). Based upon HC multiplicity, a no-observed-effect level (NOEL) for hepatocarcinogenicity could not be determined. Hepatic peroxisome proliferation was significantly increased only for 3.5 g/L DCA treatment at 26 wk. and did not correlate with the liver tumor response. The severity of hepatotoxicity increased with DCA concentration. Below 1 g/L, hepatotoxicity was mild and transient as demonstrated by the severity indices and serum lactate dehydrogenase activity. An analysis of generalized hepatocyte proliferation reflected the mild hepatotoxicity and demonstrated no significant treatment effects on the labeling index of hepatocytes outside proliferative lesions. Consequently, the induction of liver cancer by DCA does not appear to be conditional upon peroxisome induction or chemically sustained cell proliferation. Hepatotoxicity, especially at the higher doses, may exert an important influence on the carcinogenic process.

Specific [3H]retinoic acid (RA)-binding sites in nuclear and cytosolic extracts prepared from human myeloblastic leukemia HL-60 cells have been detected by sucrose density gradient sedimentation and size-exclusion highperformance liquid chromatography (HPLC) analyses. This RA-binding activity migrated as a single peak with an apparent molecular weight of 50,000 and >95% of the total binding activity was associated with the nuclear extract. Nuclear extracts prepared from COS-1 cells transfected with an expression vector for the nuclear RA receptors RARa or RARE were enriched (20-to 100-fold) with a RA-binding activity that coeluted by size-exclusion HPLC with the putative RAR from HL-60 cells. The HL-60 nuclear receptor exhibited high affinity binding of RA and its benzoic acid analogs ChS5, Ch3O, Ro 13-7410, and SRI 6409-40 and low-affinity binding of retinol, Ro 8-8717, and SRI 5442-60, correlating well with the biological activity of these compounds in HL-60 cells. Saturation binding and Scatchard plot analyses of the binding of RA to the nuclear HL-60 receptor yielded an apparent dissociation constant of -0.46 nM and 1400 ± 100 receptor sites per cell. Northern blot analyses of poly(A)+ RNA with cDNA probes specific for RARa and RARf indicated that HL-60 cells contain predominantly transcripts encoded by the RARa gene. Our results suggest that the observed nuclear RA-binding activity in HL-60 cells might mediate the action of RA in these cells.Retinoids, analogs of vitamin A, are modulators of cellular proliferation and differentiation in many cell types in vitro as well as in vivo (1-3). In human myeloblastic leukemia HL-60 cells, precursor cells that are able to undergo either myeloid or monocytic differentiation (4-8), retinoids cause terminal differentiation into morphologically mature granulocytes (9-12). This induction of differentiation is accompanied by several molecular changes, including an increase in the expression of NAD glycohydrolase (13) and transglutaminase type II activity (14) and a reduction in the levels of the protooncogene c-myc (15, 16).The mechanism by which retinoids induce differentiation in HL-60 cells remains to be established. Several studies have demonstrated that these cells do not contain detectable levels of either cellular retinol (CRBP)-or cellular retinoic acid (CRABP)-binding proteins (refs. 17,18; A.M.J. and T. R. Breitman, unpublished observations). Additional studies with a specific series of benzoic acid analogs of RA, the Ch series, showed that these retinoids, which do not bind to CRABP or CRBP, are very effective inducers of differentiation in HL-60 cells (19-21). These observations indicate that CRBP and CRABP are not involved in the mechanism of action of retinoids in HL-60 cells. It was speculated that other, high affinity receptors are involved in this signal transduction of retinoids (21,22). Recently, cDNAs of two nuclear RA receptors, RARa and RARP3, were cloned and sequenced (23)(24)(25)(26). These RARs are part of a larger gene family of ligand-re...

No abstract

Exposure of rats to diesel emissions results in the development of lung tumors. The objective of this study was to determine whether the polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs or other polycyclic organic matter adsorbed to diesel particles induces the formation of DNA adducts in the lung when compared to particles with little or no adsorbed organic matter. Rats were exposed to diesel emissions containing particles with over 30% solvent-extractable adsorbed organic matter and to particles with < 0.1% adsorbed organic matter (carbon black particles and TiO2). Wistar rats were exposed to diesel emissions (7.5 mg/m3) for 2 months, 6 months and 2 years and for 2 years to carbon black (11.3 mg/m3) and TiO2 particles (10.4 mg/m3) to compare tumorigenic response and DNA adduct formation in the lung. Two versions of the 32P-postlabeling assay for the detection of DNA adducts were used to tentatively identify nitrated-amine or arylamine adducts formed relative to other nitro PAH based on the demonstrated sensitivity of these adducts to nuclease P1 treatment. Total adduct levels were determined for peripheral lung tissue DNA as detected in a diagonal radioactive zone. One major adduct which migrated outside this region (adduct 1) and a nuclease P1-sensitive adduct (adduct 2) were quantitated separately. Adduct 1 increased significantly over time in the filtered air exposed animals but decreased markedly at the 2 year time points regardless of particle type, presumably as a result of adduct dilution through de novo cell synthesis or cell proliferation invoked in response to particle loading and/or effect on the endogenous synthesis or degradation of DNA reactive moieties. The nuclease sensitive adduct (adduct 2), possibly resulting from exposure to nitro-PAHs, was detected in diesel-exposed rats but was not detected in the rats exposed to TiO2 and carbon black. No significant elevation in PAH-derived adducts, relative to the filtered air controls, was observed in the rodents exposed to diesel emission. Our data suggest that long-term contact with these particles may result in a cell proliferative response, enhanced degradation of I-compounds not related to cell proliferation, and/or synthesis of I-compounds, irrespective of the differences in organic content associated with the three particle types. This response may be an important factor in explaining the reported similarity in tumorigenic response in rodents exposed to diesel emissions, carbon black and TiO2 particles.