Mouse zinc transporter 1 gene provides an essential function during early embryonic development

The SLC30 family of cation diffusion transporters includes at least nine members in mammals, most of which have been documented to play a role in zinc transport. The founding member of this family, Znt1, was discovered by virtue of its ability to efflux zinc from cells and to protect them from zinc toxicity. However, its physiological functions remain unknown. To address this issue, mice with targeted knockout of the Znt1 gene were generated by homologous recombination in embryonic stem cells. Heterozygous Znt1 mice were viable. In contrast, homozygous Znt1 mice died in utero soon after implantation due to a catastrophic failure of embryonic development. Although extraembryonic membranes formed around these embryos, the embryo proper failed to undergo morphogenesis past the egg cylinder stage and was amorphous by d9 of pregnancy. Expression of the Znt1 gene was detected predominantly in trophoblasts and in the maternal deciduum during the postimplantation period (d5 to d8). The failure of homozygous Znt1 embryos to develop could not be rescued by manipulating maternal dietary zinc (either excess or deficiency) during pregnancy. However, embryos in Znt1 heterozygous females were approximately 3 times more likely to develop abnormally when exposed to maternal dietary zinc deficiency during later pregnancy than were those in wildtype females. These studies suggest that Znt1 serves an essential function of transporting maternal zinc into the embryonic environment during the egg cylinder stage of development, and further suggest that Znt1 plays a role in zinc homeostasis in adult mice.     

Synthesis and structure-activity relationship studies of cinnamic acid-based novel thiazolidinedione antihyperglycemic agents

A number of 2,4-thiazolidinedione derivatives of -phenyl substituted cinnamic acid were synthesized and studied for their PPAR agonist activity. The E-isomer of cinnamic acid, 11, showed moderate PPAR transactivation. The corresponding Z-isomer, 23, and double bond reduced derivative, 15, were found to be much less potent. Although the E-isomer showed a moderate PPAR gamma transactivation, it demonstrated a strong glucose-lowering effect in a genetic rodent model of diabetes. Results of pharmacokinetic, metabolism and permeability studies are consistent with 11 being an active prodrug with an active metabolite, 14, that has similar glucose lowering and PPAR gamma agonist properties.     

RPE65 operates in the vertebrate visual cycle by stereospecifically binding all-trans-retinyl esters

RPE65 is a major protein of unknown function found associated with the retinyl pigment epithelial (RPE) membranes [Hamel, C. P., Tsilou, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Biol. Chem. 268, 15751-15757; Bavik, C. O., Levy, F., Hellman, U., Wernstedt, C., and Eriksson, U. (1993) J. Biol. Chem. 268, 20540-20546]. RPE65 knockouts fail to synthesize 11-cis-retinal, the chromophore of rhodopsin, and accumulate all-trans-retinyl esters in the RPE. Previous studies have also shown that RPE65 is specifically labeled with all-trans-retinyl ester based affinity labeling agents, suggesting a retinyl ester binding role for the protein. In the present work, we show that purified RPE65 binds all-trans-retinyl palmitate (tRP) with a K(D) = 20 pM. These quantitative experiments are performed by measuring the quenching of RPE65 fluorescence by added tRP. The binding for tRP is highly specific because 11-cis-retinyl palmitate binds with a K(D) = 14 nM, 11-cis-retinol binds with a K(D) = 3.8 nM, and all-trans-retinol (vitamin A) binds with a K(D) = 10.8 nM. This stereospecificity for tRP is to be compared to the binding of retinoids to BSA, where virtually no discrimination is found in the binding of the same retinoids. This work provides further evidence that RPE65 functions by binding to and mobilizing the highly hydrophobic all-trans-retinyl esters, allowing them to enter the visual cycle.     

Terminal uridine nick end labelling and mitosis in breast carcinoma. Correlation with tumor grade and p53 overexpression

OBJECTIVE: To study the relationship of cell death and proliferation to histologic grade and p53 expression in invasive carcinoma of the breast. STUDY DESIGN: A total of 31 cases of infiltrating duct carcinoma of the breast were randomly selected. The terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling (TUNEL) reaction and p53 immunostaining were performed on representative paraffin-embedded tissue sections. Mitotic and apoptotic indices (MI and AI) were also measured on hematoxylin-eosin-stained sections. Histologic grade of infiltrating duct carcinoma was performed with the help of the Nottingham modification of the Bloom-Richardson system. Tumor grade and p53 overexpression were correlated with MI, AI and AI detected by TUNEL. RESULTS: There were a total of 31 infiltrating duct carcinomas of the breast, of which 13 cases were grade 1 and nine cases each were grade 2 and 3. Cells with positive TUNEL showed a strong brown nuclear positivity. TUNEL showed positivity from the periphery of the nuclear margin to the central portion. AI detected by TUNEL did not correlate with tumor grade (ANOVA, P > .05). MI was significant only in grade 1 versus grade 3 and 2 versus grade 3 carcinomas (ANOVA, P < .01). The morphologic apoptotic index was significant only in grade 1 versus grade 3 carcinomas. Nine cases showed p53 overexpression, and the rest of the cases were negative for p53 immunostaining. MI, AI and TUNEL were not significantly different in p53-negative and -positive groups. Pearson's correlation coefficient showed that AI and MI were significantly related, but there was no significant relation between AI detected by TUNEL and MI. CONCLUSION: MI is still more useful than AI or AI detected by TUNEL in differentiating various grades of carcinoma of the breast.     

Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha i subunits and regulates Galpha i-mediated cell signaling

Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein alpha subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Galpha(z) subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Galpha(z,) but also with Galpha(i), as supported by in vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind (35)S-labeled Galpha(i1) protein in an AlF(4)(-)dependent manner. The interaction between RGSZ1 and Galpha(i) was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Galpha(i1) in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Galpha(i)-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an alpha(2)-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Galpha(i) and regulates Galpha(i)-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.     

Cytochrome P450 2E1 dependent catalytic activity and lipid peroxidation in rat blood lymphocytes

To investigate the similarities in the catalytic activity of blood lymphocyte P450 2E1 in blood lymphocyte with the liver isoenzyme, NADPH dependent lipid peroxidation and activity of N-nitrosodimethyamine demethylase (NDMA-d) was studied in rat blood lymphocytes. Blood lymphocytes were found to catalyse NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA). Pretreatment with ethanol or pyrazole or acetone resulted in significant increase in the NADPH dependent lipid peroxidation and the activity of NDMA-d in blood lymphocytes and liver microsomes. In vitro addition of CCl(4) to the blood lymphocytes isolated from control or ethanol pretreated rats resulted in an increase in the NADPH dependent lipid peroxidation. Significant inhibition of the basal and CCl(4) supported NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocytes isolated from control or ethanol pretreated rats by dimethyl formamide or dimethyl sulfoxide or hexane, solvents known to inhibit P450 2E1 catalysed reactions in liver and anti- P450 2E1, have indicated the role of P450 2E1 in the NADPH dependent lipid peroxidation in rat blood lymphocytes. The data indicating similarities in the NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocyte with the liver microsome have provided evidence that blood lymphocyte P450 2E1 could be used as a surrogate to monitor and predict hepatic levels of the enzyme.     

Involvement of Elk-1 in L6E9 skeletal muscle differentiation

The binding of phosphatidylinositol(4,5)-bisphosphate (PI(4,5)P(2)) to profilin at a region distinct from the actin interaction surface is demonstrated by experiments with covalently cross-linked profilin:beta-actin. The result is in agreement with observations made with several mutant profilins and provides strong evidence for two regions on mammalian profilin mediating electrostatic interaction with phosphatidylinositol lipids; one close to the binding site for poly(L-proline), and one partially overlapping with the actin-binding surface. Congruent with this, two plant profilins, which have a reduced number of positive amino acids in one of these regions, displayed a dramatically lower binding to PI(4,5)P(2) compared to human profilin I.     

Use of equivalent loss models under Section 316(b) of the Clean Water Act

Equivalent loss models encompass a variety of life table-based approaches that can be used to convert age- and life stage-specific estimates of entrainment and impingement loss to a common, easily understood currency. This common currency can be expressed in terms of numbers of individuals, yield to the fishery, or biomass to the ecosystem. These models have at least two key uses in the Section 316(b) assessment process: screening for adverse environmental impact (AEI) and determination of environmental benefits associated with intake alternatives. This paper reviews the various forms of equivalent loss models, their data input requirements, and their assumptions and limitations. In addition, it describes how these models can be used as a second-level screening tool as part of the assessment of the potential for AEI. Given their relative simplicity and ease of use, equivalent loss models should prove to be an important tool in the arsenal of impact assessment methods for Section 316(b).