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).     

Uncertainty and conservatism in assessing environmental impact under paragraph 316(b): lessons from the Hudson River case

Initially, regulation of cooling water intakes under paragraph 316(b) was extremely conservative due to the rapid increase predicted for generating capacity, and to the uncertainty associated with our knowledge of the effects of entrainment and impingement. The uncertainty arose from four main sources: estimation of direct plant effects; understanding of population regulatory processes; measurement of population parameters; and predictability of future conditions. Over the last quarter-century, the uncertainty from the first three sources has been substantially reduced, and analytical techniques exist to deal with the fourth. In addition, the dire predictions initially made for some water bodies have not been realized, demonstrating that populations can successfully withstand power plant impacts. This reduced uncertainty has resulted in less conservative regulation in some, but not all venues. New York appears to be taking a more conservative approach to cooling water intakes. The conservative approach is not based on regulations, but in a philosophy that power plant mortality is an illegitimate use of the aquatic resources. This philosophy may simplify permitting decisions, but it does not further the development of a science-based definition of adverse environmental impact.     

Evidence of two forms of poly(A) polymerase in germinated wheat embryos and their regulation by a novel protein kinase

Two forms of poly(A) polymerase (PAPI and PAPII) from germinated wheat embryos have been resolved on DEAE-cellulose ion-exchange chromatography by a linear gradient of 0-500 mM (NH(4))(2)SO(4). Further purification shows that both forms are monomeric in nature with an identical molecular weight, approximately 65 kDa. The phosphoprotein nature of PAPI and PAPII has been established by in vivo labelling with (32)P-orthophosphate. Acid hydrolysis of both (32)P-labelled purified PAPI and PAPII has revealed that phosphorylations generally take place in serine and threonine residues. PAPI and PAPII have also been characterised with respect to V(max) and K(m) for poly(A). The V(max) and K(m) values of PAPI are 28.57 and 11.37 microg, respectively, whereas 34.48 and 7.04 microg of PAPII. In vitro dephosphorylation of the purified enzyme by alkaline phosphatase leads to a significant loss of the enzyme activity, which is regained upon phosphorylation by a 65 kDa protein kinase (PK) purified from wheat embryos. The extent of phosphorylation by protein kinase shows that PK has similar affinity towards both PAPI and PAPII, whereas the phosphate incorporation in PAPII is twofold higher than PAPI suggesting their distinct chemical nature.     

Homology model of human corticosteroid binding globulin: a study of its steroid binding ability and a plausible mechanism of steroid hormone release at the site of inflammation

Corticosteroid binding globulin (CBG) and thyroxin binding globulin (TBG) both belong to the same SERPIN superfamily of serine-proteinase inhibitors but in the course of evolution CBG has adapted to its new role as a transport agent of insoluble hormones. CBG binds corticosteroids in plasma, delivering them to sites of inflammation to modify the inflammatory response. CBG is an effective drug carrier for genetic manipulation, and hence there is immense biological interest in the location of the hormone binding site. The crystal structure of human CBG (hCBG) has not been determined, but sequence alignment with other SERPINs suggests that it conforms as a whole to the tertiary structure shared by the superfamily. Human CBG shares 52.15% and 55.50% sequence similarity with alpha1-antitrypsin and alpha1-antichymotrypsin, respectively. Multiple sequence alignment among the three sequences shows 73 conserved regions. The molecular structures of alpha1-antitrypsin and alpha1-antichymotrypsin, the archetype of the SERPIN superfamily, obtained by X-ray diffraction methods are used to develop a homology model of hCBG. Energy minimization was applied to the model to refine the structure further. The homology model of hCBG contains 371 residues (His13 to Val383 ). The secondary structure comprises 11 helices, 15 turns and 11 sheets. The putative corticosteroid binding region is found to exist in a pocket between beta-sheets S4, S10, S11 and alpha helix H10. Both cortisol and aldosterone are docked to the elongated hydrophobic ligand binding pocket with the polar residues at the two extremities. A difference accessible surface area (DASA) study revealed that cortisol binds with the native hCBG more tightly than aldosterone. Cleavage at the Val379-Met380 peptide bond causes a deformation of hCBG (also revealed through a DASA study). This deformation could probably trigger the release of the bound hormone. Figure Stereoscopic view of the ribbon diagram of hCBG complexed with cortisol. The bound cortisol is shown in space filling model in blue. Helices and sheets are shown in red and magenta respectively. Turns are shown in yellow.     

Dysregulation of EGF family of growth factors and COX-2 in the uterus during the preattachment and attachment reactions of the blastocyst with the luminal epithelium correlates with implantation failure in LIF-deficient mice

Various mediators, including cytokines, growth factors, homeotic gene products, and prostaglandins (PGs), participate in the implantation process in an autocrine, paracrine, or juxtacrine manner. However, interactions among these factors that result in successful implantation are not clearly understood. Leukemia inhibitory factor (LIF), a pleiotropic cytokine, was shown to be expressed in uterine glands on day 4 morning before implantation and is critical to this process in mice. However, the mechanism by which LIF executes its effects in implantation remains unknown. Moreover, interactions of LIF with other implantation-specific molecules have not yet been defined. Using normal and delayed implantation models, we herein show that LIF is not only expressed in progesterone (P4)-primed uterine glands before implantation in response to nidatory estrogen, it is also induced in stromal cells surrounding the active blastocyst at the time of the attachment reaction. This suggests that LIF has biphasic effects: first in the preparation of the receptive uterus and subsequently in the attachment reaction. The mechanism by which LIF participates in these events was addressed using LIF-deficient mice. We observed that while uterine cell-specific proliferation, steroid hormone responsiveness, and expression patterns of several genes are normal, specific members of the EGF family of growth factors, such as amphiregulin (Ar), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin, are not expressed in LIF(-/-) uteri before and during the anticipated time of implantation, although EGF receptor family members (erbBs) are expressed correctly. Furthermore, cyclooxygenase-2 (COX-2), an inducible rate-limiting enzyme for PG synthesis and essential for implantation, is aberrantly expressed in the uterus surrounding the blastocyst in LIF(-/-) mice. These results suggest that dysregulation of specific EGF-like growth factors and COX-2 in the uterus contributes, at least partially, to implantation failure in LIF(-/-) mice. Since estrogen is essential for uterine receptivity, LIF induction, and blastocyst activation, it is possible that the nidatory estrogen effects in the P4-primed uterus for implantation are mediated via LIF signaling. However, we observed that LIF can only partially resume implantation in P4-primed, delayed implanting mice in the absence of estrogen, suggesting LIF induction is one of many functions that are executed by estrogen for implantation.