X-ray structure of methanol dehydrogenase from<Emphasis Type="Italic"> Paracoccus denitrificans</Emphasis> and molecular modeling of its interactions with cytochrome<Emphasis Type="Italic"> c</Emphasis>-551i

The X-ray structure of methanol dehydrogenase (MEDH) from Paracoccus denitrificans (MEDH-PD) was determined at 2.5 A resolution using molecular replacement based on the structure of MEDH from Methylophilus methylotrophus W3A1 (MEDH-WA). The overall structures from the two bacteria are similar to each other except that the former has a longer C-terminal tail in each subunit and shows local differences in several insertion regions. The "X-ray sequence" of the segment alphaGly444-alphaLeu452 was established, including one insertion and seven replacements compared with the reported sequence. The primary electron acceptor of MEDH-PD is cytochrome c-551i (Cyt c551i). Based on the crystal structure of MEDH-PD and of the published structure of Cyt c551i, their interactions were investigated by molecular modeling. As a guide and starting point, the covalently attached cytochrome and PQQ domains of the alcohol dehydrogenase from Pseudomonas putida HK5 (ADH2B) were used. In the modeling, two molecules of Cyt c551i could be accommodated in their interaction with the MEDH heterotetramer in accordance with the two-fold molecular symmetry of the latter. Two models are proposed, in both of which electrostatic and hydrogen bonding interactions make major contributions to inter-protein binding. One of these models involves salt bridges from alphaArg99 of MEDH to the heme propionic acids of Cyt c551i and the other involves salt bridges from alphaArg426 of MEDH to Glu112 of Cyt c551i. Both involve salt bridges from alphaLys93 of MEDH to Asp75 of Cyt c551i. The size and nature of the cytochrome/quinoprotein heterodimer interfaces and calculations of electronic coupling and electron transfer rates favor one of these models over the other.     

Autoregulation of the human liver X receptor alpha promoter

Previous work has implicated the nuclear receptors liver X receptor alpha (LXR alpha) and LXR beta in the regulation of macrophage gene expression in response to oxidized lipids. Macrophage lipid loading leads to ligand activation of LXRs and to induction of a pathway for cholesterol efflux involving the LXR target genes ABCA1 and apoE. We demonstrate here that autoregulation of the LXR alpha gene is an important component of this lipid-inducible efflux pathway in human macrophages. Oxidized low-density lipoprotein, oxysterols, and synthetic LXR ligands induce expression of LXR alpha mRNA in human monocyte-derived macrophages and human macrophage cell lines but not in murine peritoneal macrophages or cell lines. This is in contrast to peroxisome proliferator-activated receptor gamma (PPAR gamma)-specific ligands, which stimulate LXR alpha expression in both human and murine macrophages. We further demonstrate that LXR and PPAR gamma ligands cooperate to induce LXR alpha expression in human but not murine macrophages. Analysis of the human LXR alpha promoter led to the identification of multiple LXR response elements. Interestingly, the previously identified PPAR response element (PPRE) in the murine LXR alpha gene is not conserved in humans; however, a different PPRE is present in the human LXR 5'-flanking region. These results have implications for cholesterol metabolism in human macrophages and its potential to be regulated by synthetic LXR and/or PPAR gamma ligands. The ability of LXR alpha to regulate its own promoter is likely to be an integral part of the macrophage physiologic response to lipid loading.     

The Cause of the Difference in Leaf Net Photosynthetic Rate between Two Soybean Cultivars

To explore the cause of difference in photosynthetic performance between different cultivars of crops, leaf net photosynt rate (P _N) and photosystem 2 (PS2) photochemical efficiency (F_v/F_m), apparent quantum yield of carbon assimilation (_c), electron transport rate, photophosphorylation activity, etc. were measured in two soybean cultivars, Heinong 42 and Heinong 37. At pod setting and filling, significant differences in P _N between them were observed. The former with a higher P _N (from 7 to 38 %) had a significantly higher leaf thickness, leaf dry mass/area (LMA), chlorophyll content, soluble protein content, apparent quantum yield of electron transport through PS2 (_e), carboxylation efficiency (CE), and ribulose-1,5-bisphosphate carboxylase (RuBPC) activity. The significantly higher P _N of Heinong 42 is mainly due to its higher content and activity of RuBPC.     

A novel human hydroxysteroid dehydrogenase like 1 gene (HSDL1) is highly expressed in reproductive tissuesa

We report the cloning and characterization of a novel human hydroxysteroid dehydrogenase like gene (HSDL1) located on human chromosome 16q24.2. The HSDL1 cDNA is 3407 base pair in length, encoding a 309 amino acid polypeptide related to human 17-HSD3. Northern blot reveals that the HSDL1 is highly expressed in testis and ovary. In situ hybridization indicates that the expression of HSDL1 is predominantly increased in the prostate cancer tissue compared with the normal prostate tissue, which suggests that the gene expression is important to the arising of prostate cancer.     

Kappa- but not delta-opioid receptors mediate effects of ischemic preconditioning on both infarct and arrhythmia in rats

Two series of experiments were performed in the isolated perfused rat heart to determine the role of kappa- and delta-opioid receptors (OR) in cardioprotection of ischemic preconditioning (IP). In the first series of experiments, it was found that IP with two cycles of 5-min regional ischemia followed by 5-min reperfusion each reduced infarct size induced by 30-min ischemia, and the ameliorating effect of IP on infarct was attenuated with blockade of either 5 x 10(-6) mol/l nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, or 5 x 10(-6) mol/l naltrindole (NTD), a selective delta-OR antagonist. The second series showed that U50,488H, a selective kappa-OR agonist, or D-Ala(2)-D-leu(5)-enkephalin (DADLE), a selective delta-OR agonist, dose dependently reduced the infarct size induced by ischemia, which mimicked the effects of IP. The effect of 10(-5) mol/l U50,488H on infarct was significantly attenuated by blockade of protein kinase C (PKC) with specific PKC inhibitors, 5 x 10(-6) mol/l chelerythrine or 8 x 10(-7) mol/l calphostin C, as well as by blockade of ATP-sensitive K(+) (K(ATP)) channels with blockers of the channel, 10(-5) mol/l glibenclamide or 10(-4) mol/l 5-hydroxydecanoate. IP also reduced arrhythmia induced by ischemia. Nor-BNI, but not NTD, attenuated, while U50,488H, but not DADLE, mimicked the antiarrhythmic action of IP. In conclusion, the present study has provided first evidence that kappa-OR mediates the ameliorating effects of IP on infarct and arrhythmia induced by ischemia, whereas delta-OR mediates the effects only on infarct. Both PKC and K(ATP) channels mediate the effect of activation of kappa-OR on infarct.     

Distinct topologies of mono- and decavanadate binding and photo-oxidative cleavage in the sarcoplasmic reticulum ATPase

UV irradiation of the sarcoplasmic reticulum (SR) ATPase in the presence of vanadate cleaves the enzyme at either of two different sites. Under conditions favoring the presence of monovanadate, and in the presence of Ca(2+), ADP, and Mg(2+), cleavage results in two fragments of 71- and 38-kDa electrophoretic mobility. On the other hand, under conditions permitting formation of decavanadate, and in the absence of Ca(2+) and ADP, cleavage results in two fragments of 88- and 21-kDa electrophoretic mobility. The amino terminus resulting from cleavage is blocked and resistant to Edman degradation. However, the initial photo-oxidation product can be reduced with NaB(3)H(4,) resulting in incorporation of radioactive (3)H label. Extensive digestion of the labeled protein with trypsin then yields labeled peptides that are specific for the each of the photo-oxidation conditions, and can be sequenced after purification. Collection of the Edman reaction fractional products reveals the radioactive label and demonstrates that Thr(353) is the residue oxidized by monovanadate at the phosphorylation site (i.e. Asp(351)). Correct positioning of monovanadate at the phosphorylation site requires binding of Mg(2+) and ADP to the Ca(2+)-dependent conformation of the enzyme. Subsequent hydrolytic cleavage is likely assisted by the neighboring Asp(601), and yields the 71- and 38-kDa fragments. On the other hand, Ser(186) (and possibly the following three residues: Val(187), Ile(188), and Lys(189)) is the residue that is photo-oxidized by decavanadate in the absence of ADP. Hydrolytic cleavage of the oxidized product at this site is likely assisted by neighboring acidic residues, and yields the 88- and 21-kDa fragments. The bound decavanadate, which we find to produce steric interference with TNP-AMP binding, must therefore extend to the A domain (i.e. small cytosolic loop) in order to oxidize Ser(186). This protein conformation is only obtained in the absence of Ca(2+).     

Association of the mouse infertility factor DAZL1 with actively translating polyribosomes

The DAZ (Deleted in AZoospermia) gene family was isolated from a region of the human Y chromosome long arm that is deleted in about 10% of infertile men with idiopathic azoospermia. DAZ and an autosomal DAZ-like gene, DAZL1, are expressed in germ cells only. They encode proteins with an RNA recognition motif and with either a single copy (in DAZL1) or multiple copies (in DAZ) of a DAZ repeat. A role for DAZL1 and DAZ in spermatogenesis is supported by their homology to a Drosophila male infertility protein Boule and by sterility of Dazl1 knock-out mice. The biological function of these proteins remains unknown. We found that DAZL1 and DAZ bound similarly to various RNA homopolymers in vitro. We also used an antibody against the human DAZL1 to determine the subcellular localization of DAZL1 in mouse testis. The sedimentation profiles of DAZL1 in sucrose gradients indicate that DAZL1 is associated with polyribosomes, and further capture of DAZL1 on oligo(dT) beads demonstrates that the association is mediated through the binding of DAZL1 to poly(A) RNA. Our results suggest that DAZL1 is involved in germ-cell specific regulation of mRNA translation.     

A family of gamma-like calcium channel subunits

The gamma subunit was initially identified as an auxiliary subunit of the skeletal muscle calcium channel complex. Evidence for the existence of further gamma subunits arose following the characterization of a genetic defect that induces epileptic seizures in stargazer mice. We present here the first account of a family of at least five putative gamma subunits that are predominantly expressed in brain. The gamma-2 and gamma-4 subunits shift the steady-state inactivation curve to more hyperpolarized potentials upon coexpression with the P/Q type alpha(1A) subunit. The coexpression of the gamma-5 subunit accelerates the time course of current activation and inactivation of the alpha(1G) T-type calcium channel.     

Genetic immunization with lung-targeting macroaggregated polyethyleneimine-albumin conjugates elicits combined systemic and mucosal immune responses

Genetic immunization is a novel form of vaccination in which transgenes are delivered into the host to produce the foreign protein within host cells. Although systemic immune responses have been relatively easy to induce by genetic immunization, the induction of regional and mucosal immunity has often been more challenging. To address the problem of eliciting mucosal immunity in the lung, we utilized macroaggregated albumin to target plasmid DNA to the lung. Macroaggregated albumin is trapped in the lung after i. v. injection, and it is routinely used in radiolabeled form as an imaging modality to evaluate pulmonary blood flow. To couple DNA to this targeting agent, polyethyleneimine (a polycation that binds DNA and enhances transfection) was conjugated to serum albumin, and the conjugate was aggregated by heating to produce particles of 25-100 microm. The resulting particles bound plasmid DNA avidly, and when injected i.v. in mice, the particles distributed in the peripheral lung tissue in the alveolar interstitium. Particle-bound luciferase plasmid transfected a variety of cell lines in vitro, and after i.v. injection, gene expression was detected exclusively in the lung. Using human growth hormone as the encoded foreign Ag for immunization, i.v. injection of the particle-bound plasmid elicited both pulmonary mucosal and systemic immune responses, whereas naked DNA injected either i.v. or i.m. elicited only systemic responses. Thus, particle-bound plasmid DNA may have utility for genetic immunization by intravascular delivery to the lung and potentially to other organs and tissues.