Na(+) pump alpha 2-isoform specifically couples to contractility in vascular smooth muscle: evidence from gene-targeted neonatal mice

The relative expression of ₁ - and ₂-Na⁺/K⁺-ATPase isoforms found in vascular smooth muscle is developmentally regulated and under hormonal and neurogenic control. The physiological roles of these isoforms in vascular function are not known. It has been postulated that the ₁-isoform serves a "housekeeping" role, whereas the ₂-isoform localizes to a subsarcolemmal compartment and modulates contractility. To test this hypothesis, isoform-specific gene-targeted mice in which the mRNA for either the ₁- or the ₂-Na⁺/K⁺-ATPase isoform was ablated were utilized. Both of these knockouts, and , are lethal; the latter dies at birth, which allows this neonatal aorta to be studied. Isometric force in -aorta was more sensitive to contractile agonists and less sensitive to the vasodilators forskolin and sodium nitroprusside (SNP) than wild-type (WT) aorta; -aortas had intermediate values. In contrast, neonatal -aorta was similar to WT. Western blot analysis indicated a population of 70% ₁- and 30% ₂-isoforms in the WT. Thus in terms of the total Na⁺/K⁺-ATPase protein, the -aorta (at 70%) would be similar to the -aorta (at 65%) but with a dramatically different phenotype. These data suggest that individual -isoforms of the Na⁺/K⁺-ATPase differ functionally and that the ₂-isoform couples more strongly to activation-relaxation pathways. Three-dimensional image-acquisition and deconvolution analyses suggest that the ₂-isoform is distributed differently than the ₁-isoform. Importantly, these isoforms do not localize to the same regions. sodium; potassium; ATPase; contraction; transgenic     

Polar expression of ErbB-2/HER2 in epithelia. Bimodal regulation by Lin-7

ErbB-2/HER2 drives epithelial malignancies by forming heterodimers with growth factor receptors. The primordial invertebrate receptor is sorted to the basolateral epithelial surface by binding of the PDZ domain of Lin-7 to the receptor's tail. We show that all four human ErbBs are basolaterally expressed, even when the tail motif is absent. Mutagenesis of hLin-7 unveiled a second domain, KID, that binds to the kinase region of ErbBs. The PDZ interaction mediates stabilization of ErbB-2 at the basolateral surface. On the other hand, binding of KID is involved in initial delivery to the basolateral surface, and in its absence, unprocessed ErbB-2 molecules are diverted to the apical surface. Hence, distinct domains of Lin-7 regulate receptor delivery to and maintenance at the basolateral surface of epithelia.     

Immobilization of radionuclides and heavy metals through anaerobic bio-oxidation of Fe(II)

Adsorption of heavy metals and radionuclides (HMR) onto iron and manganese oxides has long been recognized as an important reaction for the immobilization of these compounds. However, in environments containing elevated concentrations of these HMR the adsorptive capacity of the iron and manganese oxides may well be exceeded, and the HMR can migrate as soluble compounds in aqueous systems. Here we demonstrate the potential of a bioremediative strategy for HMR stabilization in reducing environments based on the recently described anaerobic nitrate-dependent Fe(II) oxidation by Dechlorosoma species. Bio-oxidation of 10 mM Fe(II) and precipitation of Fe(III) oxides by these organisms resulted in rapid adsorption and removal of 55 microM uranium and 81 microM cobalt from solution. The adsorptive capacity of the biogenic Fe(III) oxides was lower than that of abiotically produced Fe(III) oxides (100 microM for both metals), which may have been a result of steric hindrance by the microbial cells on the iron oxide surfaces. The binding capacity of the biogenic oxides for different heavy metals was indirectly correlated to the atomic radius of the bound element. X-ray absorption spectroscopy indicated that the uranium was bound to the biogenically produced Fe(III) oxides as U(VI) and that the U(VI) formed bidentate and tridentate inner-sphere complexes with the Fe(III) oxide surfaces. Dechlorosoma suillum oxidation was specific for Fe(II), and the organism did not enzymatically oxidize U(IV) or Co(II). Small amounts (less than 2.5 microM) of Cr(III) were reoxidized by D. suillum; however, this appeared to be inversely dependent on the initial concentration of the Cr(III). The results of this study demonstrate the potential of this novel approach for stabilization and immobilization of HMR in the environment.     

Calcium EXAFS establishes the Mn-Ca cluster in the oxygen-evolving complex of photosystem II

The proximity of Ca to the Mn cluster of the photosynthetic water-oxidation complex is demonstrated by X-ray absorption spectroscopy. We have collected EXAFS data at the Ca K-edge using active PS II membrane samples that contain approximately 2 Ca per 4 Mn. These samples are much less perturbed than previously investigated Sr-substituted samples, which were prepared after Ca depletion. The new Ca EXAFS clearly shows backscattering from Mn at 3.4 A, a distance that agrees with that surmised from previously recorded Mn EXAFS. This result is also consistent with earlier related experiments at the Sr K-edge, using samples that contained functional Sr, that show Mn is approximately 3.5 A distant from Sr. The totality of the evidence clearly advances the notion that the catalytic center of oxygen evolution is a Mn-Ca heteronuclear cluster.     

Pathogenic poxviruses reveal viral strategies to exploit the ErbB signaling network.

Virulence of poxviruses, the causative agents of smallpox, depends on virus-encoded growth factors related to the mammalian epidermal growth factor (EGF). Here we report that the growth factors of Shope fibroma virus, Myxoma virus and vaccinia virus (SFGF, MGF and VGF) display unique patterns of specificity to ErbB receptor tyrosine kinases; whereas SFGF is a broad-specificity ligand, VGF binds primarily to ErbB-1 homodimers, and the exclusive receptor for MGF is a heterodimer comprised of ErbB-2 and ErbB-3. In spite of 10- to 1000-fold lower binding affinity to their respective receptors, the viral ligands are mitogenically equivalent or even more potent than their mammalian counterparts. This remarkable enhancement of cell growth is due to attenuation of receptor degradation and ubiquitination, which leads to sustained signal transduction. Our results imply that signal potentiation and precise targeting to specific receptor combinations contribute to cell transformation at sites of poxvirus infection, and they underscore the importance of the often ignored low-affinity ligand-receptor interactions.     

Recombinant Human Cytomegalovirus Protease with a C-Terminal (His)6Extension: Purification, Autocatalytic Release of the Mature Enzyme, and Biochemical Characterization

Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed inEscherichia coli.The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site. The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at amino acids 256–257 constitutes a site naturally cleaved by the protease during capsid maturation. The 268-amino-acid polypeptide with the (His)₆tag was expressed at high levels inE. colias inclusion bodies. After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni²⁺affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256–257 to remove 12 amino acids including the (His)₆tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus. Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy. The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein inE. coli.In addition, the refolded CMV PR could be crystallized for X-ray diffraction.     

Cloning and Expression of OX-DAPRO Degrading Genes from Soil Microbe

The strain BYK 1, capable ot utilizing DAP or OX-DAPRO as a sole source ot nitrogen and carbon was Identitied as Pseudomonas stutzeri by various microbial and biochemical tests. Transtormation experiments showed that the OX-DAPRO utilization property Is encoded by the plasmid. Restriction of the plasmid DNA toll owed by cloning ot fragments and screening on OX-DAPRO medium led to Isolation of a clone with DNA Insert of ～ 2.2 kb (designated as BSKS) which encoded OX-DAPRO degrading gel)e. The growth and OX-DAPRO utilization with this fragment was similar to wild type strain.     

Regional Blood-Brain Barrier Permeability to Amino Acids After Portacaval Anastomosis

Abstract: The influx of phenylalanine, tryptophan, leucine, and lysine across the blood-brain barrier of individual brain structures was studied in rats 7–8 weeks after a portacaval shunt or sham operation. The method involved a brief infusion of labeled amino acid in tracer quantity and quantitative autoradiography. The clearance rates of phenylalanine, tryptophan, and leucine were increased in proportion to each other in every region examined, but not by the same factor. Tryptophan clearance increased the most (about 200%) and leucine the least (about 30%), compared with phenylalanine (about 80%). This was unexpected, as all three amino acids are believed to be transported by the same mechanism. The changes were most marked in several limbic structures and the reticular formation, whereas the hypothalamus was least affected. Plasma clearance of lysine was decreased in all areas by about 70%. Since the circulating lysine concentration was decreased by 13%, the actual rate of lysine influx was even more reduced. The results demonstrate specific alterations in two different amino acid transport systems. The resulting excess brain neutral amino acids, some of which are neurotransmitter precursors, as well as reduced basic amino acid availability, may be of etiological significance in hepatic encephalopathy.