KIN, a mammalian nuclear protein immunologically related to E. coli RecA protein

A polypeptide of about 120 kDa, called KIN, has been identified in rat FR 3T3 cells by immunoblotting using affinity-purified antibodies against the RecA protein of Escherichia coli (38 kDa). The KIN protein as shown by fluorescent light microscopy and electron microscopy is essentially concentrated in the nucleus. Its level is higher in proliferating than in quiescent cells. Cell treatment with mitomycin C increases the level of the KIN protein. We sought similar proteins in other mammalian cells. Proteins with the same electrophoretic mobility were detected in mouse, monkey and human cell lines as well as in rat and mouse embryos.     

Isocitrate dehydrogenase kinase/phosphatase

In Escherichia coli, isocitrate dehydrogenase (IDH) is regulated by phosphorylation. This phosphorylation cycle is catalyzed by an unusual, bifunctional protein:IDH kinase/phosphatase. IDH kinase/phosphatase is expressed from a single gene, aceK, and both activities are catalyzed by the same polypeptide. The amino acid sequence of IDH kinase/phosphatase does not exhibit the characteristics which are typical of other protein kinases, although it does contain a consensus ATP binding site. The available evidence suggests that the IDH kinase and IDH phosphatase reactions occur at the same active site and that the IDH phosphatase reaction results from the back reaction of IDH kinase tightly coupled to ATP hydrolysis. The function of the IDH phosphorylation cycle is to control the flux of isocitrate through the glyoxylate bypass. This pathway is essential for growth on acetate because it prevents the quantitative loss of the acetate carbons as CO2 in the Krebs' cycle. IDH kinase/phosphatase monitors general metabolism by responding to the levels of a wide variety of metabolites, many of which activate IDH phosphatase and inhibit IDH kinase. The ability of IDH kinase/phosphatase to monitor general metabolism allows. the IDH phosphorylation cycle to compensate for substantial perturbations of the system, such as a 15-fold overproduction of IDH. The significance of the cellular level of IDH kinase/phosphatase has also been evaluated. The level of this protein is in great excess of that required for steady-state growth on acetate. In contrast, IDH kinase/phosphatase is, in some cases, rate-limiting for the dephosphorylation of IDH which results when preferred carbon sources are added to cultures growing on acetate.     

Photolabeling of a O2-. generating protein in bovine polymorphonuclear neutrophils by an arylazido NADP+ analog

A plasma membrane fraction of bovine polymorphonuclear neutrophils enriched in NADPH-dependent, O2-. generating oxidase activity, and a number of fractions solubilized in detergent, recovered during the course of the purification of this oxidase have been tested for their ability to react with radiolabeled N-4-azido-2-nitrophenyl aminobutyryl NADP+ (arylazido NADP+ or NAP4-NADP+). In the dark, NAP4-NADP+ and its reduced form NAP4-NADPH, were found to inhibit competitively the NADPH-dependent O2-. generating oxidase activity of the plasma membrane fraction of bovine neutrophils activated by phorbol myristate acetate. The nitrene derivative formed upon photoirradiation of NAP4-NADP(H) bound covalently to different proteins of the plasma membrane. Photolabeling of these proteins was prevented by preincubation with an excess of NADPH. Photolabeling of a protein of 65,000 Mr was decreased by omission of phorbol myristate acetate as activating agent of the respiratory burst in neutrophils or by addition of micromolar amounts of Cibacron Blue and mersalyl which are known to inhibit the production of O2-. by the plasma membrane fraction. During the course of the purification procedure, the 65000 Mr protein emerged as the preferentially photolabeled protein. These data, in agreement with previous findings concerning the purification of an NADPH-dependent, O2-. generating oxidase protein of Mr 65000 from bovine neutrophils (Doussière, J. and Vignais, P.V. (1985) Biochemistry 24, 7231-7239), strongly suggest that a single protein of Mr 65000, located in the plasma membrane fraction of bovine neutrophils, is able to act both as an NADPH deshydrogenase and as an oxygen reductase to generate O2-.     

Neointima formation after vascular injury is angiotensin II mediated.

Smooth muscle proliferation of injured blood vessels leads to pathologically significant stenosis in animals and humans. We report here the pharmacological confirmation of an involvement of angiotensin II in this process as a major, necessary mediator of neointima formation. In the rat carotid artery, an animal model of post-angioplastic restenosis, we have obtained by local intraluminal infusion of peptidic angiotensin II antagonist after balloon catheterization, suppression of neointima formation and preservation of the luminal integrity. Sham operated control animals treated without medication and operated control animals treated simultaneously with angiotensin converting enzyme inhibitor and with agonistic angiotensin II, suffered major stenosis through the myoproliferative response of the injured vessel. These results prove that angiotensin II plays a key role as a mediator of vascular neointima formation.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Fermentation and isolation of C10-deacetylase for the production of 10-deacetylbaccatin III from baccatin III

10-Deacetylabaccatin III (10 DAB), an important precursor for paclitaxel semisynthesis, is enhanced in yew extracts using C10-deacetylase and C13-deacylase enzymes.(4) C10-deacetylase is an intracellular enzyme produced by the fermentation of a soil microorganism, Nocardioides luteus (SC 13912). During the fermentation of Nocardioides luteus, the growth of cells reaches a maximum growth at 28 h. C10-deacetylase enzyme activity starts at 26 h and peaks at 38 h of the fermentation. The cells are recovered by centrifugation. The C10-deacetylase enzyme was purified from the Nocardioides luteus cells. The enzyme was purified 190-fold to near homogeneity. The purified enzyme appeared as a single band on 12.5% SDS-PAGE analysis with a molecular weight of 40,000 daltons. (c) 1995 John Wiley & Sons, Inc.     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.