bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequences related to HLA-DR alpha chain on human chromosome 6: restriction enzyme polymorphism detected with DC alpha chain probes.
Authors: Trowsdale J , Lee J , Carey J , Grosveld F , Bodmer J , Bodmer W
Published: May 2006

Three sets of cosmid clones--containing the HLA-DR alpha chain gene and two additional related genes--were isolated from human genomic DNA libraries by using a cDNA probe for the HLA-DR alpha chain. Southern blot analysis using DNA from somatic cell hybrids indicated that all of the clones mapped to chromosome 6. Partial sequence analysis showed that the two additional related genes were highly homologous to each other, and to the HLA-DR alpha chain, in parts of the exon that encoded the alpha 2 domain but were more divergent in intron sequences. One of the genes corresponds to the HLA-DR-related DC series. DNA probes made from this gene revealed marked restriction enzyme polymorphism when hybridized to genomic DNA from HLA-DR typed homozygous cell lines. The patterns obtained from a number of homozygous and heterozygous cell lines correlated with the HLA-DR crossreactive serotypes and also indicated that there is a further sequence in the haploid human genome that is closely homologous with the DC alpha chain sequence. One family was studied and showed the expected HLA-DR-associated inheritance of restriction enzyme patterns. No polymorphism has yet been demonstrated in restriction enzyme fragments that include the other cloned sequence, which may correspond to the SB alpha chain gene or to a novel HLA-DR-related gene. These experiments indicate that there are at least three sequences in the human genome related, but not identical, to the HLA-DR alpha chain gene.
Stabilization of proteins by a bacteriophage T4 gene cloned in Escherichia coli.
Authors: Simon L D , Randolph B , Irwin N , Binkowski G
Published: May 2006

The cloned bacteriophage T4 pin gene functions to stabilize several different kinds of proteins in Escherichia coli bacteria. Incomplete proteins such as puromycyl polypeptides, abnormal but complete proteins such as the lambda phage tsO protein, and labile eukaryotic proteins encoded by genes cloned in E. coli such as mature human fibroblast interferon are stabilized in cells in which the T4 pin gene is expressed. The cloned T4 pin gene does not seem to affect the turnover of normal E. coli proteins.
Isolation of cellular DNA sequences that allow expression of adjacent genes.
Authors: Fried M , Griffiths M , Davies B , Bjursell G , La Mantia G , Lania L
Published: May 2006

We have employed a strategy for the isolation and identification of cellular control (expression) sequences dependent on their ability to confer expression on a selectable gene devoid of its own expression sequences. The polyoma virus (Py) Hae II-BamHI DNA fragment, which comprises 84% of the intact viral DNA and contains the Py transforming region but lacks Py 5' expression sequences, was decreased markedly in its transformation of rat cells. Hae II-cleaved mouse cellular DNA was ligated to the Py Hae II-BamHI fragment. A transformed colony (H1) isolated after transfection of the ligated DNA onto rat cells was found to contain multiple inserts of Py DNA, most of which were biologically inactive. A transformed colony (H2) isolated after transfection of rat cells with total H1 DNA was found to contain a single insert of Py DNA. The H2 cells are highly tumorigenic and synthesize the three Py tumor antigens. Initiation of transcription of the Py early mRNAs in H2 cells occurs at the same Py nucleotides as in complete Py DNA. The viral and adjacent cellular DNA sequences were cloned from H2 cellular DNA. The transforming efficiency of the cloned Py transforming region and adjacent H2 cellular DNA was 20-40% of that of the viral DNA containing Py expression sequences. By BAL-31 deletion mapping it was observed that the first 58 base pairs of H2 cellular DNA were sufficient for the expression of the Py-transforming region. The sequence of the first 149 base pairs of the H2 cellular DNA was determined and does not show any striking similarities to upstream 5' sequences of a number of viral and host structural genes. Features of the H2 cellular sequence are discussed.
Cooperative model for the binding of Xenopus transcription factor A to the 5S RNA gene.
Authors: Hanas J S , Bogenhagen D F , Wu C W
Published: May 2006

7S particles containing 5S RNA and the transcription regulatory protein factor A have been purified to near homogeneity from Xenopus laevis oocytes. The binding of the transcription factor to the Xenopus borealis somatic 5S RNA gene has been monitored by quantitating the DNase I protection patterns of the protein-DNA interaction. Under stoichiometric binding conditions--i.e., when all added DNA binds to the factor--two protein molecules are required to saturate the 5S RNA gene. Under equilibrium binding conditions, titration of the 5S RNA gene with factor A results in a sigmoidal binding isotherm suggesting a cooperative interaction; half-saturation of binding is observed at a free factor A concentration of 1 nM. Cooperative binding between factor A and the 5S RNA gene may contribute to the stability of the transcription apparatus and its maintenance during cell division.
Evidence for phosphorylation/dephosphorylation of rat liver acyl-CoA:cholesterol acyltransferase.
Authors: Gavey K L , Trujillo D L , Scallen T J
Published: May 2006

Acyl-coenzyme A:cholesterol O-acyltransferase (ACATase; EC 2.3.1.26) is a membrane-bound microsomal enzyme that catalyzes the formation of long-chain fatty-acyl cholesterol esters in rat liver and other tissues. This enzyme is important in regulating the concentration of unesterified cholesterol in the cell. Having recently demonstrated that rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; EC 1.1.1.34), the major regulatory enzyme in cholesterol biosynthesis, undergoes in vivo phosphorylation and inactivation after a single cholesterol meal, we decided to test the hypothesis that the enzyme ACATase, important in cholesterol utilization and storage, is also subject to regulation by phosphorylation/dephosphorylation. The results show that rat liver ACATase can be reversibly inactivated/activated, in vitro, by incubation conditions that favor dephosphorylation/phosphorylation. Activation was also achieved by using a partially purified protein kinase extracted from microsomes. It is significant that HMG-CoA reductase is inactivated by phosphorylation whereas ACATase is activated by phosphorylation. ACATase is, therefore, regulated by phosphorylation in a manner exactly opposite to that of HMG-CoA reductase. We propose that the coordinate regulation of ACATase and HMG-CoA reductase by phosphorylation/dephosphorylation provides a mechanism for short-term intracellular cholesterol homeostasis.
Sequence dependence of hydrogen exchange kinetics in DNA duplexes at the individual base pair level in solution.
Authors: Patel D J , Ikuta S , Kozlowski S , Itakura K
Published: May 2006

The kinetics for hydrogen exchange at individual base pairs in self-complementary deoxydodecanucleotide duplexes have been estimated from NMR saturation recovery measurements on the resolved imino protons as a function of temperature. The imino protons of dA . dT base pairs in the center of the fully alternating d(C-G-C-G-T-A-T-A-C-G-C-G) duplex exchange a factor of 2- to 3-fold faster than the corresponding protons at the same positions in the partially alternating d(C-G-C-G-A-A-T-T-C-G-C-G) duplex. These exchange parameters are a direct measure of the rate constants for transient opening of individual dA . dT base pairs in the dodecanucleotide duplexes and demonstrate faster opening kinetics for the "TATA" box region compared to the related "AATT" segment.
Total synthesis of insulin-like growth factor I (somatomedin C).
Authors: Li C H , Yamashiro D , Gospodarowicz D , Kaplan S L , Vliet G V
Published: May 2006

Human insulin-like growth factor I (somatomedin C) with 70 amino acid residues and three disulfide bridges has been synthesized by the solid-phase method. The synthetic product behaves as a pure polypeptide in paper electrophoresis, isoelectric focusing, and high-performance liquid chromatography, Its elution behavior in Sephadex G-50, isoelectric point, amino acid composition, and growth-stimulating activity in bovine adrenal cortical cells or granulosa cells are comparable to those reported for the natural product. In radioimmunoassay, the synthetic product is indistinguishable from the natural hormone when either synthetic or natural product is used as the labeled ligand.
Isolation of an intact DNA polymerase-primase from embryos of Drosophila melanogaster.
Authors: Kaguni L S , Rossignol J M , Conaway R C , Lehman I R
Published: May 2006

A procedure has been devised for the purification of intact DNA polymerase alpha from early embryos of Drosophila melanogaster. The purified enzyme consists of at least three polypeptides with Mrs of 182,000, 60,000, and 50,000. These are related antigenically to the alpha (Mr 148,000), beta (Mr 58,000), and gamma (Mr 46,000) subunits, respectively, of the DNA polymerase described previously [Banks, G. R., Boezi, J. A. & Lehman, I. R. (1979) J. Biol. Chem. 254, 9886-9892]. The alpha subunit (Mr 182,000) has a molecular weight indistinguishable from that observed in extracts of freshly harvested embryos and presumably present in vivo. As in the previous preparation, the alpha subunit is required for DNA polymerase activity and is very likely the catalytic subunit of the enzyme. The ratio of primase to polymerase remains constant throughout the purification. Thus, the primase is very likely an integral component of the Drosophila DNA polymerase alpha. The purified DNA polymerase-primase contains no detectable endo- or exodeoxyribonuclease and has pH, MgCl2, (NH4)2SO4, and NaCl optima identical to those reported previously. In contrast, the Km for dTTP is 3.7 microM as compared with 17.5 microM for the previous enzyme. Sensitivities to aphidicolin and N-ethylmaleiimide and resistance to dideoxy TTP are unchanged.
Down-regulation of epidermal growth factor receptor correlates with plasminogen activator activity in human A431 epidermoid carcinoma cells.
Authors: Gross J L , Krupp M N , Rifkin D B , Lane M D
Published: May 2006

Human A431 epidermoid carcinoma cells in culture exhibit epidermal growth factor (EGF)-induced "down-regulation" of cell-surface and total cellular (Triton X-100 extractable) EGF receptors caused entirely by an enhanced rate (4-fold) of receptor inactivation [Krupp, M. N., Connolly, D. T. & Lane, M. D. (1982) J. Biol. Chem. 257, 11489-11496]. The following observations show that this enhanced rate of EGF receptor inactivation is closely correlated with an increased cellular activity of plasminogen activator (PA), a serine protease. First, EGF-induced down-regulation of cell-surface and total cellular EGF receptors and the concomitant increase in cellular PA activity occur with identical kinetics, the t 1/2 for both processes being 3-3.5 hr. Second, the EGF dose-response curves for down-regulation of total cellular EGF receptor and increased PA activity are similar. The EGF concentrations for half-maximal responses of both processes are 10-15 nM and 20 nM, respectively. Third, the removal of EGF from previously down-regulated cells results in the recovery of total cellular EGF binding activity with a concurrent loss of cellular PA activity. Fourth, blocking PA synthesis or activity with cycloheximide or dexamethasone prevents down-regulation of the EGF receptor. Fifth, the addition of leupeptin, an inhibitor of PA and plasmin action, blocks EGF-induced receptor down-regulation as well as the increase of PA activity. That EGF receptor down-regulation is independent of plasminogen per se in the culture medium suggests that PA-mediated events may initiate the rapid inactivation of the EGF receptor that occurs during down-regulation.
Recurrence of 49-base decamers, nonomers, and octamers within mouse C mu gene of Ig heavy chain and its primordial building block.
Authors: Yazaki A , Ohno S
Published: May 2006

Within the published 2,168-base-long mouse C mu gene of Ig heavy chain consisting of four coding and four noncoding segments, 2 base decamers, 8 nonomers, and 39 octamers recurred. Recurring base heptamers (about 100) and hexamers (about 350) were simply too numerous to merit individual identification. In spite of extensive overlaps between these recurring base decamers to hexamers, they occupied nearly the entire length of mouse Ig C mu gene. As with other genes of the beta-sheet-forming beta 2-microglobulin family, the Ig C mu gene (flanking and intervening noncoding sequences included) is not a unique sequence but rather it is degenerate repeats of the 45-base-long primordial building-block sequence uniquely its own. This primordial building block must originally have specified the 15-amino-acid-residue-long primordial arm of beta-sheet-forming loops, the characteristics of the beta 2-microglobulin family of polypeptides.
Neuronal production, migration, and differentiation in a vocal control nucleus of the adult female canary brain.
Authors: Goldman S A , Nottebohm F
Published: May 2006

The vocal control nucleus designated HVc (hyperstriatum ventrale, pars caudalis) of adult female canaries expands in response to systemic testosterone administration, which also induces the females to sing in a male-like manner. We became interested in the possibility of neurogenesis as a potential basis for this phenomenon. Intact adult female canaries were injected with [3H]thymidine over a 2-day period. Some birds were given testosterone implants at various times before thymidine. The birds were sacrificed 5 wk after hormone implantation, and their brains were processed for autoradiography. In parallel control experiments, some birds were given implants of cholesterol instead of testosterone. All birds showed considerable numbers of labeled neurons, glia, endothelia, and ventricular zone cells in and around HVc. Ultrastructural analysis confirmed the identity of these labeled neurons. Cholesterol- and testosterone-treated birds had similar neuronal labeling indices, which ranged from 1.8% to 4.0% in HVc. Thus, neurogenesis occurred in these adults independently of exogenous hormone treatment. Conversely, both glial and endothelial proliferation rates were markedly stimulated by exogenous testosterone treatment. We determined the origin of the thymidine-incorporating neurons by sacrificing two thymidine-treated females soon after their thymidine injections, precluding any significant migration of newly labeled cells. Analysis of these brains revealed no cells of neuronal morphology present in HVc but a very heavily labeled ventricular zone overlying HVc. We conclude that neuronal precursors exist in the HVc ventricular zone that incorporate tritiated thymidine during the S phase preceding their mitosis; after division these cells migrate into, and to some extent beyond, HVc. This ventricular zone neurogenesis seems to be a normally occurring phenomenon in intact adult female canaries.
Pattern of methylation of two genes coding for housekeeping functions.
Authors: Stein R , Sciaky-Gallili N , Razin A , Cedar H
Published: May 2006

The distribution of sites that can be methylated was analyzed in the Chinese hamster adenine phosphoribosyl-transferase (aprt) gene and the patterns of methylation of this gene and the mouse dihydrofolate reductase (dhfr) gene were studied by using CpG restriction enzymes. Both genes were found to be unmethylated completely at their 5'-end region and methylated heavily throughout the rest of the gene. Because the hamster aprt gene can be inhibited by DNA methylation in vivo, the results suggest that 5' undermethylation of this gene may be a necessary condition for its expression. The pattern of methylation of each of these two genes was similar in sperm and all other somatic tissue DNAs. This is in contrast to many tissue-specific genes that were found to be highly methylated in sperm DNA and undermethylated in the tissue in which they are expressed. This result is consistent with the fact that both aprt and dhfr are key enzymes in the biosynthesis of nucleotides and therefore expected to be synthesized in all cells.
Amino acid sequence of the catalytic subunit of aspartate transcarbamoylase from Escherichia coli.
Authors: Konigsberg W H , Henderson L
Published: May 2006

We propose a primary structure for the catalytic subunit of aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) from Escherichia coli based on amino acid sequences of fragments obtained by cyanogen bromide cleavage, by tryptic digestion of the succinylated polypeptide, and by chymotryptic and proteinase C digestion of the intact catalytic chain. The protein contains 310 amino acids and has a calculated molecular weight of 33,944. The negatively and positively charged residues are distributed uniformly, and there is no indication of charge clustering in the linear sequence.
Promoters regulated by the glnG (ntrC) and nifA gene products share a heptameric consensus sequence in the -15 region.
Authors: Ow D W , Sundaresan V , Rothstein D M , Brown S E , Ausubel F M
Published: May 2006

We have determined the nucleotide sequences of the Klebsiella pneumoniae nifL (regulation of N2 fixation genes) and the Escherichia coli glnA (glutamine synthetase) promoters. We compared these sequences with the published sequences of three other promoters that, like the nifL and glnA promoters, are activated by the general nitrogen regulators glnF (ntrA) and glnG (ntrC). The three promoters are the argTr (arginine transport) and dhuA (histidine transport) promoters of Salmonella typhimurium and the nifH (nitrogenase) promoter of Rhizobium meliloti. All five sequences (with at most one mismatch) contain the heptameric consensus sequence T-T-T-T-G-C-A. In the R. meliloti nifH and K. pneumoniae nifL promoters, in which the transcription initiation sites have been determined, the consensus sequence is situated in the -15 region. We recently reported that the K. pneumoniae nifA product, which activates nif genes, can substitute for the glnG (ntrC) product in activating promoters of several genes involved in nitrogen assimilation, including the nifL, the glnA, and the R. meliloti nifH promoters. It is likely that nifA also activates the S. typhimurium argTr and dhuA promoters. In contrast, the glnG product cannot substitute for the nifA product in the activation of the K. pneumoniae nifH (nitrogenase) promoter. Consistent with this latter observation, and supporting the conclusion that the T-T-T-T-G-C-A sequence is a regulatory site for glnG product activation, the K. pneumoniae nifH promoter (C-C-C-T-G-C-A) has only partial similarity with the T-T-T-T-G-C-A consensus sequence in the -15 region.
Mutations in lambda repressor's amino-terminal domain: implications for protein stability and DNA binding.
Authors: Hecht M H , Nelson H C , Sauer R T
Published: May 2006

The DNA binding properties of 52 different single-amino acid substitutions in lambda repressor's amino-terminal domain have been characterized. Seven proteins bearing mutations that change solvent-exposed side chains have been purified. The amino-terminal domains of these mutant repressors are folded and are comparable to the wild-type amino-terminal domain in thermal stability. In contrast, a purified mutant repressor bearing a substitution in a buried side chain contains an amino-terminal domain with decreased thermal stability. We argue that mutations that alter solvent-exposed wild-type side chains define residues that form the operator DNA binding surface of lambda repressor whereas completely or partially buried mutations exert their effect by decreasing protein stability.
Short-latency local actions of nerve growth factor at the growth cone.
Authors: Seeley P J , Greene L A
Published: May 2006

Cultures of neurite-bearing pheochromocytoma (PC12) cells and of sympathetic neurons have been examined by time-lapse video microscopy. In the presence of nerve growth factor (NGF), the neurites of such cultures elongated and their growth cones changed geometry, via microspike and lamellipodial motion, on a time scale of minutes. Withdrawal of NGF caused process extension to cease and a progressive reduction in growth-cone area as a result of retraction of lamellipodia and microspikes. By approximately equal to 4 hr after NGF withdrawal, most neurite tips were smooth sided, devoid of conical expansions at their termini, and virtually immobile. Addition of NGF to cultures from which it had been withdrawn induced motion of microspikes and projections from the upper surface of growth cones within 2 min, while lamellipodial spreading and neurite reextension were induced after approximately equal to 20 min. For PC12 cells, these responses to replacement of NGF could not be mimicked by addition of dibutyryl cAMP (less than or equal to 2 mM) or the Ca2+ ionophore A23187 (less than or equal to 5 microM) to NGF-deprived cultures nor inhibited by the presence of EGTA (less than or equal to 2 mM) or calcium antagonists in the culture medium. Since neurite fragments formed by transection of processes of PC12 cells deprived of NGF responded to its replacement in a manner similar to intact neurites, it is concluded that the effects are focal to the neurite and growth cone and independent of the cell body. This influence of NGF on growth-cone shape and motility represents short-term local activation of this structure and has significance for control of neurite extension.
Modification of DNA by reducing sugars: a possible mechanism for nucleic acid aging and age-related dysfunction in gene expression.
Authors: Bucala R , Model P , Cerami A
Published: May 2006

Reducing sugars react nonenzymatically with protein amino groups to initiate a process called nonenzymatic browning. Long-lived proteins, such as collagen and the lens crystallins, accumulate sufficient modification in vivo that they acquire many of the chemical properties characteristic of aged proteins. We have obtained evidence that nucleic acids also can undergo nonenzymatic modification by sugars. Incubation of DNA or nucleotides with glucose 6-phosphate (Glc-6-P) produces spectral changes similar to those described for nonenzymatic browning proteins. The occurrence of chemical modification was verified by measuring the transfection efficiency of viral DNA after incubation with glucose and Glc-6-P. A loss of transfection potential occurred that was first order with respect to time and sugar concentration. The rate of inactivation by Glc-6-P was 25 times that of glucose; 8 days of incubation with 150 mM Glc-6-P decreased transfection by 4 orders of magnitude. Glc-6-P also produced strand scission in a time- and concentration-dependent manner. We conclude that glucose, Glc-6-P, and possibly other sugars can react with DNA to produce significant structural and biological alterations. Since nucleic acids are long-lived molecules in the resting cell, the accumulation of these addition products might be a mechanism for the decreased genetic viability characteristic of the aged organism.
Cell surface antigens of human bladder cancer defined by mouse monoclonal antibodies.
Authors: Fradet Y , Cordon-Cardo C , Thomson T , Daly M E , Whitmore W F , Lloyd K O , Melamed M R , Old L J
Published: May 2006

Mouse monoclonal antibodies were obtained by immunization with cultured human bladder cancer or lysates of bladder papilloma. They identify 11 distinct antigenic systems as defined by serological analysis of cultured cells and studies of antigen distribution in normal and neoplastic tissues. The most restricted of these antigens, Om5, defines a subset of bladder tumors. Om5 is not detected in normal bladder urothelium or in any other normal or malignant tissue. T101 and JP165 are also subset markers for bladder cancer that are not detected in normal tissues. T16, T43, T87, and J143 (antigens represented on many cultured cells) are found in specific areas of the normal urinary tract and in a distinctive range of other normal and malignant cell types--e.g., T16 expression in pluristratified epithelium of skin, exocervix, and esophagus. T138 antigen is also a common feature of cultured cell lines, but its expression in sections of normal tissues is restricted to endothelial cells. In contrast, T110 is poorly represented on cultured cells but can be detected in culture supernatants. Localization of T110 in normal tissues showed that it is a component of the extracellular matrix. All determinants detected by this series of antibodies are heat labile and not related to A, B, H, I, Lewis blood group antigens. Six of the antibodies immunoprecipitated glycoproteins from radiolabeled cell lysates: AbT16 (Mrs 48,000 and 42,000), AbT87 (Mr 60,000), AbT43 (Mr 85,000), AbJ143 (Mr 140,000, 120,000, 60,000), AbT43 (Mr 85,000), AbJ143 (Mr 140,000, 120,000, and 30,000), and AbT110 (Mr 240,000).
Intramitochondrial movement of adrenal sterol carrier protein with cholesterol in response to corticotropin.
Authors: Conneely O M , Headon D R , Olson C D , Ungar F , Dempsey M E
Published: May 2006

Sterol carrier protein (SCP) is a highly abundant, ubiquitous, low molecular weight protein that has a rapid turnover and multifunctional roles in lipid metabolism and transport. It is also known as the fatty acid-binding protein. These and other characteristics led to studies on the possible role of SCP in mediation of steroidogenic responses of the adrenal gland to corticotropin. To quantitate the level of SCP in adrenal tissue and subcellular fractions, a specific immunochemical assay was developed using an antibody to homogeneous liver SCP. SCP is a major protein in adrenal cells (greater than 100 micrograms/mg of total protein). The bulk of it is present in the soluble (60%) and mitochondrial (35%) fractions. Nearly all mitochondrial SCP is present in the inner membrane. Adrenal SCP undergoes a dramatic diurnal variation, varying from 2% to 12% of total protein. When corticotropin was administered in vivo in the presence of aminoglutethimide, an inhibitor of steroidogenesis, there was a parallel movement of SCP with cholesterol to the inner mitochondrial membrane. Other work showed mitochondrial SCP levels increase only in situations where there is an increase in cholesterol levels. These findings strongly suggest that one of the functions of adrenal SCP is to participate in teh corticotropin-stimulated movement of cholesterol to the inner mitochondrial membrane for steroidogenesis.
Human liver alcohol dehydrogenase: amino acid substitution in the beta 2 beta 2 Oriental isozyme explains functional properties, establishes an active site structure, and parallels mutational exchanges in the yeast enzyme.
Authors: Jörnvall H , Hempel J , Vallee B L , Bosron W F , Li T K
Published: May 2006

The homodimeric Oriental beta 2 beta 2 isozyme of human liver alcohol dehydrogenase, corresponding to an allelic variant at the ADH2 gene locus, was studied in order to define the amino acid exchange in relation to the beta 1 beta 1 isozyme, the predominant allelic form among Caucasians. Sequence analysis reveals that the amino acid substitution occurs at position 7 of the largest CNBr fragment, corresponding to position 47 of the whole protein chain. Here, the beta 2 form has a histidine residue, while, in common with other characterized mammalian liver alcohol dehydrogenases, the beta 1 form has an arginine residue. This exchange does not affect the adjacent cysteine-46 residue, which is a protein ligand to the active-site zinc atom, thus clarifying previously inconsistent results. The histidine/arginine-47 mutational replacement corresponds to a position that binds the pyrophosphate group of the coenzyme NAD(H); this explains the functional differences between the beta 1 beta 1 and beta 2 beta 2 isozymes, including both a lower pH optimum and higher turnover number of beta 2 beta 2, which is likely to be the mutant form. The exchange demonstrates the existence of parallel but separate mutations in the evolution of alcohol dehydrogenases because these mammalian enzymes differ at exactly the same position by the same type of substitution as is found between a mutant and the wild-type constitutive forms of the corresponding yeast enzyme.
Cooperative transient trapping of photosystem II protons by the integral membrane portion (CF0) of chloroplast ATP-synthase after mild extraction of the four-subunit catalytic part (CF1)
Authors: Junge Wolfgang , Hong Yu Qun , Qian Lu Ping , Viale Alexandro
Published: May 2006

The ATP-synthase in chloroplasts is built from two blocks, CF0, which is integral to the thylakoid membrane and which serves as a proton channel, and CF1, attached to CF0, which is catalytically active. This study is aimed at understanding proton conduction through CF0. By a mild procedure we extracted <10% of total CF1, predominantly the four-subunit CF1 without the δ subunit. Extracted chloroplasts were excited with short flashes of light and the time course of the transmembrane potential and of the pH changes in both phases was measured spectrophotometrically. Mild extraction of CF1 caused two effects. (
i
) Up to 50% of the protons rapidly released from water oxidation transiently escaped detection in the thylakoid interior. (
ii
) The initial extent of the transmembrane potential was decreased by some 10% (20-μs resolution). Protons that were not detected inside appeared in the external phase after having passed the thylakoid membrane. pH titrations of the transient loss of protons produced an extremely sharp transition (near pH 7.5) as if six protons were buffered in a strictly cooperative manner. These effects were reversed upon addition of
N,N′
-dicyclohexylcarbodiimide, which, among other actions, blocks the proton channel through CF0. We interpret these observations as follows. (
i
) CF0 incorporates proton binding groups, which can act in a hexacooperative way. These groups are located near the middle of the membrane. (
ii
) After extraction of CF1, protons produced during water oxidation have very rapid access to these groups, but they pass the full span of the membrane more slowly: buffering precedes conduction through CF0.
Occurrence of caldesmon (a calmodulin-binding protein) in cultured cells: comparison of normal and transformed cells.
Authors: Owada M K , Hakura A , Iida K , Yahara I , Sobue K , Kakiuchi S
Published: May 2006

Caldesmon is a calmodulin-binding and F-actin-binding protein originally purified from chicken gizzard smooth muscle. This protein binds to F-actin filaments in a Ca2+- and calmodulin-dependent "flip-flop" fashion, thereby regulating the function of actin filaments. Here we report that various lines of cultured cells contain a Mr 77,000 protein that specifically reacts with the affinity-purified caldesmon antibody raised against chicken gizzard caldesmon . Among the fibroblast proteins that had been pulse-labeled with [35S]methionine, the Mr 77,000 protein was the only protein band detected on the NaDodSO4 gel that reacted with the anticaldesmon . The subcellular distribution of the Mr 77,000 protein was investigated by the indirect immunofluorescence technique using the anticaldesmon . In all fibroblast cell lines examined, the immunofluorescence localized along the cellular stress fibers and in leading edges of the cell. In Rous sarcoma virus-transformed cells (S7-1), however, the distribution of the fluorescence changed to a diffuse and blurred appearance. These staining patterns of anticaldesmon obtained with the normal and transformed cells coincided with those of antiactin in the corresponding states, strongly suggesting the functional linkage between the Mr 77,000 protein and actin filaments. We propose to refer to this Mr 77,000 protein as caldesmon 77. The cellular level of caldesmon 77 in transformed S7-1 cells decreased to about one-third of that in their normal counterparts (cell line no. 7). Essentially the same result was obtained with normal rat kidney cells infected with the temperature-sensitive transformation mutant Schmidt-Ruppin strain of Rous sarcoma virus (68 N2 clone). The cellular level of caldesmon 77 observed at a permissive temperature (35 degrees C) was about one-third of that at a nonpermissive temperature (38.5 degrees C). These changes of caldesmon 77 in transformed cells may correlate with the loss of Ca2+ regulation in the transformed state.
6-Deoxyacyclovir: a xanthine oxidase-activated prodrug of acyclovir.
Authors: Krenitsky T A , Hall W W , de Miranda P , Beauchamp L M , Schaeffer H J , Whiteman P D
Published: May 2006

Acyclovir [9-[(2-hydroxyethoxy)methyl]guanine] is an acyclic guanine nucleoside analogue that is widely used clinically as an antiherpetic agent. Its limited absorption in humans after oral administration prompted the search for prodrugs. A congener, referred to as 6- deoxyacyclovir [2-amino-9-[(2-hydroxyethoxy)methyl]-9H-purine], was synthesized and found to be 18 times more water soluble than was acyclovir. Surprisingly, this congener was readily oxidized to acyclovir by xanthine oxidase (EC 1.2.3.2). It was also oxidized by aldehyde oxidase (EC 1.2.3.1) largely to 8-hydroxy-6- deoxyacyclovir [2-amino-8-hydroxy-9-[(2-hydroxyethoxy)methyl]-9H-purine] and then to 8- hydroxyacyclovir [2-amino-6,8-dihydroxy-9[(2-hydroxyethoxy)methyl]-9H-purine]. 6- Deoxyacyclovir and the major products of its oxidation by aldehyde oxidase lacked appreciable activity against herpes simplex type I in vitro. On the basis of these results, it was apparent that the success of 6- deoxyacyclovir as a prodrug in vivo would depend upon how well its desired activation by xanthine oxidase competed with the nonactivating oxidations by aldehyde oxidase. In rats dosed orally with 6- deoxyacyclovir , absorption was extensive and the major urinary metabolite was acyclovir. In two human volunteers, urinary excretions of acyclovir were 5-6 times greater than those typically observed after administration of equivalent doses of acyclovir itself. The areas under the plasma concentration-time curves for acyclovir were also 5-6 times greater. Plasma levels of acyclovir peaked soon after ingestion of the prodrug, indicating rapid absorption and metabolic conversion. These results suggested that 6- deoxyacyclovir might have clinical usefulness as a prodrug of acyclovir suitable for oral administration.
Intrinsic GABAergic system of adrenal chromaffin cells.
Authors: Kataoka Y , Gutman Y , Guidotti A , Panula P , Wroblewski J , Cosenza-Murphy D , Wu J Y , Costa E
Published: May 2006

Histochemical and biochemical studies demonstrate that gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (EC 4.1.1.15), and GABA aminotransferase (EC 2.6.1.19) are present in bovine adrenal chromaffin cells. Moreover, [3H]GABA can be taken up and stored by primary cultures of adrenal chromaffin cells. Nicotinic receptor stimulation or KCl depolarization releases the [3H]GABA taken up by these cell cultures. GABA and benzodiazepine recognition sites located in chromaffin cells interact with each other with modalities similar to those described for GABA and benzodiazepine recognition sites located in synaptic membranes prepared from brain tissue. Bicuculline facilitates the release of catecholamine from chromaffin cells induced by nicotinic receptor stimulation but it fails to influence the release of catecholamine evoked by K+ depolarization. Since the GABA-benzodiazepine receptor system appears to modulate nicotinic receptor function, it is suggested that GABA transmission might participate in modulating responsiveness of chromaffin cells to incoming cholinergic stimuli.
Glycine-directed peptide amidation: presence in rat brain of two enzymes that convert p-Glu-His-Pro-Gly-OH into p-Glu-His-Pro-NH2 (thyrotropin-releasing hormone).
Authors: Kizer J S , Busby W H , Cottle C , Youngblood W W
Published: May 2006

To study the possibility of glycine-directed amidation in rat brain, we synthesized the substrate p-Glu-His-Pro-Gly-OH. Adult and neonatal rat brain and adult rat pituitary were sonicated, frozen and thawed, and fractionated by gel permeation chromatography, and fractions from each tissue were assayed for enzymatic activity capable of converting this model substrate into thyrotropin-releasing hormone. We report the presence in rat brain and rat pituitary of two enzymes catalyzing conversion of p-Glu-His-Pro-Gly-OH into thyrotropin-releasing hormone. Based on the differing chemical and physical properties of these two enzymes and their differing affinities for a number of p-Glu-His-Pro-aa-OH analogs (in which aa = glycine, beta-alanine, gamma-butyric acid, and delta-aminovaleric acid), we conclude that there are two distinct enzymatic processes for the terminal amidation of peptides in brain and that COOH-terminal extensions other than glycine are capable of directing COOH-terminal amidation.
Nitroaromatic radiation sensitizers substitute for oxygen in neocarzinostatin-induced DNA damage.
Authors: Kappen L S , Goldberg I H
Published: May 2006

The ability of neocarzinostatin (NCS) chromophore to damage DNA, as manifested by strand breaks and base release, is markedly decreased under anaerobic conditions but can be restored by nitroaromatic radiation sensitizers, which by themselves have no effect. The effectiveness of these compounds is correlated with their electron affinity as measured by their one-electron reduction potentials and is inversely related to the concentration of thiol used to activate the NCS. Whereas strand breaks with thymidine 5'-aldehyde at the 5' end and released thymine are the main DNA damage products in O2, under anaerobic conditions misonidazole causes a marked increase in the release of thymine and in the formation of breaks with 5'- phosphate ends. In both cases the 3' end of the break carries a phosphate group, and the attack-site specificity of spontaneous and alkali-labile DNA strand breakage and base release are identical. In O2, misonidazole does not affect the extent of DNA damage or alter the distribution of DNA damage products found with NCS alone. The data do not distinguish whether the nitroaromatic compounds function by interacting with NCS-induced nascent damage on the DNA, by being converted by activated NCS into a DNA-damaging species, or by participating in the activation of NCS to a DNA-damaging species. The implications of these results for the treatment of hypoxic tumor cells with the combined use of radiomimetic drugs and radiation sensitizers are discussed.
Primary structure of the gene encoding rat preprosomatostatin.
Authors: Montminy M R , Goodman R H , Horovitch S J , Habener J F
Published: May 2006

The somatostatins are peptides of 14 and 28 amino acids that are produced in a variety of endocrine and nonendocrine tissues. These peptides inhibit the secretion of many different pituitary, pancreatic, and gastrointestinal hormones. Previously, we have reported the isolation and nucleotide sequence of a cDNA derived from a rat medullary thyroid carcinoma that encoded preprosomatostatin , a 116-amino-acid precursor of somatostatin. We now report the structural characterization of the rat somatostatin gene isolated from recombinant bacteriophage libraries prepared from rat liver DNA. The gene spans 1.2 kilobases and is interrupted within the coding sequence of prosomatostatin by a single intron of 630 bases. A sequence characteristic of a Goldberg- Hogness promoter ("TATA" box), T-T-T-A-A-A-A, is located 31 bases upstream from the transcriptional initiation site. A repetitive DNA sequence, highly reiterated in the rat genome, is located in the 5' flanking region of the gene within 900 bases of the initiation site.
Identification of a 34,000-dalton mitogenic protein associated with plasma membranes from human A431 epidermoid carcinoma cells.
Authors: Bishayee S , Matesic D , Das M
Published: May 2006

We present evidence for a discrete 34,000-Da polypeptide with mitogenic activity, associated with plasma membranes from human A431 carcinoma cells. Plasma membranes from A431 cells are highly mitogenic for quiescent fibroblasts. A significant fraction of the membrane-associated activity can be released by treatment with a high concentration of salt and is relatively acid stable. Incubation of 125I-labeled salt-extracted proteins with target fibroblasts results in preferential binding of a 34,000-Da protein--i.e., greater than 90% of the cell-bound radioactivity is associated with the 34,000-Da polypeptide. Studies correlating the mitogenic activity with the cell-binding 34,000-Da protein indicate that this protein is the acid-stable, peripherally attached, mitogenically active component of A431 membranes. Available data suggest that this protein may be mitogenically active in the nanomolar concentration range. Some properties of this protein are described.
Inherited predisposition to iridocyclitis with juvenile rheumatoid arthritis: selectivity among HLA-DR5 haplotypes.
Authors: Miller M L , Fraser P A , Jackson J M , Larson M G , Petersen R A , Chylack L T , Glass D N
Published: May 2006

HLA-DR5 is associated with a chronic iridocyclitis and juvenile rheumatoid arthritis with onset in early childhood. Previously published data provided indirect evidence for selective linkage between two HLA-B alleles and HLA-DR5. To test this observation further, 38 families, the probands of which have chronic iridocyclitis and juvenile rheumatoid arthritis, were HLA typed so that haplotypes associated with disease could be established. HLA-DR5 was linked to HLA-Bw44 or to HLA-Bw35 and to HLA-Cw4 in the majority of haplotypes obtained in the probands. Both HLA-Bw44, -DR5 and HLA-Bw35, -Cw4, -DR5 occurred more commonly in the proband haplotypes than in the control haplotypes. The frequency of the haplotype HLA-Bw44, -DR5 was 0.133 compared with 0.007 (X2 = 27.04, P less than 10(-6] and for HLA-Bw35, -Cw4, -DR5, it was 0.093 compared with 0.11 (X2 = 13.83, P = 0.0002). The relative risks were 20.77 and 9.23, respectively, versus 3.52 for HLA-DR5 alone. HLA-Bw44 occurred much more commonly in proband HLA-DR5 haplotypes (0.455) compared with control HLA-DR5 haplotypes (0.067) (X2 = 8.26, P less than 0.01). Not all HLA-DR5-bearing haplotypes predisposed equally to chronic iridocyclitis and early-onset pauciarticular juvenile rheumatoid arthritis.
Toward an analogue of alcoholism in mice: scale factors in the model.
Authors: Dole V P , Gentry R T
Published: May 2006

Mice of the C57BL strain, given continuous access to 10% alcohol and plain water, with unlimited food and no stress, frequently drink enough alcohol to produce intoxicating levels in the blood. Nevertheless, this behavior does not appear to replicate the essential features of human alcoholism since the drinking lacks serious toxic effects and the intoxication occurs only as transient episodes in association with homeostatic consumption of fluid and food. It is suggested that continuous monitoring of intake and estimation of the concentration of alcohol in blood, which are now technically feasible, will permit distinction between alcoholic-type drinking and a simple licking for the flavor of alcohol in beverage concentration.
Complete amino acid sequence of human plasma beta 2-glycoprotein I.
Authors: Lozier J , Takahashi N , Putnam F W
Published: May 2006

We have determined the complete amino acid sequence of beta 2-glycoprotein I (Mr, congruent to 50,000), a human plasma protein that is associated with lipids and binds to platelets but whose function is not yet known. The protein consists of 326 amino acids and has five attached glucosamine-containing oligosaccharides. The protein is rich in cysteine and proline, and the sequence is notable for the frequent occurrence of Cys-Pro linkages at regular intervals. Computerized analysis of the sequence reveals five consecutive homologous segments in which cysteine, proline, and tryptophan appear to be highly conserved. This suggests that beta 2-glycoprotein I may have evolved by repeated duplications of a gene coding for a 60-amino acid segment of protein.
Active-site carbamate formation and reaction-intermediate-analog binding by ribulosebisphosphate carboxylase/oxygenase in the absence of its small subunits
Authors: Andrews T. John , Ballment Beth
Published: May 2006

Even though depleted of more than 90% of its small subunits, ribulose 1,5-bisphosphate carboxylase/oxygenase from
Synechococcus
ACMM 323 still formed a stable complex with 2-carboxyarabinitol 1,5-bisphosphate from which exchange of the activator CO
2
molecule was prevented. The stoichiometry between nonexchangeable CO
2
and large subunits was unchanged regardless of the presence or absence of small subunits. The small-subunit-depleted enzyme was also “activated” by exposure to CO
2
and Mg
2+
, although it was necessary for the small subunits to be bound before this “activation” could be expressed. Binding of small subunits occurred rapidly, its rate depending on subunit concentration. The initial rate of “activation” was not slowed in the absence of small subunits but its extent at equilibrium was reduced. These observations are not consistent with an obligate role for the small subunits in the activation process. Their necessity in catalysis must stem from a more subtle involvement in the catalytic mechanism itself.
Proteolysis and structure of skeletal muscle actin.
Authors: Mornet D , Ue K
Published: May 2006

Under standard conditions, G-actin has been submitted to nine proteases of varying specificity, and in each case the pattern of fragments produced has been studied by NaDodSO4 gel electrophoresis. The results suggest that the actin monomer consists of a large region (ca. 33 kilodaltons) and a small, easily degraded region (ca. 9 kilodaltons). The COOH terminus is in the large region. Consideration of primary sequence homologies, medium resolution maps of actin crystals, and certain reactions of actin suggests that the NH2 terminus is in the small region, as is the negative sequence to which a divalent metal cation is normally chelated, but that the nucleotide-binding site is on the large region near the junction between the regions. From analysis of these results, numerous properties of actin are understandable.
Characterization of a cDNA coding for human factor X.
Authors: Leytus S P , Chung D W , Kisiel W , Kurachi K , Davie E W
Published: May 2006

A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human factor X, a plasma protein participating in the middle phase of the blood coagulation cascade. Ten positive clones were isolated from 2 X 10(6) phage and plaque purified. The cDNA in the phage containing the largest insert has been sequenced and shown to code for human factor X. This cDNA insert contained 1137 base pairs coding for a portion of the light chain of the molecule, a connecting region, the heavy chain, a stop codon, a short 3' noncoding region, and a poly(A) tail. The sequence of A-T-T-A-A-A, which functions as a potential recognition site for polyadenylylation or processing, was present in the 3' end of the coding sequence and preceded the stop codon of TGA by 1 base pair and the poly(A) tail by 14 base pairs. The amino acid sequence deduced from the cDNA indicated that factor X is synthesized as a single-chain polypeptide containing the light and heavy chains connected by an Arg-Lys-Arg tripeptide. The single-chain molecule is then converted to the light and heavy chains by cleavage of two (or more) internal peptide bonds. In plasma, these two chains are linked together by a disulfide bond. The DNA sequence coding for the active site of human factor X showed a high degree of identity with prothrombin and factor IX, two other vitamin K-dependent serine proteases that participate in blood coagulation. These data along with the protein sequence data previously published for the light chain of human factor X establish the complete amino acid sequence for the mature protein present in plasma.