bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleotide sequence for yeast dihydrolipoamide dehydrogenase.
Authors: Browning K S , Uhlinger D J , Reed L J
Published: May 2006

Rabbit antiserum to the dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) component of the pyruvate dehydrogenase complex from bakers' yeast was used to screen plaques produced by a lambda gt11 yeast cDNA library. A 2.1-kilobase insert was isolated that also hybridized to a 17-base mixed oligonucleotide probe corresponding to the amino-terminal sequence of the yeast dihydrolipoamide dehydrogenase. The cDNA has a coding sequence of 499 amino acids that corresponds to a 21-residue signal peptide and a 478-residue mature protein (Mr = 51,558). Computer analysis shows that yeast dihydrolipoamide dehydrogenase has about 41% amino acid identity with Escherichia coli dihydrolipoamide dehydrogenase. Particularly striking is the conservation of sequence in the active site region of the dihydrolipoamide dehydrogenases from E. coli, yeast, and pig heart.
Class I major histocompatibility complex proteins diffuse isotropically on immune interferon-activated endothelial cells despite anisotropic cell shape and cytoskeletal organization: application of fluorescence photobleaching recovery with an elliptical beam.
Authors: Stolpen A H , Pober J S , Brown C S , Golan D E
Published: May 2006

Interferon gamma induces striking phenotypic alterations in confluent cultures of human vascular endothelial cells (HEC), including cell shape change from polygonal to elongated and cytoskeletal actin rearrangement from dense peripheral bands to longitudinal bundles of stress fibers. Since many transmembrane proteins, including class I major histocompatibility complex (MHC) proteins, interact with cytoskeletal actin, an interferon-gamma-induced anisotropic arrangement of stress fibers might cause anisotropic lateral diffusion of HEC class I MHC proteins. To test this hypothesis, we adapted the fluorescence photobleaching recovery technique to allow measurement of anisotropic diffusion of fluorescently labeled molecules on two-dimensional surfaces. A highly eccentric elliptical Gaussian laser beam was used to photobleach the sample and to monitor fluorescence recovery. In this technique, named "line fluorescence photobleaching recovery," lateral diffusion is measured along that axis of the sample that is perpendicular to the major axis of the elliptical beam. The lateral diffusion coefficient and fractional mobility are obtained by fitting the experimental data to a theoretical recovery curve, the form of which is determined by the solution to a modified version of the diffusion equation in which a tensor is used to describe diffusion in two orthogonal directions. Fluorescein-conjugated murine monoclonal antibodies were used to label class I MHC proteins on interferon-gamma-treated HEC and human dermal fibroblasts. These two cultured human cell types were found to be similar in their elongated shape and anisotropic stress fiber organization. Class I MHC protein lateral mobility was compared to that of fluorescein-labeled phosphatidyl-ethanolamine, a membrane phospholipid probe. Class I MHC proteins diffused anisotropically on human dermal fibroblasts, whereas fluorescein-labeled phosphatidylethanolamine diffused isotropically on this cell type. In contrast, both class I MHC proteins and fluorescein-labeled phosphatidylethanolamine diffused isotropically on interferon-gamma-treated HEC. These data suggest that neither elongated shape nor anisotropic stress fiber arrangement is sufficient to induce anisotropic diffusion of proteins on the HEC plasma membrane.
Type-specific neutralization of the human immunodeficiency virus with antibodies to env-encoded synthetic peptides.
Authors: Palker T J , Clark M E , Langlois A J , Matthews T J , Weinhold K J , Randall R R , Bolognesi D P , Haynes B F
Published: May 2006

A synthetic peptide (SP-10-IIIB) with an amino acid sequence [Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro -Pro-Gly-(Tyr); amino acids 303-321] from the human immunodeficiency virus (HIV) isolate human T-cell lymphotropic virus type III (HTLV-III) HTLV-IIIB envelope glycoprotein gp120 was coupled to tetanus toxoid and used to raise goat antibodies to HIV gp120. Goat anti-SP-10-IIIB serum bound to the surface of HTLV-IIIB-infected CEM T cells but not to the surface of HTLV-IIIRF-infected or uninfected CEM T cells. Anti-SP-10-IIIB antibodies also selectively bound to gp120 from lysates of HTLV-IIIB cells in immunoblot assays. Twenty-one percent of sera (28 of 175) from patients seropositive for HIV contained antibodies that reacted with SP-10-IIIB in RIA. Human anti-SP-10-IIIB antibodies affinity purified from acquired immunodeficiency syndrome (AIDS) patient serum bound to HTLV-IIIB-infected cells and immunoprecipitated gp120. Goat antibodies to SP-10-IIIB neutralized HTLV-IIIB (80% neutralization titer of 1/600), inhibited HTLV-IIIB-induced syncytium formation, but did not neutralize HIV isolates HTLV-IIIRF or HTLV-IIIMN or inhibit syncytium formation with these isolates. Also, goat antiserum to an homologous synthetic peptide [SP-10-IIIRF(A), (Cys)-Arg-Lys-Ser-Ile-Thr-Lys-Gly-Pro-Gly-Arg-Val-Ile-Tyr] from gp120 of HIV isolate HTLV-IIIRF inhibited syncytium formation by HTLV-IIIRF, but did not inhibit syncytium formation by HTLV-IIIB or by HTLV-IIIMN. Thus, the amino acid sequences of SP-10-IIIB and SP-10-IIIRF(A) define homologous regions of gp120 that are important in type-specific virus neutralization. The identification of these type-specific neutralizing epitopes should facilitate the design of a polyvalent, synthetic vaccine for AIDS.
Intragenic cis-acting art gene-responsive sequences of the human immunodeficiency virus.
Authors: Rosen C A , Terwilliger E , Dayton A , Sodroski J G , Haseltine W A
Published: May 2006

The art gene product of the human immunodeficiency virus is required for the expression of virion capsid and envelope gene proteins. The experiments presented here show that sequences located within the coding region of the envelope gene exert a negative effect on the expression of heterologous genes and that the negative effect of these sequences can be relieved by the art gene product. This region in the env gene contains negative regulatory sequences that inhibit gene expression, as well as a sequence necessary for the art gene product-dependent relief of repression. The experiments define the cis- and trans-acting components of a regulatory system that permits differential expression of human immunodeficiency virus virion structural and regulatory proteins.
Geometry of the simian virus 40 large tumor antigen-DNA complex as probed by protease digestion.
Authors: Simmons D T
Published: May 2006

I have mapped the regions of simian virus 40 (SV40)-encoded large tumor (T) antigen that remained associated with origin-containing SV40 DNA after digestion with various concentrations of Pronase E, a nonspecific protease. Immunoaffinity-purified, labeled T antigen was bound to SV40 DNA-cellulose and treated with Pronase E. A "core" region representing amino acids 140 to about 281 was identified by peptide mapping in the fraction that remained bound to the DNA-cellulose after treatment with a high (135 micrograms/ml) concentration of enzyme. This region corresponds to the DNA-binding domain of the protein molecule. After treatment with Pronase E at 66 micrograms/ml, the bound fraction consisted of the DNA-binding domain and a region that extends to residue 371. This larger protein segment binds more stably to the viral DNA than does the core by itself. At lower concentrations of Pronase E, additional sequences from the NH2-terminal region of T antigen and from the COOH-half of the molecule were observed in the bound fractions. Linear maps of resistant regions, generated for each concentration of protease used, provide information about the geometry of the protein molecule associated with the DNA. I suggest that regions that are easily cleaved by the protease are exposed in the DNA-protein complex, whereas those that remain bound to the DNA at increasing concentrations of the enzyme represent segments that are in progressively closer proximity to the viral DNA origin.
CDC7-dependent protein kinase activity in yeast replicative-complex preparations.
Authors: Jazwinski S M
Published: May 2006

A protein kinase activity was identified in preparations of DNA-replicative complex from the budding yeast Saccharomyces cerevisiae. The activity phosphorylated only a few of the endogenous proteins in the replicative fraction, and it displayed a marked preference for a 48-kDa polypeptide. Despite this relative specificity, the protein kinase activity was capable of utilizing exogenously added histone as substrate. The 48-kDa polypeptide was phosphorylated on serine residue(s) exclusively by the endogenous activity in the replicative-complex preparation. The activity was not stimulated by cAMP, cGMP, Ca2+/phosphatidylserine/diacylglycerol, or Ca2+/calmodulin. It did not utilize Ca2+ or Zn2+ in the place of Mg2+, and Mn2+ was only 22% as effective in fulfilling the divalent-cation requirement. Most importantly, the protein kinase activity was heat-sensitive in replicative fractions from the cell division cycle 7 (cdc7) mutant, which arrests at or close to the G1/S boundary of the cell cycle at restrictive temperature. Thus, the activity is CDC7-dependent. An effect of heat treatment on replicating activity in the replicative fraction from cdc7 cells was also found. This result and the finding that the protein kinase activity copurified with replicating activity in the preparations suggest that the CDC7 gene product and the protein kinase activity, whether or not they are the same entity, interact with yeast replicative complex. All of these results raise the possibility that phosphorylation of components of the replication machinery may play a role in the control of initiation of DNA replication during the cell cycle. It is possible that the phosphorylation observed is part of a protein kinase cascade that regulates progress through the G1 phase of the cell cycle.
A cDNA clone for cytotactin contains sequences similar to epidermal growth factor-like repeats and segments of fibronectin and fibrinogen.
Authors: Jones F S , Burgoon M P , Hoffman S , Crossin K L , Cunningham B A , Edelman G M
Published: May 2006

Cytotactin is an extracellular glycoprotein that influences neuron-glia interactions. It has been shown to appear in multiple forms that are differentially expressed in neural and non-neural tissues during vertebrate development. We report here the isolation and characterization of a cytotactin cDNA clone (lambda C801) that encodes 933 amino acids, equivalent to about half of a cytotactin polypeptide. Clone lambda C801 is an authentic cytotactin cDNA: it encodes a polypeptide that reacts with a monoclonal anti-cytotactin antibody and its deduced amino acid sequence is identical for 15 amino acids to the directly determined sequence of a CNBr fragment that reacted with the same antibody. Southern blot analyses with fragments of lambda C801 suggested that there may be only one cytotactin gene, but RNA transfer blots detected multiple mRNAs ranging in size from 6.5 to 8.0 kilobases. An 8.0-kilobase message and a Mr 240,000 cytotactin polypeptide were present in embryonic gizzard but not brain, while a 7.2-kilobase message and a Mr 220,000 polypeptide were present in brain but not gizzard. These results indicate that differential splicing of primary transcripts of the cytotactin gene yields various site-specific polypeptides. Sequence analyses of lambda C801 indicated that it specifies a region with extensive similarities to other proteins: the sequence begins with four consecutive epidermal growth factor-like repeats that are followed by eight segments that closely resemble each other and the type III repeats in fibronectin, and it ends with a 66 amino acid sequence similar to part of the beta and gamma chains of fibrinogen. One fibronectin-like repeat contains a single Arg-Gly-Asp sequence. The similarities with all three of these apparently unrelated proteins are extensive, suggesting that cytotactin has an evolutionary and possibly a functional relationship to each.
An indicator gene to demonstrate intracellular transposition of defective retroviruses.
Authors: Heidmann T , Heidmann O , Nicolas J F
Published: May 2006

An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neoRT). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) deleted for the envelope gene to test for intracellular transposition of defective retroviruses [Mo-MLV(neo)RT]. NeoRT contains the selectable neo gene (which confers resistance to the drug G418), inactivated by a polyadenylylation sequence inserted between the neo promotor and coding sequence. The polyadenylylation sequence is flanked (on the antisense strand of the DNA) by a donor and an acceptor splice site so as to be removed upon passage of the provirus through an RNA intermediate. 3T3 cells transfected with the defective Mo-MLV(neo)RT provirus are sensitive to G418. After trans-complementation with Mo-MLV, viral transcripts confer resistance to G418 upon infection of test cells. In the resistant cells, the polyadenylylation sequence has been removed, as a result in most cases of precise splicing of the intronic domain. Retrotransposition of the defective Mo-MLV(neo)RT provirus was demonstrated by submitting transfected G418-sensitive clones to selection. Between 1 and 10 G418-resistant clones were obtained per 10(7) cells. Several possess additional copies, with evidence for precise removal of the intronic domain. By using target test cells in coculture experiments, extracellular intermediates of retrotransposition could not be detected.
Enhancement of acute allergic inflammation by indomethacin is reversed by prostaglandin E2: apparent correlation with in vivo modulation of mediator release.
Authors: Raud J , Dahlén S E , Sydbom A , Lindbom L , Hedqvist P
Published: May 2006

Intravital microscopy and determination of in vivo histamine release revealed that the cyclooxygenase inhibitor indomethacin reduced antigen-induced vasodilation while enhancing plasma extravasation, leukocyte accumulation, and histamine release in cheek pouches of immunized hamsters. Topical application of prostaglandin E2 (PGE2, 30 nM) totally reversed the indomethacin-induced potentiation of the inflammatory reaction to antigen challenge and suppressed both the histamine release and plasma leakage also in the absence of indomethacin. On the other hand, PGE2, which per se caused vasodilation, markedly potentiated the postcapillary leakage of plasma induced by histamine or leukotriene C4, as well as the leukocyte activation and subsequent plasma extravasation evoked by leukotriene B4. Taken together, the data indicate that PGE2 reduced the antigen response by suppression of mediator release from the numerous mast cells present in the cheek pouch. Moreover, the PGE2-sensitive potentiation by indomethacin of the antigen response suggests that endogenous vasodilating prostaglandins (possibly PGE2) predominantly were anti-inflammatory.
Receptor-mediated release of endothelium-derived relaxing factor and prostacyclin from bovine aortic endothelial cells is coupled.
Authors: de Nucci G , Gryglewski R J , Warner T D , Vane J R
Published: May 2006

Bovine aortic endothelial cells were grown on microcarrier beads and were perfused with Krebs-Ringer solution. Endothelium-derived relaxing factor (EDRF) was bioassayed on a cascade of four strips of rabbit aorta, and prostacyclin was analyzed by RIA of 6-oxo-prostaglandin F1 alpha. The endothelial cells released EDRF and prostacyclin when stimulated with bradykinin and its analogues, or with ADP, ATP, arachidonic acid, and phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3). The detection of EDRF was potentiated by superoxide dismutase, and the relaxation of rabbit aortic strips induced by EDRF was antagonized by methylene blue. The release of EDRF and prostacyclin was inhibited by phorbol myristate acetate, R59022 (a diacylglycerol kinase inhibitor), and gentamycin. We suggest that the release of EDRF and prostacyclin is coupled and the initial common step is activation of a phospholipase C.
Immunological lesions in human uracil DNA glycosylase: association with Bloom syndrome.
Authors: Seal G , Brech K , Karp S J , Cool B L , Sirover M A
Published: May 2006

Three monoclonal antibodies that react with uracil DNA glycosylase of normal human placenta were tested to determine whether one of the antibodies could be used as a negative marker for Bloom syndrome. As defined by enzyme-linked immunosorbent assay, monoclonal antibody 40.10.09, which reacts with normal human glycosylase, neither recognized nor inhibited native uracil DNA glycosylase from any of five separate Bloom syndrome cell strains. Immunoblot analyses demonstrated that the denatured glycosylase protein from all five Bloom syndrome cell strains was immunoreactive with the 40.10.09 antibody. Further, each native enzyme was immunoreactive with two other anti-human placental uracil DNA glycosylase monoclonal antibodies. In contrast, ELISA reactivity was observed with all three monoclonal antibodies in reactions of glycosylases from 5 normal human cell types and 13 abnormal human cell strains. These results experimentally verify the specificity of the aberrant reactivity of the Bloom syndrome uracil DNA glycosylase. The possibility arises that determination of the lack of immunoreactivity with antibody 40.10.09 may have value in the early diagnosis of Bloom syndrome.
Improved tools for biological sequence comparison.
Authors: Pearson W R , Lipman D J
Published: May 2006

We have developed three computer programs for comparisons of protein and DNA sequences. They can be used to search sequence data bases, evaluate similarity scores, and identify periodic structures based on local sequence similarity. The FASTA program is a more sensitive derivative of the FASTP program, which can be used to search protein or DNA sequence data bases and can compare a protein sequence to a DNA sequence data base by translating the DNA data base as it is searched. FASTA includes an additional step in the calculation of the initial pairwise similarity score that allows multiple regions of similarity to be joined to increase the score of related sequences. The RDF2 program can be used to evaluate the significance of similarity scores using a shuffling method that preserves local sequence composition. The LFASTA program can display all the regions of local similarity between two sequences with scores greater than a threshold, using the same scoring parameters and a similar alignment algorithm; these local similarities can be displayed as a "graphic matrix" plot or as individual alignments. In addition, these programs have been generalized to allow comparison of DNA or protein sequences based on a variety of alternative scoring matrices.
N-methylation: potential mechanism for metabolic activation of carcinogenic primary arylamines.
Authors: Ziegler D M , Ansher S S , Nagata T , Kadlubar F F , Jakoby W B
Published: May 2006

Two amine N-methyltransferases isolated from rabbit liver catalyze S-adenosylmethionine-dependent N-methylation of benzidine and 4-aminobiphenyl but not of 4-aminoazobenzene or 2-aminobiphenyl. The enzymatic reaction products were analyzed and found to be identical to synthetic N-methylbenzidine and N-methyl-4-aminobiphenyl. N-Methylation may be a critical step in the metabolic activation of primary arylamines because N-methylarylamines, unlike primary arylamines, are readily N-oxygenated by the NADPH- and oxygen-dependent microsomal flavin-containing monooxygenase. Kinetic studies carried out with the purified porcine liver monooxygenase demonstrate that, while activity with primary arylamines could not be detected, N-methyl derivatives of benzidine, 4-aminoazobenzene, and 4-aminobiphenyl are substrates. Products formed from N-methyl-4-aminobiphenyl had the properties of the hydroxylamine and/or nitrone in that the enzyme- and time-dependent incubation product(s) reduced Fe3+ to Fe2+, and formaldehyde was formed during the course of the reaction. These data suggest that N-methyl-4-aminobiphenyl is oxidized to N-hydroxy-N-methyl-4-aminobiphenyl, which can undergo further oxidation to a nitrone that hydrolyzes to formaldehyde and N-hydroxy-4-aminobiphenyl.
Thymine dimers bend DNA.
Authors: Husain I , Griffith J , Sancar A
Published: May 2006

A 32-base-pair DNA fragment containing a thymine photodimer was constructed and ligated head-to-tail to obtain multimers of this sequence in which thymine dimers were in phase with the helix screw axis (approximately equal to 3 turns apart). The ligation products were analyzed by one- and two-dimensional gel electrophoresis and quantitative electron microscopy. These analyses show that the thymine photodimer introduces a bend of approximately equal to 30 degrees in DNA, which causes anomalously slow migration of DNA fragments in polyacrylamide gels and facilitates the formation of small covalent circles. Repair of thymine dimers by DNA photolyase abolishes the anomalous migration.
Cell expansion and single-cell separation induced by colchicine in suspension-cultured soybean cells
Authors: Hayashi Takahisa , Yoshida Keiichiro
Published: May 2006

Single plant cells have been obtained without the preparation of protoplasts by culturing pieces of soybean callus tissue with colchicine. Cell expansion and separation were evoked by colchicine (1 mM) within a week of culture. Microscopic observation showed that cells took on a spherical shape in the presence of colchicine and then separated into single cells. Addition of colchicine to the culture medium did not affect the composition of cell wall polysaccharides, but a uronic acid-rich extracellular polysaccharide appeared during cell expansion and separation. Addition of microtubule stabilizers, glycerol (300 mM) or dimethyl sulfoxide [3% (vol/vol)], inhibited the secretion of the polysaccharide as well as cell expansion and separation. The extracellular polysaccharide elicited by colchicine was isolated by ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sepharose CL-6B from the conditioned medium of colchicine-treated soybean cells. The purified 18-kDa polysaccharide immediately enhanced cell expansion and separation when added to soybean callus tissue cultured in medium containing colchicine, even at low concentrations (0.1 mM). The polysaccharide was composed of galacturonic acid and, after digestion with a pectinase preparation, had no effect on the cells. Methylation analysis suggests that the polysaccharide consists of ≈100 sequential α-1,4-galacturonic acids. The galacturonan increased the viability of separated cells cultured in medium containing colchicine, and the single cells obtained did not produce a wound-response callose. (Aminoethoxyvinyl)glycine, a specific inhibitor of ethylene production, extensively decreased the cell expansion and separation but did not inhibit the formation of the extracellular polysaccharide, suggesting that the polysaccharide may exert its effect by stimulating ethylene production.
pH-induced changes in Rhodospirillum rubrum cytochrome c2 and subsequent renaturation: an 15N NMR study.
Authors: Yu L P , Smith G M
Published: May 2006

The 15N-enriched ferrocytochrome c2 from Rhodospirillum rubrum was studied by 15N NMR at different solvent pH values. The mobility and chemical shift of the N-terminal glutamic acid (335.4 ppm at pH 5.1) were found to depend on pH. It was least mobile between pH 8 and 9.0, which is explained in terms of pH-dependent conformational changes and formation of salt linkages and/or hydrogen bonds. The resonances of the lysine side chains are centered around 341.7 ppm at low pH and move upfield with pH by about 8.4 ppm with pKa values of 10.8. The exchange rates of the epsilon NH protons are lowest near their pKa values. The protein is very stable in the pH range between 4.9 and 10.0 but unfolds abruptly at pH 10.5-11. Denaturation was verified by the measurement of several parameters by NMR. The renaturation of the protein demonstrates that the folding begins with reformation of heme coordination and establishment of a hydrophobic core, followed by positioning of side chains and peptide backbones linking the nucleation centers. The repositioning processes had time scales of minutes to hours in contrast to the reported values of seconds in some studies.
Purification and affinity labeling of dihydropyridine receptor from rabbit skeletal muscle membranes.
Authors: Kanngiesser U , Nalik P , Pongs O
Published: May 2006

Undegraded dihydropyridine (DHP)-receptor (putatively a voltage-gated Ca2+ channel) has been purified as a 340-kDa protein complex to approximately 80% homogeneity (2.4 nmol of DHP-receptor per mg of protein) from rabbit skeletal muscle by a rapid purification protocol. Transverse-tubule membranes were prepared in high yield by Ribi-press treatment. The DHP-receptor complex was solubilized in 1% digitonin followed by a two-step chromatographic purification procedure. The equilibrium dissociation constant of [3H](+)-PN200-110 binding (Kd; 0.9 nM) was not significantly changed by solubilization or purification. The purified DHP-receptor is composed of two subunits with apparent molecular masses of 148 kDa and 195 kDa migrating in polyacrylamide gels under nonreducing conditions as a single moiety of approximately 300 kDa. The 195-kDa subunit was affinity-labeled with [3H]azidopine in both transverse-tubule membranes and purified DHP-receptor preparations. The subunit can be degraded by high-energy irradiation to a 26-kDa peptide and by proteolysis to a 32-kDa peptide. Thus, it is probably due to proteolytic cleavage and/or photolysis that neither purification nor affinity-labeling studies have previously identified a DHP-receptor subunit of comparable molecular mass (195 kDa).
Cholesterol aggregation and interaction with cholesterol oxidase in supercritical carbon dioxide.
Authors: Randolph T W , Clark D S , Blanch H W , Prausnitz J M
Published: May 2006

High-pressure EPR spectroscopy indicates that cholesterol forms aggregates in supercritical carbon dioxide. In pure carbon dioxide, changes in cholesterol aggregate size or packing structure are observed with changing pressure. Near the critical point of carbon dioxide, cholesterol solubility is too low to permit significant aggregation, and monomeric cholesterol is observed. Addition of small amounts of dopants to supercritical carbon dioxide strongly affects cholesterol aggregation. Branched butanols (2-methyl-1-propanol and 2-methyl-2-propanol) and ethanol (to a lesser degree) promote cholesterol aggregation, while methanol, acetone, and 1-butanol do not. Cosolvents that promote aggregation also increase the rate at which cholesterol oxidase from Gloeocysticum chrysocreas catalyzes the oxidation of cholesterol. In supercritical carbon dioxide solutions, the EPR spectroscopy reveals little or no conformational change in cholesterol oxidase as 2-methyl-2-propanol or methanol is added. Damp cholesterol oxidase binds multiple cholesterol molecules; dry enzyme loses the ability to bind cholesterol. When molecular oxygen is the oxidizing agent, the rate of enzymatic cholesterol oxidation is greatly reduced in bone-dry carbon dioxide compared to that in water-saturated carbon dioxide.
Isolation and sequence of a cDNA clone encoding the 31-kDa subunit of bovine kidney vacuolar H+-ATPase.
Authors: Hirsch S , Strauss A , Masood K , Lee S , Sukhatme V , Gluck S
Published: May 2006

We have isolated a cDNA encoding the 31-kDa subunit of the bovine kidney vacuolar H+-ATPase. The composite sequence contains 1219 base pairs, which includes the entire 678-base-pair coding region. A lysine-rich sequence previously found in the Na+, K+-ATPase alpha subunit and the H+,K+-ATPase was identified in the 31-kDa subunit. An RNA blot and an immunoblot demonstrated variable 31-kDa subunit expression and immunoreactivity in different tissues; the highest levels were observed in kidney medulla and brain with both types of analysis. The isolation of a cDNA for the 31-kDa subunit is an important step in understanding this subunit's role in the function and regulation of the vacuolar H+-ATPase.
Receptor binding sites for substance P, but not substance K or neuromedin K, are expressed in high concentrations by arterioles, venules, and lymph nodules in surgical specimens obtained from patients with ulcerative colitis and Crohn disease.
Authors: Mantyh C R , Gates T S , Zimmerman R P , Welton M L , Passaro E P , Vigna S R , Maggio J E , Kruger L , Mantyh P W
Published: May 2006

Several lines of evidence indicate that tachykinin neuropeptides [substance P (SP), substance K (SK), and neuromedin K (NK)] play a role in regulating the inflammatory and immune responses. To test this hypothesis in a human inflammatory disease, quantitative receptor autoradiography was used to examine possible abnormalities in tachykinin binding sites in surgical specimens from patients with inflammatory bowel disease. Surgical specimens of colon were obtained from patients with ulcerative colitis (n = 4) and Crohn disease (n = 4). Normal tissue was obtained from uninvolved areas of extensive resections for carcinoma (n = 6). In all cases, specimens were obtained less than 5 min after removal to minimize influences associated with degradation artifacts and were processed for quantitative receptor autoradiography by using 125I-labeled Bolton-Hunter conjugates of NK, SK, and SP. In the normal colon a low concentration of SP receptor binding sites is expressed by submucosal arterioles and venules and a moderate concentration is expressed by the external circular muscle, whereas SK receptor binding sites are expressed in low concentrations by the external circular and longitudinal muscle. In contrast, specific NK binding sites were not observed in any area of the human colon. In colon tissue obtained from ulcerative colitis and Crohn disease patients, however, very high concentrations of SP receptor binding sites are expressed by arterioles and venules located in the submucosa, muscularis mucosa, external circular muscle, external longitudinal muscle, and serosa. In addition, very high concentrations of SP receptor binding sites are expressed within the germinal center of lymph nodules, whereas the concentrations of SP and SK binding sites expressed by the external muscle layers are not altered significantly. These results demonstrate that receptor binding sites for SP, but not SK or NK, are ectopically expressed in high concentrations (1000-2000 times normal) by cells involved in mediating inflammatory and immune responses. These data suggest that SP may be involved in the pathophysiology of inflammatory bowel disease and might provide some insight into the interaction between the nervous system and the regulation of inflammation and the immune response in human inflammatory disease.
Immortalization of mouse neural precursor cells by the c-myc oncogene.
Authors: Bartlett P F , Reid H H , Bailey K A , Bernard O
Published: May 2006

Immortalized cell lines have been generated from embryonic mouse neuroepithelium by infection with a retrovirus containing the c-myc oncogene. The morphology and the antigenic phenotype of the cloned cell lines are characteristic of normal neuroepithelium. Although the cell lines are stable and do not spontaneously differentiate, morphological changes can be induced with both acidic and basic fibroblast growth factor. Fibroblast growth factor at 5 ng/ml stimulates differentiation of the neuroepithelial cells, and it has been shown that the cloned cell line 2.3D can differentiate into astrocytes, containing glial fibrillary acidic protein, and neurons, expressing the A2B5 marker and neurofilaments. This indicates that some cells in the neuroepithelium at embryonic day 10 are multipotent and are not restricted to either the glial or neuronal cell lineage. The cell lines also can be induced with interferon gamma to express class I and class II histocompatibility antigens. The response of the c-myc-immortalized cell lines to these two factors is similar to that observed with freshly isolated neuroepithelium and suggests that such immortalized precursor populations are representative of the cells found in the developing neuroepithelium.
Identification and purification of a Saccharomyces cerevisiae protein with the DNA binding specificity of mammalian activating transcription factor.
Authors: Lin Y S , Green M R
Published: May 2006

Activating transcription factor (ATF) is a mammalian transcriptional activator, which is involved in the expression of many viral E1a-inducible and cellular cAMP-inducible genes. Here we identify from the yeast Saccharomyces cerevisiae a previously uncharacterized protein whose DNA binding specificity is like mammalian ATF. We purify this protein (yATF) and show that it is a 66-kDa polypeptide. Finally, we demonstrate that a mammalian ATF site can function as an upstream activating sequence in S. cerevisiae. Taken together, our results suggest that yATF is a previously uncharacterized S. cerevisiae transcriptional activator.
Characterization of hydrogen-uptake activity in the hyperthermophile Pyrodictium brockii.
Authors: Pihl T D , Schicho R N , Kelly R M , Maier R J
Published: May 2006

Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 105 degrees C. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have characterized P. brockii hydrogen-uptake activity with regard to temperature, ability to couple hydrogen oxidation to artificial electron acceptor reduction, sensitivity to O2, and cellular localization. The hydrogen-uptake activity was localized predominantly in a particulate fraction, was reversibly inhibited by O2, and coupled H2 uptake to the reduction of positive potential artificial electron acceptors. Comparisons between these results and those of the well-studied hydrogen-uptake hydrogenase from the mesophile Bradyrhizobium japonicum showed the two enzymes to be similar despite the very different natural environments of the organisms. However, the optimum temperature for activity differed greatly in the two organisms. We have also used immunological and genetic probes specific to the 65-kDa subunit of B. japonicum hydrogenase to assay crude extracts and genomic DNA, respectively, from P. brockii and found the enzymes to be similar in these respects as well. In addition, we report a formulation for artificial seawater capable of sustaining the growth of P. brockii.
Lymphocyte antigen Leu-19 as a molecular marker of regeneration in human skeletal muscle.
Authors: Schubert W , Zimmermann K , Cramer M , Starzinski-Powitz A
Published: May 2006

Antigen Leu-19 (Leu19-Ag), a 200- to 220-kDa surface glycoprotein, was originally identified on a subset of human peripheral lymphocytes exhibiting non-major histocompatibility complex-restricted cytotoxicity. Here we report that monoclonal antibody Leu-19 (mAb-Leu19) labels structures in human skeletal muscle: (i) satellite cells, which form the stem cell pool of muscle fiber regeneration, both in normal and diseased muscle; (ii) myotubes and myotube projections in regions of muscle fiber repair; (iii) periodically organized fibrillar structures in areas of regeneration; (iv) the surface of myoblasts and developing myotubes in culture. mAb-Leu19 precipitated a protein of approximately 200 kDa from cultured muscle cells. Our data show that Leu19-Ag is expressed on muscle-specific components of myosegments in repair and thus represents a molecular marker of muscle regeneration. On the basis of this molecular marker and using laser scan microscopy, it is possible to visualize at the light microscopic level hitherto undetectable details of muscle regeneration in routine cryostat sections.
Characterization and expression of a cDNA encoding the human androgen receptor.
Authors: Tilley W D , Marcelli M , Wilson J D , McPhaul M J
Published: May 2006

We report the isolation of a cDNA that encodes the complete human androgen receptor. The cDNA predicts a protein of 917 amino acids with a molecular weight of 98,918. Introduction of the cDNA into heterologous mammalian cells caused expression of high levels of a protein that binds dihydrotestosterone with the affinity, specificity, and sedimentation properties characteristic of the native human androgen receptor. Comparisons with the amino acid sequence of previously cloned steroid hormone receptors revealed a high degree of sequence conservation with the progesterone, glucocorticoid, and mineralocorticoid receptors in the putative hormone and DNA-binding domain regions.
Marked in vivo antiretrovirus activity of 9-(2-phosphonylmethoxyethyl)adenine, a selective anti-human immunodeficiency virus agent.
Authors: Balzarini J , Naesens L , Herdewijn P , Rosenberg I , Holy A , Pauwels R , Baba M , Johns D G , De Clercq E
Published: May 2006

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a potent and selective inhibitor of the replication of human immunodeficiency virus (HIV) in vitro in human T-lymphocyte MT-4, H9, and ATH8 cells. PMEA also inhibits Moloney murine sarcoma virus (Mo-MSV)-induced transformation of murine C3H embryo fibroblasts. Moreover, PMEA causes a dose-dependent suppression of tumor formation and associated mortality in mice inoculated with Mo-MSV. At a dose of 50 or 20 mg/kg per day PMEA effected a 90-100% protection of the mice against Mo-MSV-induced tumor formation and mortality. Even with a PMEA dose as low as 1 to 5 mg/kg per day, tumor formation was significantly delayed and the survival rate was significantly enhanced. In parallel experiments, azidothymidine exhibited a comparable inhibitory effect on Mo-MSV-induced tumor formation and associated death only at a 25-fold higher dose than PMEA. Because PMEA has stronger in vivo antiretrovirus potency and selectivity than azidothymidine and various other compounds currently being subjected to clinical trials, PMEA studies should be pursued to assess the potential of this compound in the treatment of acquired immunodeficiency syndrome (AIDS) and other retrovirus infections in humans.
Change in desensitization of cat muscle acetylcholine receptor caused by coexpression of Torpedo acetylcholine receptor subunits in Xenopus oocytes.
Authors: Sumikawa K , Miledi R
Published: May 2006

Cat muscle acetylcholine receptors (AcChoR) expressed in Xenopus oocytes desensitized more slowly than Torpedo electric organ AcChoRs, also expressed in oocytes. To examine the bases for the different degrees of desensitization, cat-Torpedo AcChoR hybrids were formed by injecting oocytes with cat denervated muscle mRNA mixed with a large excess of cloned Torpedo AcChoR subunit mRNAs. Hybrid AcChoRs formed by coinjection of cat muscle mRNA with the Torpedo beta or delta subunit mRNAs desensitized as slowly as cat AcChoR. In contrast, the hybrid AcChoRs expressed by coinjection with the Torpedo gamma subunit mRNA desensitized much more rapidly than cat AcChoR. The AcChoRs expressed in oocytes injected with cat muscle mRNA together with the Torpedo beta, gamma, and delta subunit mRNAs desensitized as rapidly as Torpedo AcChoR, indicating that the cat alpha subunit does not play an important role in determining the slow rate of desensitization. It is concluded that the difference in the rates of desensitization of cat and Torpedo AcChoRs is determined mainly by differences in their respective gamma subunits.
Purification of Tetrahymena actin reveals some unusual properties.
Authors: Hirono M , Kumagai Y , Numata O , Watanabe Y
Published: May 2006

Actin from Tetrahymena pyriformis has been purified by monitoring the presence of the actin gene product with an antiserum against a synthetic N-terminal peptide deduced from the nucleotide sequence of the Tetrahymena actin gene that we cloned previously. This highly purified Tetrahymena actin shares many essential properties with ubiquitous actin, including ion-dependent polymerization to microfilaments, binding with muscle heavy meromyosin to form arrowheads, and activation of the Mg2+-ATPase of muscle myosin subfragment 1. On the other hand, some properties of this purified Tetrahymena actin clearly differ from those of muscle actin: (i) Tetrahymena actin has 8 times less ability to activate the Mg2+-ATPase of muscle myosin subfragment 1 than muscle actin; (ii) Tetrahymena actin did not bind to phalloidin at all; (iii) Tetrahymena actin did not inhibit DNase I activity at all. In general, Tetrahymena actin has very unusual properties when compared to other actins described so far. This actin is expected to provide important clues for elucidating problems concerning the relationships between the structural and functional domains in an actin molecule.
Affinity labeling of
Avena
phytochrome with ATP analogs
Authors: Wong Yum-Shing , Lagarias J. Clark
Published: May 2006

The presence of ATP-dependent, polycation-stimulated protein kinase activity in highly purified phytochrome preparations [Wong, Y.-S., Cheng, H.-C., Walsh, D. A. & Lagarias, J. C. (1986)
J. Biol. Chem.
261, 12089-12097] has renewed the hypothesis that the phytochrome photoreceptor possesses enzymatic activity. A prerequisite for protein kinase function is the presence of an ATP binding site. Here we present evidence for a nucleoside triphosphate binding site(s) in the phytochrome molecule. Two ATP analogs, 5′-
p
-fluorosulfonylbenzoyladenosine and 8-azidoadenosine 5′-triphosphate, were used to affinity label purified
Avena
phytochrome. Labeling with both reagents is stimulated by the polycations poly(Lys
75
,Ala
25
) and histone H1. Coincubation with ATP inhibits the polycation-stimulated labeling of phytochrome. In similar experiments GTP, CTP, UTP, ADP, and pyrophosphate, but not adenosine or AMP, also prevent photoaffinity labeling of phytochrome.
Human nuclear NAD+ ADP-ribosyltransferase: localization of the gene on chromosome 1q41-q42 and expression of an active human enzyme in Escherichia coli.
Authors: Herzog H , Zabel B U , Schneider R , Auer B , Hirsch-Kauffmann M , Schweiger M
Published: May 2006

The gene for human nuclear NAD+ ADP-ribosyltransferase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosetransferase, EC 2.4.2.30; pADPRT] was localized to chromosome 1 at q41-q42 by in situ hybridization with a pADPRT-specific cDNA probe. Expression of a pADPRT cDNA under control of the lac promoter in Escherichia coli induces the synthesis of a group of related proteins that were immunoreactive with pADPRT antibody and that had catalytic properties very similar to those of the human enzyme. Purification of this enzymatic activity was performed essentially as described for the human enzyme. The Km, pH optimum, optimal reaction temperature, and inhibition by 3-aminobenzamide and 3-methoxybenzamide were found to be similar for the recombinant and the human enzymes. The purified recombinant enzyme consists of two major proteins of Mr 99,000 and Mr 89,000. Both proteins show pADPRT activity in activity gel analysis with [32P]NAD+ as substrate. Microsequencing of these two proteins isolated by denaturing gel electrophoresis and deletion mutagenesis of the pADPRT expression plasmid shows that the Mr 99,000 and Mr 89,000 proteins derive from initiation of translation at internal translational start signals located within the pADPRT cDNA.
Ribonuclease H activities associated with viral reverse transcriptases are endonucleases.
Authors: Krug M S , Berger S L
Published: May 2006

A series of test substrates have been synthesized to establish the effect of termini on the putative exoribonuclease H activity of reverse transcriptase. Recombinant reverse transcriptase from human immunodeficiency virus, natural enzyme from avian myeloblastosis virus, and a known endonuclease, Escherichia coli ribonuclease H, cleaved relaxed, circular, covalently closed plasmids in which 770 consecutive residues of one strand were ribonucleotides. The avian enzyme also deadenylated capped globin mRNA with a covalently attached oligo(dT) tail at the 3' end. These results resolve a long-standing controversy--that the viral enzymes are obligatory exonucleases in vitro, based on their failure to cleave certain substrates for E. coli ribonuclease H, including circular poly(A).linear poly(T) and ribonucleotide-substituted supercoiled plasmids, but resemble endonucleases in vivo, based on their ability to degrade RNA in complex DNA.RNA hybrids. The data strongly suggest that the viral enzymes are endonucleases with exquisite sensitivity to the conformation of heteroduplexes. Inhibition of viral, but not cellular, ribonuclease H with ribonucleoside-vanadyl complexes further distinguishes these enzymes.
Aspartyl beta-hydroxylase: in vitro hydroxylation of a synthetic peptide based on the structure of the first growth factor-like domain of human factor IX.
Authors: Gronke R S , VanDusen W J , Garsky V M , Jacobs J W , Sardana M K , Stern A M , Friedman P A
Published: May 2006

beta-Hydroxylation of aspartic acid is a post-translational modification that occurs in several vitamin K-dependent coagulation proteins. By use of a synthetic substrate comprised of the first epidermal growth factor-like domain in human factor IX and either mouse L-cell extracts or rat liver microsomes as the source of enzyme, in vitro aspartyl beta-hydroxylation was accomplished. Aspartyl beta-hydroxylase appears to require the same cofactors as known alpha-ketoglutarate-dependent dioxygenases. The hydroxylation reaction proceeds with the same stereospecificity and occurs only at the aspartate corresponding to the position seen in vivo. Further purification and characterization of this enzymatic activity should now be possible.
Mechanism of programmed cell death in the blastocyst.
Authors: Pierce G B , Lewellyn A L , Parchment R E
Published: May 2006

The malignant growth potential of embryonal carcinoma cells may be controlled by environmental factors. For example, embryonal carcinoma cells placed into normal blastocysts may not exhibit the continued growth expected of malignant cells but rather may lose all aspects of the malignant phenotype and become apparently normal embryonic cells. Loss of the malignant phenotype of embryonal carcinoma cells occurs early in these injected blastocysts and has been used as the basis of assays to study the mechanisms of regulation of embryonal carcinoma by the blastocyst. In this regard, P19, an embryonal carcinoma that makes midgestation chimeras, was regulated by blastocele fluid plus contact with trophectoderm but not by blastocele fluid plus contact with inner cell mass (ICM). In contrast, ECa 247, which makes trophectoderm, was regulated by exposure to blastocele fluid plus contact with trophectoderm or ICM. During the course of these experiments, dead embryonal carcinoma and ICM cells were observed, and blastocele fluid was then shown to kill ECa 247 and normal ICM cells of early blastocysts with trophectodermal potential. P19 cells and ICM cells with potential to make the embryo were not killed by blastocele fluid. Programmed cell death occurs in the ICM of the blastocyst during the transition from early (when ICM has the potential to make trophectoderm) to late (when the ICM lacks the potential to make trophectoderm). It is postulated that this programmed cell death is designed to eliminate redundant ICM cells with trophectodermal potential, and its mechanism of action is mediated by epigenetic factors in blastocele fluid.
GDP stimulates the phosphorylation of a 36-kDa membrane protein in Dictyostelium discoideum.
Authors: Anschutz A , Howlet A , Klein C
Published: May 2006

Increasing effort is directed toward elucidating the mechanisms by which guanine nucleotide-binding proteins regulate specific cellular processes. A common feature of this class of proteins is that GTP induces a transition from an inactive to an active conformation. The latter is limited by the hydrolysis of GTP and the coincident production of GDP. Here we provide evidence that guanine nucleotides may regulate biological processes by inducing the phosphorylation of specific proteins. In particular, we report a GDP-dependent phosphorylation of p36, a 36-kDa protein of Dictyostelium discoideum plasma membranes.