bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased expression of the epidermal growth factor receptor gene in malignant gliomas is invariably associated with gene amplification.
Authors: Wong A J , Bigner S H , Bigner D D , Kinzler K W , Hamilton S R , Vogelstein B
Published: May 2006

Primary malignant gliomas from 63 patients were analyzed to determine the relationship between amplification of the gene encoding the epidermal growth factor receptor (EGFR) and expression of the corresponding mRNA. Twenty-four tumors were found to have amplified the EGFR gene and amplification of other genes occurred in three additional tumors. Hybridization with synthetic RNA probes was used to quantitate mRNA levels in situ. All 24 tumors with amplification of the EGFR gene had high levels of expression of this gene, while none of the 39 tumors without amplification had increased levels. This shows that, in human gliomas, large increases in the expression of the EGFR gene are invariably associated with alterations in gene structure.
Familial defective apolipoprotein B-100: low density lipoproteins with abnormal receptor binding.
Authors: Innerarity T L , Weisgraber K H , Arnold K S , Mahley R W , Krauss R M , Vega G L , Grundy S M
Published: May 2006

Previous in vivo turnover studies suggested that retarded clearance of low density lipoproteins (LDL) from the plasma of some hypercholesterolemic patients is due to LDL with defective receptor binding. The present study examined this postulate directly by receptor binding experiments. The LDL from a hypercholesterolemic patient (G.R.) displayed a reduced ability to bind to the LDL receptors on normal human fibroblasts. The G.R. LDL possessed 32% of normal receptor binding activity (approximately equal to 9.3 micrograms of G.R. LDL per ml were required to displace 50% of 125I-labeled normal LDL, vs. approximately equal to 3.0 micrograms of normal LDL per ml). Likewise, the G.R. LDL were much less effective than normal LDL in competing with 125I-labeled normal LDL for cellular uptake and degradation and in stimulating intracellular cholesteryl ester synthesis. The defect in LDL binding appears to be due to a genetic abnormality of apolipoprotein B-100: two brothers of the proband possess LDL defective in receptor binding, whereas a third brother and the proband's son have normally binding LDL. Further, the defect in receptor binding does not appear to be associated with an abnormal lipid composition or structure of the LDL: the chemical and physical properties of the particles were normal, and partial delipidation of the LDL did not alter receptor binding activity. Normal and abnormal LDL subpopulations were partially separated from plasma of two subjects by density-gradient ultracentrifugation, a finding consistent with the presence of a normal and a mutant allele. The affected family members appear to be heterozygous for this disorder, which has been designated familial defective apolipoprotein B-100. These studies indicate that the defective receptor binding results in inefficient clearance of LDL and the hypercholesterolemia observed in these patients.
Red light-induced formation of ubiquitin-phytochrome conjugates: Identification of possible intermediates of phytochrome degradation
Authors: Shanklin John , Jabben Merten , Vierstra Richard D.
Published: May 2006

Phytochrome is the photoreceptor that controls red light-mediated morphogenesis in higher plants. It exists in two photointerconvertible forms, a red light-absorbing form, Pr, and a far-red light-absorbing form, Pfr. Because photoconversion of Pr to Pfr by a brief light pulse decreases the
in vivo
half-life of this chromoprotein by a factor of ≈100, this system offers a unique way to modulate the turnover rate of a specific protein and hence study the mechanisms responsible for selective protein degradation. In etiolated oat [
Avena sativa
(L.)] seedlings, degradation of phytochrome as Pfr follows zero-order kinetics as measured both spectrally and by ELISA, with 50% of Pfr lost in ≈130 min at 27°C. Immunoblot analysis of the destruction process with anti-oat phytochrome immunoglobulins reveals that degradation involves the loss of the 124-kDa phytochrome monomer and that proteolytic intermediates of apparent molecular mass lower than 124 kDa do not accumulate to detectable levels
in vivo
(<0.015% of total phytochrome). The latter observation suggests that proteolytic breakdown of the protein is extremely rapid. However, a series of polypeptides with higher apparent molecular mass and recognized by anti-phytochrome immunoglobulins (principally 129 and 134 kDa) appears after photoconversion to Pfr. These polypeptides represent no more than a few percent of the total immunologically detectable phytochrome pool and have incremental differences in apparent molecular mass of 5 kDa. They appear within 5 min after Pfr formation, reach maximal levels between 90 and 180 min, and decline thereafter. These polypeptides and others of apparent molecular mass up to 160 kDa are also detectable with immunoglobulins directed against either oat or human ubiquitin, indicating that they are ubiquitin-phytochrome conjugates. Since ubiquitin conjugation is involved in intracellular protein turnover and since formation and degradation of Pfr-ubiquitin conjugates coincide with the turnover of Pfr, these data suggest that the Pfr form of phytochrome is degraded via a ubiquitin-dependent proteolytic pathway.
his operons of Escherichia coli and Salmonella typhimurium are regulated by DNA supercoiling.
Authors: Rudd K E , Menzel R
Published: May 2006

The hisW mutations of Salmonella typhimurium are highly pleiotropic mutations that elevate his operon expression, reduce ilv gene expression, alter stable RNA metabolism, and confer defective growth properties. The hisW mutations are highly linked to a naladixic acid-resistant gyrA mutation of S. typhimurium. Multicopy recombinant plasmids containing the Escherichia coli gyrA gene are able to complement both the growth defects and the elevated his operon expression associated with the hisW mutations. We conclude that hisW mutations are alleles of the gyrA gene. The hisU1820 mutant of S. typhimurium exhibits many of the same phenotypes as hisW mutants. Several lines of evidence, including high transduction linkage to recF, suggest that hisU1820 is an allele of gyrB. Finally, well-characterized gyrA and gyrB alleles of E. coli are also his regulatory mutations. We propose that a wild-type degree of chromosomal superhelicity is required for maximal production of histidyl-tRNA and normal his operon regulation.
Kinetoplast DNA minicircles: regions of extensive sequence divergence.
Authors: Rogers W O , Wirth D F
Published: May 2006

Previous work has shown that the kinetoplast minicircle DNA of Leishmania species exhibits species-specific sequence divergence and this observation has led to the development of a DNA probe-based diagnostic test for leishmaniasis. In the work reported here, we demonstrate that the minicircle is composed of three types of DNA sequences with differing specificities reflecting different rates of DNA sequence change. A library of cloned fragments of kinetoplast DNA (kDNA) from Leishmania mexicana amazonensis was prepared and the cloned subfragments were found to contain DNA sequences with different taxonomic specificities based on hybridization analysis with various species of Leishmania. Four groups of subfragments were found, those that hybridized with a large number of Leishmania sp. as well as sequences unique to the species, subspecies, or isolate. Analysis of nested deletions of a single, full-length minicircle demonstrates that these different taxonomic specificities are contained within a single minicircle. This implies that different regions of a single minicircle have DNA sequences that diverge at different rates. These sequences represent potentially valuable tools in diagnostic, epidemiologic, and ecological studies of leishmaniasis and provide the basis for a model of kDNA sequence evolution.
Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose.
Authors: Aebersold R H , Leavitt J , Saavedra R A , Hood L E , Kent S B
Published: May 2006

We have developed a general two-step method for obtaining peptide fragments for sequence analysis from picomole quantities of proteins separated by gel electrophoresis. After separation by one- or two-dimensional polyacrylamide gel electrophoresis, proteins are electrophoretically transferred (electroblotted) onto nitrocellulose, the protein-containing regions are detected by reversible staining and are cut out, and each protein is digested in situ by proteolytic enzymes such as trypsin or staphylococcal V-8 protease. The resulting peptide fragments are separated by narrow-bore reverse-phase HPLC, collected, and sequenced in a gas-phase sequenator. Excellent peptide recoveries and the absence of extraneous contaminants in the separation of the peptide fragment mixture allow the generation of extensive internal sequence information from picomole amounts of protein. The method thus overcomes the problem of obtaining amino acid sequence data from N-terminally blocked proteins and provides multiple, independent stretches of sequence that can be used to generate oligonucleotide probes for molecular cloning and/or used to search sequence data bases for related proteins. This method has been successfully applied to the routine amino acid sequence analysis of a wide range of proteins isolated from one- and two-dimensional polyacrylamide gels.
Binding of the virion protein mediating alpha gene induction in herpes simplex virus 1-infected cells to its cis site requires cellular proteins.
Authors: McKnight J L , Kristie T M , Roizman B
Published: May 2006

In herpes simplex virus 1-infected cells, the transcription of alpha genes, the first set of genes to be expressed, is induced by a virion component, the alpha-trans-induction factor, and requires a cis site. Homologs of the cis site are present in the promoter-regulatory domains of all alpha genes and bind two cellular proteins designated as alpha H1 and alpha H2-alpha H3. We report that alpha-trans-induction factor, synthesized in vitro or present in nuclear extracts of infected cells, forms complexes with viral DNA fragments containing its cis-acting site only in the presence of cellular proteins and only under conditions that also enable the binding of the alpha H1 protein to the DNA. The induction of alpha genes by alpha-trans-induction factor appears, therefore, to be mediated by the interaction of the viral protein with cellular proteins at its cis-acting site.
High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling.
Authors: Sipe D M , Murphy R F
Published: May 2006

The kinetics of acidification of diferric human transferrin in BALB/c mouse 3T3 cells were determined by flow cytometry using a modification of the fluorescein-rhodamine fluorescence ratio technique. For cells labeled at 0 degrees C and warmed to 37 degrees C, the minimum pH observed was 6.1 +/- 0.2, occurring 5 min after warmup. This step was followed by a slower alkalinization to the pH of the external medium, occurring with a half-time of 5 min. Warmup to 24 degrees C or 17 degrees C resulted in slowing of the time of onset of acidification such that the minimum pH was 6.3 +/- 0.2, attained 15 and 25 min after warmup, respectively; the alkalinization step was completely blocked. The limited acidification observed for transferrin corresponds to the initial phase of acidification normally observed for other (nonrecycled) ligands. Since transferrin is not further acidified, the results confirm the existence of two phases of acidification during endocytosis. Measurements of transferrin dissociation at neutral pH after exposure to mildly acidic pH support the conclusion that the transferrin cycle may be completed without exposure of transferrin to a pH below 6. The mildly acidic pH of the endocytic compartments involved in recycling may play a role in regulating enzymatic processing of endocytosed material.
High rate of recombination and double crossovers in the mouse pseudoautosomal region during male meiosis.
Authors: Soriano P , Keitges E A , Schorderet D F , Harbers K , Gartler S M , Jaenisch R
Published: May 2006

The recombination rate in meiosis between the mouse X and Y chromosomes was analyzed. Mice heterozygous at two pseudoautosomal alleles, the steroid sulfatase gene and the Mov-15 provirus marker, were crossed. The provirus in the Mov-15 transgenic mouse strain had been previously shown to be carried in the pseudoautosomal region of the sex chromosomes. Recombination frequencies were shown to be 7-fold higher in this region in male meiosis than in female meiosis. Three-point crosses indicated the occurrence in male meiosis of double recombination events in the pseudoautosomal region, with little or no crossover interference, which is in marked contrast to observations made on the similar region of the human sex chromosomes. This result is contrary to a previous model, which predicted a single crossover event in male meiotic pairing of mammalian sex chromosomes.
Pancreastatin and islet hormone release.
Authors: Efendić S , Tatemoto K , Mutt V , Quan C , Chang D , Ostenson C G
Published: May 2006

The effect of pancreastatin on the release of insulin, glucagon, and somatostatin was studied in the isolated perfused rat pancreas. After an initial equilibration period (-20 to 0 min) with a basal glucose concentration (3.3 mM), the pancreata were perfused with either 16.7 mM glucose (0-40 min) or with 20 mM arginine (0-20 min). Pancreastatin was introduced 10 min prior to and throughout the administration of the high glucose and arginine and continued during their perfusion. As expected, the glucose and the arginine augmented insulin and somatostatin release. Pancreastatin (1 and 10 nM) markedly suppressed the first phase of insulin release with both insulinogogues used, while the early somatostatin secretion was not significantly decreased. However, the peak incremental somatostatin response to arginine was reduced by 50% (P less than 0.05). Conversely, the peptide (10 nM) tended to augment arginine-induced glucagon release. Pancreastatin (100 nM) also suppressed glucose-stimulated insulin release from isolated rat islets. These pancreastatin-mediated alterations in islet hormone release are reminiscent of those known to characterize non-insulin-dependent diabetes. Therefore, the significance of pancreastatin in islet physiology and pathophysiology deserves special consideration.
HbXL99 alpha: a hemoglobin derivative that is cross-linked between the alpha subunits is useful as a blood substitute.
Authors: Snyder S R , Welty E V , Walder R Y , Williams L A , Walder J A
Published: May 2006

Under deoxygenated conditions, bis(3,5-dibromosalicyl) fumarate reacts with hemoglobin selectively to cross-link the alpha subunits between Lys-alpha 1 99 and Lys-alpha 2 99. We have characterized further the properties of this recently described hemoglobin and have demonstrated its utility as a blood substitute. The oxygen transport characteristics of the cross-linked derivative are very similar to those of whole blood. Under physiological conditions, the partial pressure of oxygen at half-saturation of hemoglobin is increased to 29 mm Hg (1 mm Hg = 133.3 kPa), compared to 12 mm Hg for hemoglobin A, fully compensating for the absence of 2,3-bisphosphoglycerate outside of the erythrocyte. The Hill coefficient is 2.9. The dependence of the oxygen affinity of HbXL99 alpha on CO2 is also identical to that of hemoglobin A. The cross-link between the alpha subunits blocks dissociation of oxyhemoglobin into alpha beta dimers and thereby prevents renal excretion of the modified hemoglobin. In the rat, the half-life of HbXL99 alpha in plasma, at a 15% volume exchange, is increased to 3.3 hr, compared to 90 min for hemoglobin A. Cross-linking HbXL99 alpha intermolecularly with bis(sulfosuccinimidyl) suberate to form predominantly a mixture of dimers and trimers further increased the half-life of the hemoglobin within the circulation by about 2-fold. The rate of autooxidation of the transfused hemoglobin was found to be markedly reduced because of the presence of an endogenous reducing system in plasma.
Reversible, calcium-dependent membrane association of human leukocyte 5-lipoxygenase.
Authors: Rouzer C A , Samuelsson B
Published: May 2006

Maximal activity of human leukocyte 5-lipoxygenase requires Ca2+, ATP, a microsomal membrane preparation, and two cytosolic stimulatory factors. We report here some effects of Ca2+ on the physical properties of the 5-lipoxygenase. When leukocytes were homogenized in the presence of 2 mM EDTA, 5-lipoxygenase was found to be a soluble enzyme. However, when Ca2+ was added to homogenization buffers at 0-1 mM in excess of EDTA, increasing quantities of the enzyme were recovered in the microsomal membrane fraction (100,000 X g pellet). The membrane-associated enzyme was resolubilized by washing pellet preparations in buffers containing 2 mM EDTA and was partially purified by anion-exchange chromatography. Studies of the stimulatory-factor requirements of the membrane-associated, resolubilized, and partially purified enzyme indicated that one of the cytosolic 5-lipoxygenase stimulatory factors exhibited a reversible, Ca2+-dependent membrane association, similar to that of the enzyme itself. Ca2+ also caused a destabilization of the 5-lipoxygenase. Homogenates prepared in the presence of Ca2+ contained lower total enzyme activity, and retention of activity in these samples over time was also diminished.
Induction of
Bradyrhizobium japonicum
common
nod
genes by isoflavones isolated from
Glycine max
Authors: Kosslak Renee M. , Bookland Roger , Barkei John , Paaren Herbert E. , Appelbaum Edward R.
Published: May 2006

The early events in legume nodulation by
Rhizobium
spp. involve a conserved gene cluster known as the common
nod
region. A broad-host-range plasmid (pEA2-21) containing a
Bradyrhizobium japonicum nodDABC-lacZ
translational fusion was constructed and used to monitor
nod
gene expression in response to soybean root extract. Two inducing compounds were isolated and identified. Analysis using ultraviolet absorption spectra, proton nuclear magnetic resonance, and mass spectrometry showed that the two inducers were 4′,7-dihydroxyisoflavone (daidzein) and 4′,5,7-trihydroxyisoflavone (genistein). Induction was also seen with some, but not all, of the flavonoid compounds that induce
nod
genes in fast-growing
Rhizobium
strains that nodulate clover, alfalfa, or peas. When pEA2-21 was introduced into
Rhizobium trifolii
, it was inducible by flavones but not by daidzein and genistein. In
Rhizobium fredii
, pEA2-21 was induced by isoflavones and flavones. Thus, the specificity of induction appears to be influenced by the host-strain genome.
Bioactivation mechanism of the cytotoxic and nephrotoxic S-conjugate S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine.
Authors: Dekant W , Lash L H , Anders M W
Published: May 2006

The bioactivation of S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC) was studied with purified bovine kidney cysteine conjugate beta-lyase and with N-dodecylpyridoxal bromide in cetyltrimethylammonium bromide micelles as a pyridoxal model system. The beta-lyase and the pyridoxal model system converted CTFC to chlorofluoroacetic acid and inorganic fluoride, which were identified by 19F NMR spectrometry. 2-Chloro-1,1,2-trifluoroethanethiol and chlorofluorothionoacetyl fluoride were formed as metabolites of CTFC and were trapped with benzyl bromide and diethylamine, respectively, to yield benzyl 2-chloro-1,1,2-trifluoroethyl sulfide and N,N-diethyl chlorofluorothioacetamide, which were identified by gas chromatography/mass spectrometry. The bioactivation mechanism of CTFC therefore involves the initial formation of the unstable thiol 2-chloro-1,1,2-trifluoroethanethiol, which loses hydrogen fluoride to form the acylating agent chlorofluorothionoacetyl fluoride; hydrolysis of the thionoacyl fluoride affords the stable, terminal metabolites chlorofluoroacetic acid and inorganic fluoride. The intermediate acylating agent and chlorofluoroacetic acid may contribute to the cytotoxic effects of CTFC.
Inhibition of hydroxymethylglutaryl-coenzyme A synthase by L-659,699.
Authors: Greenspan M D , Yudkovitz J B , Lo C Y , Chen J S , Alberts A W , Hunt V M , Chang M N , Yang S S , Thompson K L , Chiang Y C
Published: May 2006

A beta-lactone isolated from Fusarium sp. has been shown to be a potent specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase [(S)-3-hydroxy-3-methylglutaryl-CoA acetoacetyl-CoA-lyase (CoA-acetylating), EC, 4.1.3.5] from rat liver. The structure of this beta-lactone, termed L-659,699, is (E,E)-11-[3-(hydroxy-methyl)-4-oxo-2-oxytanyl]-3,5,7-trimethyl-2,4 - undecadienenoic acid. A partially purified preparation of cytoplasmic HMG-CoA synthase from rat liver was inhibited by L-659,699 with an IC50 of 0.12 microM. The enzyme HMG-CoA reductase, beta-ketoacyl-CoA thiolase, acetoacetyl-CoA synthetase, and fatty acid synthase were not inhibited to any extent by this compound. In cultured Hep G2 cells, the compound inhibited the incorporation of [14C]acetate into sterols with an IC50 of 6 microM, while incorporation of [3H]mevalonate into sterols in these cells was not affected. The activity of HMG-CoA reductase in the cultured Hep G2 cells was induced in a dose-dependent manner by incubation with L-659,699. A 37-fold increase in reductase was observed after a 24-hr incubation with 62 microM L-659,699. The effect of a number of analogs of L-659,699 on HMG-CoA synthase is also discussed.
Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.
Authors: Tomkinson B , Wernstedt C , Hellman U , Zetterqvist O
Published: May 2006

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.
Carcinogen-induced mdr overexpression is associated with xenobiotic resistance in rat preneoplastic liver nodules and hepatocellular carcinomas.
Authors: Fairchild C R , Ivy S P , Rushmore T , Lee G , Koo P , Goldsmith M E , Myers C E , Farber E , Cowan K H
Published: May 2006

We have previously reported the isolation of a human breast cancer cell line resistant to doxorubicin (adriamycin; AdrR MCF-7 cells) that has also developed the phenotype of multidrug resistance (MDR). MDR in this cell line is associated with increased expression of mdr (P glycoprotein) gene sequences. The development of MDR in AdrR MCF-7 cells is also associated with changes in the expression of several phase I and phase II drug-detoxifying enzymes. These changes are remarkably similar to those associated with development of xenobiotic resistance in rat hyperplastic liver nodules, a well-studied model system of chemical carcinogenesis. Using an mdr-encoded cDNA sequence isolated from AdrR MCF-7 cells, we have examined the expression of mdr sequences in rat livers under a variety of experimental conditions. The expression of mdr increased 3-fold in regenerating liver. It was also elevated (3- to 12-fold) in several different samples of rat hyperplastic nodules and in four of five hepatomas that developed in this system. This suggests that overexpression of mdr, a gene previously associated with resistance to antineoplastic agents, may also be involved in the development of resistance to xenobiotics in rat hyperplastic nodules. In addition, although the acute administration of 2-acetylaminofluorene induced an 8-fold increase in hepatic mdr-encoded RNA, performance of a partial hepatectomy either before or after administration of 2-acetylaminofluorene resulted in a greater than 80-fold increase in mdr gene expression over that in normal untreated livers. This represents an important in vivo model system in which to study the acute regulation of this drug resistance gene.
Cell-specific activity of the constituent elements of the simian virus 40 enhancer.
Authors: Nomiyama H , Fromental C , Xiao J H , Chambon P
Published: May 2006

We have compared the activation of transcription from the early promoter of simian virus 40 brought about by the enhancer during transient expression in several cell lines. The enhancer stimulated RNA synthesis approximately 400-, 100-, 100-, and 300-fold in human HeLa cells, the mouse plasmacytoma B cells MPC11, and the nondifferentiated and retinoic acid-differentiated mouse embryonal carcinoma F9 cells, respectively. The contribution to enhancer activity of its multiple constituent motifs has been investigated in the different cell types using the pA series of recombinants mutated throughout the enhancer. A comparison of the activity of these recombinants in the four cell lines indicates that the activity of the various enhancer motifs is cell specific. The activity of a given motif in a given cell line correlates well with the known presence in this cell line of a protein(s) that binds specifically to that motif in vitro.
Human placental Na+,K+-ATPase alpha subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization.
Authors: Chehab F F , Kan Y W , Law M L , Hartz J , Kao F T , Blostein R
Published: May 2006

A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na+,K+-ATPase alpha subunit was cloned from human placenta and its sequence was identical to that encoding the alpha subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na+,K+-ATPase gene from various human tissues and cell lines revealed only one band (approximately 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine greater than placenta greater than liver greater than pancreas, and in the cell lines the levels were human erythroleukemia greater than butyrate-induced colon greater than colon greater than brain greater than HeLa cells. mRNA was undetectable in reticulocytes, consistent with our failure to detect positive clones in a size-selected (greater than 2 kilobases) lambda gt11 reticulocyte cDNA library. DNA analysis revealed a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the alpha subunit is on the short arm (band p11-p13) of chromosome 1.
Involvement of a nucleolar component, perichromonucleolin, in the condensation and decondensation of chromosomes.
Authors: Shi L J , Ni Z M , Zhao S , Wang G , Yang Y
Published: May 2006

A component of the nucleolar material, perichromonucleolin (PCN), was identified by a specific antiserum against nucleoli obtained from a scleroderma patient. The distribution and changes of PCN during the mitotic cycle were followed using this antiserum and indirect immunofluorescence microscopy. Based upon the behavior of PCN during mitosis, it could be differentiated into chromosomal and nonchromosomal portions. During prophase the former gradually associates with the surface of the condensing chromosomes and forms the coat or pellicle around the metaphase and anaphase chromosomes. This pellicle PCN is carried by the anaphase chromosomes to the daughter nuclei. During the time from telophase to interphase, the pellicle PCN dissociates from the decondensing chromosomes and is incorporated into the new nucleoli. The nonchromosomal PCN, after the breakdown of the nuclear membrane, distributes in the cytoplasmic region around the condensed chromosomes and diffuses into the cytoplasm during anaphase. Preliminary biochemical analysis by immunoblotting showed that the PCN consists of two main proteins with molecular masses of 36 kDa and 30 kDa.
Fractionation of HeLa cell nuclear extracts reveals minor small nuclear ribonucleoprotein particles.
Authors: Krämer A
Published: May 2006

Upon chromatographic fractionation of HeLa cell nuclear extracts, small RNAs of 145 and 66/65 nucleotides, respectively, were detected that are distinct from the abundant small RNAs present in the extract. These RNAs are precipitated by antibodies directed against the trimethylguanosine cap structure, characteristic for small nuclear RNAs (snRNAs) of the U type. The RNAs of 145 and 66/65 nucleotides appear to be associated with at least one of the proteins common to the major small nuclear ribonucleoprotein particles U1 to U6, since they are specifically bound by anti-Sm antibodies. These criteria characterize the RNAs that are 145 and 66/65 nucleotides in length as U-type snRNAs. Upon gel filtration, the RNAs are found within particles of molecular weights approximately equal to 150,000 and 115,000, respectively. The RNA of 145 nucleotides represents a different minor snRNA, designated U11, whereas the RNA of 66/65 nucleotides may correspond to either mammalian U7 or U10 RNA.
Two enzymes involved in biosynthesis of the host-selective phytotoxin HC-toxin
Authors: Walton Jonathan D.
Published: May 2006

Cochliobolus carbonum
race 1 produces a cyclic tetrapeptide HC-toxin, which is necessary for its exceptional virulence on certain varieties of maize. Previous genetic analysis of HC-toxin production by the fungus has indicated that a single genetic locus controls HC-toxin production. Enzymes involved in the biosynthesis of HC-toxin have been sought by following the precedents established for the biosynthetic enzymes of cyclic peptide antibiotics. Two enzymatic activities from
C. carbonum
race 1 were found, a D-alanine- and an L-proline-dependent ATP/PP
i
exchange, which by biochemical and genetic criteria were shown to be involved in the biosynthesis of HC-toxin. These two activities were present in all tested race 1 isolates of
C. carbonum
, which produce HC-toxin, and in none of the tested race 2 and race 3 isolates, which do not produce the toxin. In a genetic cross between two isolates of
C. carbonum
differing at the
tox
locus, all
tox
+
progeny had both activities, and all
tox
-
progeny lacked both activities.
Identification of a gene that regulates expression of M protein, the major virulence determinant of group A streptococci.
Authors: Caparon M G , Scott J R
Published: May 2006

By using Tn916 insertional mutagenesis, we have identified mry (M protein RNA yield), a gene required for high-level expression of the M protein, an essential virulence determinant of the group A streptococcus. The mry::Tn916 mutation causes a reduction by a factor of approximately equal to 50 in the amount of M protein produced, in the original strain and in a nonmutagenized host to which the mutation was transferred by transduction. The insertion is located approximately 1.8 kilobases upstream of the structural gene for M protein (emm) and its promoter. The genomic region including mry::Tn916 and emm was cloned on a cosmid vector and introduced into Escherichia coli. When the transposon excised precisely from the chimeric cosmid in E. coli, the resulting streptococcal DNA showed the same restriction pattern as the homologous chromosomal region in the parental nonmutagenized streptococcus. The sequence of the promoter for emm was not altered in the mry mutant. The reduction in protein correlates with a decrease in the amount of M protein-specific mRNA, indicating that mry regulates transcription of emm.
Grafted noradrenergic neurons suppress seizure development in kindling-induced epilepsy.
Authors: Barry D I , Kikvadze I , Brundin P , Bolwig T G , Björklund A , Lindvall O
Published: May 2006

Norepinephrine-rich cell suspensions, prepared from the locus coeruleus region of rat fetuses, were grafted bilaterally into the hippocampus of rats made hypersensitive to hippocampal kindling by a neurotoxic lesion of the central catecholamine system. The animals with grafts showed a marked suppression of the onset and progression of kindling-induced epilepsy, and this effect was correlated with the degree of graft-derived noradrenergic innervation of the host hippocampal formation. We conclude that grafted neurons can modulate the excitability of epileptic brain regions.
Tail shock produces inhibition as well as sensitization of the siphon-withdrawal reflex of Aplysia: possible behavioral role for presynaptic inhibition mediated by the peptide Phe-Met-Arg-Phe-NH2.
Authors: Mackey S L , Glanzman D L , Small S A , Dyke A M , Kandel E R , Hawkins R D
Published: May 2006

Recent studies have shown that, in addition to being modulated by presynaptic facilitation, the sensory neurons of the gill- and siphon-withdrawal reflex of Aplysia are also capable of being modulated by transient presynaptic inhibition produced by the peptide Phe-Met-Arg-Phe-NH2. These two modulatory effects involve different second-messenger systems: the facilitation is mediated through cAMP-dependent protein phosphorylation, and the inhibition is mediated through the lipoxygenase pathway of arachidonic acid. To explore the behavioral function of this inhibition, we have carried out a parametric analysis of the effect of tail shock on the siphon-withdrawal reflex. In addition to producing sensitization of the withdrawal reflex, tail shock also transiently inhibits the reflex. The inhibition is produced by relatively weak shock, whereas sensitization is more prominent and may mask the inhibition with stronger shock. Furthermore, inhibition is not observed after habituation training. Cellular studies suggest that the behavioral inhibition is mediated, at least in part, by presynaptic inhibition of transmitter release from the siphon sensory neurons. Moreover, we have identified an interneuron within the left pleural ganglion (LPL16) that shows Phe-Met-Arg-Phe-NH2 immunoreactivity, is activated by tail shock, and simulates the presynaptic inhibitory actions produced by tail shock. Therefore, our results suggest that presynaptic inhibition mediated by Phe-Met-Arg-Phe-NH2 and its lipoxygenase second messenger contributes to behavioral inhibition of the siphon-withdrawal reflex.
Biosynthesis and intracellular transport of the receptor for platelet-derived growth factor.
Authors: Claesson-Welsh L , Rönnstrand L , Heldin C H
Published: May 2006

The biosynthesis of the receptor for platelet-derived growth factor (PDGF) was examined in metabolically labeled human foreskin fibroblasts. The receptor was synthesized as a 145-kDa precursor, which, when incubated with endo-beta-N-acetylglucosaminidase H (endo H), underwent a 15-kDa decrease in molecular mass. This indicates that the size of the core protein is about 130 kDa and that the 145-kDa form represents a receptor precursor carrying high-mannose N-linked oligosaccharide groups. Within 15 min after synthesis, the receptor was converted to a 165-kDa form. This form was entirely resistant to endo H treatment and probably represents a receptor molecule that has undergone further posttranslational modification, including O-linked glycosylation. Subsequently, within 30 min, a molecule of 170 kDa--i.e., the size of the mature receptor--appeared. A slightly larger molecule, of 175 kDa, which could be immunoprecipitated from PDGF-stimulated 32P-labeled cells, probably represents a receptor further modified by autophosphorylation. The 170-kDa molecule had an isoelectric point of about 4.5. Addition of PDGF increased the turnover rate of the 170-kDa PDGF receptor.
Isolation and sequencing of the gene encoding delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni: overexpression of the protein.
Authors: Kuliopulos A , Shortle D , Talalay P
Published: May 2006

We describe the cloning, sequencing, and overexpression of the steroid isomerase (3-oxosteroid delta 5-delta 4-isomerase, EC 5.3.3.1) gene of Pseudomonas testosteroni. A genomic library of P. testosteroni total DNA constructed from partial EcoRI digests ligated to a lambda gtWES vector was probed with a 23-base oligonucleotide mixture [ATGAAC(T)ACC(A,T)CCG(C,A)GAG(A)CAC(T)ATGAC] corresponding to the NH2-terminal sequence of steroid isomerase. Subclones derived from a recombinant phage containing a 5400-base-pair insert were sequenced and found to contain the expected 375-nucleotide open reading frame flanked at both ends by in-frame TGA termination codons. The DNA sequence agreed with the above 125-amino acid sequence except for codons 22, 24, 33, and 38, all of which encoded Asp rather than Asn, and codon 77, which encoded Glu rather than Gln. A 1370-base-pair fragment was inserted into pUC 19 plasmid vector and used to construct a strain of Escherichia coli JM 101 that overexpressed the isomerase gene in the presence of isopropyl beta-D-thiogalactopyranoside. Cytosolic extracts of this strain contained a major soluble protein that migrated with the steroid delta-isomerase subunit on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. These cytosolic extracts had 10-50% of the specific activity of crystalline isomerase, depending on the method of preparation. The recombinant enzyme was crystallized in both monoclinic (flat plates) and hexagonal (bipyramids) crystal forms, described previously for the enzyme isolated from P. testosteroni. The kinetic properties of the crystalline recombinant enzyme, including specific activity, Km for 5-androstene-3,17-dione (340 microM), and Ki for the competitive inhibitor 19-nortestosterone (11.9 microM), agreed closely with the values reported for the isolated enzyme.
Importance of disulfide bridges in the structure and activity of Escherichia coli enterotoxin ST1b.
Authors: Gariépy J , Judd A K , Schoolnik G K
Published: May 2006

A 13-amino acid sequence of the Escherichia coli heat-stable enterotoxin ST1b encodes its receptor-binding and diarrheal functions. This sequence includes six cysteines involved in three intramolecular disulfide bridges. To determine the importance of disulfide bridges to the biological activity of ST1b, we synthesized 15 analogues of the tridecapeptide representing all possible replacements of two of the six cysteines by alanines. Only 2 analogues--namely, A6,11ST1b-(6-18) and A10,18ST1b(6-18)--could inhibit the binding of a radiolabeled analogue of ST1b to rat intestinal cells. The purified peptides were, respectively, 4200 and 130 times less effective as inhibitors than ST1b(6-18), the sequence that includes all six cysteines. In addition, both peptides produce diarrhea when given orally to suckling mice. These analogues share in common only two cysteines (Cys-7 and Cys-15), suggesting that four cysteines, two of which are Cys-7 and Cys-15, are necessary for activity. A pattern of disulfide linkages is proposed where Cys-7 is paired to Cys-15, Cys-6 to Cys-11, and Cys-10 to Cys-18, the preceding disulfide bridges being ranked in descending order of importance in terms of their respective contribution to the activity of the enterotoxin. Using this disulfide bridge arrangement and constraints derived from NMR spectroscopy, we propose a folding pattern for the toxic domain of ST1b.
Genetic transfer in
Desulfovibrio desulfuricans
Authors: Rapp Barbara J. , Wall Judy D.
Published: May 2006

An apparently defective bacteriophage capable of mediating transduction has been identified in culture filtrates of
Desulfovibrio desulfuricans
(American Type Culture Collection 27774). Phage-mediated intraspecies transfer of antibiotic resistance markers occurs with a frequency of 10
-5
to 10
-6
per recipient cell. The vector contains linear fragments of double-stranded DNA of about 13.5 kilobase pairs, which appear to be random pieces of bacterial DNA. As yet, neither induction nor plaque formation has been observed. To our knowledge, a system of genetic exchange has not been described before for a member of the sulfate-reducing bacteria.
From behavior to membranes: testosterone-induced changes in action potential duration in electric organs.
Authors: Bass A H , Volman S F
Published: May 2006

The electric organ of mormyrid fishes consists of action potential-generating cells called electrocytes, which together produce a pulse-like electric organ discharge (EOD). The appearance of an EOD depends, in part, on the characteristic features of a single electrocyte's action potentials. In some species, gonadal steroid hormones induce increases in EOD duration, which mimic natural sex differences. We now show that testosterone-induced changes in EOD duration are associated with a 2- to 3-fold increase in the duration of action potentials generated by single electrocytes. Together with other anatomical and biochemical data, the results emphasize the exquisite interrelationship between steroid hormone action and the cellular machinery determining the electrical properties of single cells that underlie sexually dimorphic and seasonal behaviors.
Human neutrophil elastase releases cell surface mucins from primary cultures of hamster tracheal epithelial cells.
Authors: Kim K C , Wasano K , Niles R M , Schuster J E , Stone P J , Brody J S
Published: May 2006

Primary hamster tracheal epithelial cells growing on a collagen gel matrix produce high molecular weight mucins indistinguishable from mucins produced in vivo. Using a modified version of these confluent cultures, we have demonstrated here that (i) release of mucins can be stimulated by human neutrophil elastase (HNE; EC 3.4.21.37); (ii) HNE can degrade mucins, and both mucin release and degradation by HNE require an active catalytic site; and (iii) there are at least two pools of mucins in these cells: one is a rapidly turning-over spontaneously releasable constitutive pool, the other is a slowly turning-over HNE-releasable pool. We provide evidence that the HNE-releasable mucins are membrane bound and associated with the secretory cell apical surface.
Human lymphocyte Fc receptor for IgE: sequence homology of its cloned cDNA with animal lectins.
Authors: Ikuta K , Takami M , Kim C W , Honjo T , Miyoshi T , Tagaya Y , Kawabe T , Yodoi J
Published: May 2006

We have purified the human lymphocyte Fc receptor specific for IgE (Fc epsilon receptor) and its soluble form by using the anti-Fc epsilon receptor monoclonal antibody H107. Using an oligonucleotide probe corresponding to the partial amino acid sequence of the soluble Fc epsilon receptor related to IgE binding factor, we cloned, sequenced, and expressed a cDNA for the receptor. The Fc epsilon receptor has 321 amino acid residues with no NH2-terminal signal sequence. The receptor was separated into two domains by a putative 24-amino acid residue transmembrane region located near the NH2-terminal end. The Fc epsilon receptor showed a marked homology with animal lectins including human and rat asialoglycoprotein receptors, chicken hepatic lectin, and rat mannose binding proteins.
Cell division promoting activity of naturally occurring dehydrodiconiferyl glucosides: do cell wall components control cell division?
Authors: Binns A N , Chen R H , Wood H N , Lynn D G
Published: May 2006

Naturally occurring isomers of the dehydrodiconiferyl glucosides have been isolated from Vinca rosea crown gall tumors and have been tested for cell division promoting activities in the tobacco pith and leaf assay systems. The enantiomeric isomers A and B are active, although they are required at concentrations up to 2 orders of magnitude higher than zeatin riboside to promote comparable growth. We estimate that the active dehydrodiconiferyl glucosides are present in rapidly growing tissues (tumor tissue, habituated tissue, cultured nontransformed tissue) in micromolar concentrations. In quiescent tobacco pith tissue, the levels of these compounds are reduced by a factor of 100. These results suggest that cytokinin may exert control of cell division through the accumulation of molecules (the dehydrodiconiferyl glucosides) that are apparent cell wall components.
Chromatin structure is required to block transcription of the methylated herpes simplex virus thymidine kinase gene.
Authors: Buschhausen G , Wittig B , Graessmann M , Graessmann A
Published: May 2006

Inhibition of herpes simplex virus (HSV) thymidine kinase (TK) gene transcription (pHSV-106, pML-BPV-TK4) by DNA methylation is an indirect effect, which occurs with a latency period of approximately equal to 8 hr after microinjection of the DNA into TK- rat 2 and mouse LTK- cells. We have strong evidence that chromatin formation is critical for the transition of the injected DNA from methylation insensitivity to methylation sensitivity. Chromatin was reconstituted in vitro by using methylated and mock-methylated HSV TK DNA and purified chicken histone octamers. After microinjection, the methylated chromatin was always biologically inactive, as tested by autoradiography of the cells after incubation with [3H]thymidine and by RNA dot blot analysis. However, in transformed cell lines, reactivation of the methylated chromatin occurred after treatment with 5-azacytidine. Furthermore, integration of the TK chromatin into the host genome is not required to block expression of the methylated TK gene. Mouse cells that contained the pML-BPV-TK4 chromatin permanently in an episomal state also did not support TK gene expression as long as the TK DNA remained methylated.
The E2 "gene" of bovine papillomavirus encodes an enhancer-binding protein.
Authors: Moskaluk C , Bastia D
Published: May 2006

The E2 early open reading frame (presumably gene) of bovine papillomavirus-1 was fused in frame with the collagen-beta-galactosidase-encoding region of the vector pJG200 and was expressed in and partially purified from Escherichia coli. The hybrid protein specifically bound to the enhancer region of bovine papillomavirus at several sites. DNase I-cleavage protection analysis of one such site revealed the protected sequence. A comparison of the protected sequence with the remainder of the DNA sequences that also have affinity for the protein revealed a consensus sequence having the motif AATTGGCGGNNCG, in which N is any nucleotide. The protected region also includes a sequence with 2-fold rotational symmetry--ATCGGTG/CACCGAT.
The Thy-1 antigen exhibits rapid lateral diffusion in the plasma membrane of rodent lymphoid cells and fibroblasts.
Authors: Ishihara A , Hou Y , Jacobson K
Published: May 2006

Thy-1 is a plasma membrane protein, but its primary structure lacks the typical membrane-spanning sequence. Recent studies revealed that a glycophospholipid is covalently bound to the carboxyl terminus, suggesting that the protein is integrated into the plasma membrane by this lipid moiety. Lateral diffusion of Thy-1 was measured in mouse thymocytes, lymphoma cells, and fibroblasts by the fluorescence recovery after photobleaching technique. Thy-1 was labeled with rhodamine-conjugated anti-Thy-1 monoclonal antibodies. Diffusion coefficients of 2-4 X 10(-9) cm2/sec were obtained for the antigen-antibody complex in all the cell types. About 50% of the Thy-1 was mobile. The diffusion coefficient for the mobile fraction of Thy-1 is considerably larger than the diffusion coefficients of many other plasma membrane proteins. Rather, the diffusion coefficient of Thy-1 is similar to those of lipid analogs embedded in the same membrane, providing strong support for the suggested lipid anchoring of this antigen.