bluuebunny/crossref_metadata_2025 huggingface dataset:
Subcellular compartmentalization of 1-methyl-4-phenylpyridinium with catecholamines in adrenal medullary chromaffin vesicles may explain the lack of toxicity to adrenal chromaffin cells.
Authors: Reinhard J F , Diliberto E J , Viveros O H , Daniels A J
Published: May 2006

Cultures of bovine adrenomedullary chromaffin cells accumulated 1-methyl-4-phenylpyridinium (MPP+) in a time- and concentration-dependent manner by a process that was prevented by desmethylimipramine. The subcellular localization of the incorporated [methyl-3H]MPP+ was examined by differential centrifugation and sucrose density gradient fractionation and was found to be predominantly colocalized with catecholamines in chromaffin vesicles, and negligible amounts were detected within the mitochondrial fraction. When chromaffin cell membranes were made permeable with the detergent digitonin in the absence of calcium, there was no increase in the release of [3H]MPP+, indicating that there is negligible accumulation of the neurotoxin in the cytosol. Simultaneous exposure to digitonin and calcium induced cosecretion of MPP+ and catecholamines. Stimulation of the cells with nicotine released both catecholamines and MPP+ at identical rates and percentages of cellular content in a calcium-dependent manner. Last, when cells were incubated with MPP+ in the presence of tetrabenazine (an inhibitor of vesicular uptake), the chromaffin cell toxicity of MPP+ was potentiated. We submit that the ability of the chromaffin cells to take up and store MPP+ in the chromaffin vesicle prevents the toxin's interaction with other structures and, thus, prevents cell damage. As an extension of this hypothesis, the relative resistance of some brain monoaminergic neurons to the toxic actions of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine may result from the subcellular sequestration of MPP+ in the storage vesicle.
Selecting the highest of three binomial probabilities
Authors: Zybert Patricia , Levin Bruce
Published: May 2006

A sequential elimination procedure for selecting the highest probability in binomial trials, proposed by Levin and Robbins [Levin, B. & Robbins, H. (1981)
Proc. Natl. Acad. Sci. USA
78, 4663-4666], is examined further in the special case of trials involving three probabilities. A conjectured inequality relating ratios of selection probabilities to odds ratios is shown to hold only under certain necessary and sufficient conditions. Weaker conjectured inequalities involving the probability of correct selection are shown to hold without restriction.
Primary structure of rat cardiac beta-adrenergic and muscarinic cholinergic receptors obtained by automated DNA sequence analysis: further evidence for a multigene family.
Authors: Gocayne J , Robinson D A , FitzGerald M G , Chung F Z , Kerlavage A R , Lentes K U , Lai J , Wang C D , Fraser C M , Venter J C
Published: May 2006

Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene.
Escherichia coli replication factor Y, a component of the primosome, can act as a DNA helicase.
Authors: Lee M S , Marians K J
Published: May 2006

The primosome is a mobile multienzyme DNA replication-priming complex that requires seven Escherichia coli proteins for assembly (the products of the dnaB, dnaC, dnaG, and dnaT genes as well as proteins n and n" and replication factor Y). It has been shown previously that the primosome, in combination with the E. coli DNA polymerase III holoenzyme, can form replication forks in vitro that move at rates similar to those measured in vivo and that the primosome and one of the components of the primosome, the DNA B protein, have DNA helicase activity. Evidence is presented here that another component of the primosome, replication factor Y, possesses DNA helicase activity as well. Factor Y helicase activity requires the presence of E. coli single-stranded DNA binding protein, Mg2+, and hydrolyzable ATP or dATP. Helicase activity is stimulated 15-fold when the enzyme is actively loaded onto single-stranded DNA through a primosome assembly site, and duplex DNA is unwound unidirectionally, 3'----5', along the DNA strand to which the protein is bound.
Cell-cycle-specific interaction of nuclear DNA-binding proteins with a CCAAT sequence from the human thymidine kinase gene.
Authors: Knight G B , Gudas J M , Pardee A B
Published: May 2006

Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, we have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene.
Correlation between the 32-kDa sigma factor levels and in vitro expression of Escherichia coli heat shock genes.
Authors: Skelly S , Coleman T , Fu C F , Brot N , Weissbach H
Published: May 2006

S-30 extracts from Escherichia coli cells were used to express heat shock (HS) and non-HS genes in vitro in a DNA-directed protein synthesis system. The S-30 extracts prepared from cells that have been shifted to 45 degrees C express HS genes in vitro approximately 8 times better than extracts from cells at 33 degrees C. In contrast, the expression of non-HS genes in extracts from heat-induced cells is only 40% of that seen in extracts from cells at 33 degrees C. These results correlate well with the levels of HS sigma factor and normal sigma factor bound to RNA polymerase. Thus, there was an 8-fold increase in the HS sigma factor and a 60% decrease in the normal sigma factor associated with RNA polymerase at the higher temperature. Part of the increase in the level of the HS sigma factor could be accounted for by a 3-fold increase in the level of HS sigma factor mRNA during heat induction.
Theoretical studies on oscillations in microtubule polymerization.
Authors: Chen Y D , Hill T L
Published: May 2006

Oscillations in the polymerization of microtubules have been studied theoretically, using differential equations and (more realistically) Monte Carlo simulations. There is gross qualitative agreement between theory and experiment but a really satisfactory model has not been found as yet.
Solubilization of a membrane factor that stimulates levels of substance P and choline acetyltransferase in sympathetic neurons.
Authors: Wong V , Kessler J A
Published: May 2006

The choice of which neurotransmitters will be produced by a developing neuron is influenced by the microenvironment of the neuron. In this study we show that neuronal contact with membrane-associated molecules promotes expression of peptidergic and cholinergic traits. Treatment of cultured neonatal rat sympathetic neurons with plasma membranes derived from adult rat spinal cord or sympathetic ganglia induced expression of the peptide transmitter substance P and increased levels of the cholinergic biosynthetic enzyme choline acetyltransferase. The transmitter-stimulating activity could be solubilized from spinal cord membranes by the detergent octyl glucoside but not by Triton X-100. The choline acetyltransferase- and substance P-stimulating activity also could be extracted from spinal cord membranes by 4 M sodium chloride, suggesting that the active material is membrane associated rather than an intrinsic structural membrane molecule. Trypsin or heat treatment of the extract destroyed the transmitter-stimulating activity, indicating that the factor contains a protein. Activity also was destroyed by hyaluronidase treatment, suggesting that the active material may contain a glycosaminoglycan. The choline acetyltransferase-stimulating activity in the 4 M NaCl extract was eluted in a single peak from a calibrated Sephadex G-75 column with a retention time slightly less than that of a 25-kDa standard. NaDodSO4/polyacrylamide gel electrophoresis of the active peak revealed a predominant band at 29 kDa. Thus, contact-mediated stimulation of substance P and choline acetyltransferase activity in sympathetic neurons results from neuronal exposure to a 29-kDa membrane-associated factor.
Ribonucleolytic activity of angiogenin: essential histidine, lysine, and arginine residues.
Authors: Shapiro R , Weremowicz S , Riordan J F , Vallee B L
Published: May 2006

The homology of angiogenin and pancreatic RNase A provides a compelling reason to systematically compare the characteristics of the two proteins using the chemical modification approaches that proved essential to understanding the action of RNase. Reagents specific for histidine, lysine, and arginine markedly decrease the ribonucleolytic activity of angiogenin, much as has been observed for RNase A. Activity is abolished by reduction of the disulfide bonds and is restored by reoxidation. Methionine, tyrosine, and carboxyl group reagents have no significant effect. From the point of view of reactivity, the histidine and lysine residues in angiogenin are severalfold less susceptible to modification than those in RNase A. Arginine reagents, on the other hand, inactivate angiogenin considerably faster than RNase A. Considering specificity, bromoacetate inactivates angiogenin at pH 5.5 by modifying 1.5 histidines, but lysine and arginine reagents are less specific. Thus, 3.8 and 6.3 residues, respectively, are modified by 1-fluoro-2,4-dinitrobenzene and by formaldehyde plus cyanoborohydride, under conditions where activity decreases by approximately 80% in both cases. With phenylglyoxal, 6.7 arginines are lost when there is 92% inactivation. Poly(G) prevents inactivation by lysine and arginine reagents, and phosphate protects against the effects of lysine modification. Thus, the functional consequences of these modifications likely reflect the loss of critical residues rather than general conformational effects.
Structure and formation of microplasmin.
Authors: Wu H L , Shi G Y , Wohl R C , Bender M L
Published: May 2006

The structure of human microplasmin, prepared from plasmin in alkaline solution, has been studied. Microplasmin consists of two polypeptide chains connected by disulfide bonds. One polypeptide is the B chain of plasmin consisting of 230 amino acids, and the other peptide is the COOH-terminal portion of the A chain of plasmin consisting of 31 amino acid residues. Microplasmin has a molecular weight of 28,635, calculated from its primary sequence. It is slightly more positively charged than plasminogen and is a more hydrophobic molecule. The proposed scheme for the formation of microplasmin involves autolysis at specific peptide bonds and scrambling of especially sensitive disulfide bonds in alkaline solution.
Mode of substrate carboxyl binding to the [4Fe-4S]+ cluster of reduced aconitase as studied by 17O and 13C electron-nuclear double resonance spectroscopy.
Authors: Kennedy M C , Werst M , Telser J , Emptage M H , Beinert H , Hoffman B M
Published: May 2006

The active form of aconitase has a diamagnetic [4Fe-4S]2+ cluster. A specific iron ion (Fea, which is lost during inactivation) is the binding site for substrate, as shown by Mössbauer spectroscopy. We have studied the mode of substrate and analogue binding at equilibrium to the paramagnetic [4Fe-4S]+ cluster of the reduced active form by 17O and 13C electron-nuclear double resonance spectroscopy with specifically labeled substrates. The data show that with substrate, only the carboxyl at C-2 of the propane backbone is strongly bound in addition to H2O or OH- (HxO) from the solvent, whereas in an isocitrate analogue that has a nitro group at C-2, the carboxyl and hydroxyl at C-1 are bound along with solvent HxO. We conclude from these data that, on addition of any one of the three substrates, cis-aconitate is the predominant species bound to Fea of the cluster along with solvent HxO and that cis-aconitate is bound in the citrate mode (carboxyl at C-2). The results with the nitro analogue show that the enzyme can also bind a substrate-like ligand to the cluster in the alternative isocitrate mode (carboxyl at C-1), as is implicit in models proposed for the aconitase reaction.
Periodic mitotic events induced in the absence of DNA replication.
Authors: Schlegel R , Pardee A B
Published: May 2006

We have discovered and report here a means of separating a mitotic "subcycle" from the G1- and S-phase events of the mammalian cell cycle. Time-lapse videomicroscopy of Syrian hamster fibroblast (BHK) cells revealed that caffeine could induce multiple entries into mitosis while cells were blocked in DNA synthesis. As with normal mitoses, the abundance of mitosis-specific phosphoproteins was coupled with the condensation of chromatin. The BHK temperature-sensitive mutant tsBN2 also completed multiple entries into mitosis while arrested during DNA replication and raised to the restrictive temperature. Periodic mitotic events occurred even when BHK cells were exposed to low concentrations of serum or cycloheximide, conditions that prevent the cycling of BHK cells by blocking their entry into S phase. These results suggest that an oscillation governing the activation and inactivation of mitotic factors can be generated in mammalian cells and uncoupled from the G1 and DNA replication events of the normal cell cycle. This system will be useful for examining the molecular nature of mitotic factors.
Expression of high and low molecular weight caldesmons during phenotypic modulation of smooth muscle cells.
Authors: Ueki N , Sobue K , Kanda K , Hada T , Higashino K
Published: May 2006

We investigated the expression of two molecular weight forms of caldesmon in a wide range of tissues and cells. The distribution of high molecular weight caldesmon (h-caldesmon, Mr 120,000-150,000) was restricted to smooth muscles where it was found in large quantity. The low molecular weight protein (l-caldesmon, Mr 70,000-80,000) was widely distributed in nonmuscle tissues and cells. Therefore, the expression of h-caldesmon might be much more specific to smooth muscles. We then examined the expressional changes of two caldesmons during phenotypic modulation of smooth muscle cells (SMCs). In developing gizzards, the expression of caldesmons switched from the l- to the h-form. Contrarily, the expression turned from h- to l-caldesmon in association with dedifferentiation of aortic SMCs in primary culture. In agreement with these observations, the levels of those mRNAs that direct the synthesis of both caldesmons were apparently in proportion to the quantities of protein, as determined by use of an in vitro translation system. In addition, h-caldesmon in smooth muscle-like BC3H1 cells increased in its amount with a concomitant reduction of l-caldesmon following serum-depleted and contact-inhibited cytodifferentiation. These results suggest that the expressional changes of two caldesmons are closely correlated with the phenotypic modulation of SMCs.
Molecular cloning and characterization of the structural gene for protein I, the major outer membrane protein of Neisseria gonorrhoeae.
Authors: Carbonetti N H , Sparling P F
Published: May 2006

Protein I (P.I) is the major outer membrane protein of Neisseria gonorrhoeae and serves as a porin. By using oligonucleotide probes derived from the known amino-terminal sequence of the mature protein, we have cloned the gene encoding the P.I of gonococcal strain FA19 in three overlapping fragments and determined the DNA sequence. The gene sequence predicts a protein with characteristics typical of the porins of other Gram-negative bacteria. A clone expressing P.I in Escherichia coli was obtained by removing a portion of the P.I gene promoter and reconstructing the entire P.I gene in a position just downstream from a phage T7 promoter. Expression of P.I was then achieved by introducing this recombinant plasmid into an E. coli strain containing an inducible T7 polymerase gene. The clone produced a protein that was identical in size to native P.I and reacted with anti-P.I monoclonal antibodies. Prolonged expression of the protein apparently was lethal for E. coli, possibly explaining failures to clone an intact P.I gene with its own promoter.
Constitutive expression of the yeast HEM1 gene is actually a composite of activation and repression.
Authors: Keng T , Guarente L
Published: May 2006

We show that HEM1 (encoding 5-aminolevulinate synthase) expression, while constitutive under all steady-state growth conditions tested, is activated by the HAP2-HAP3 global activation system that controls expression of apocytochromes. This finding creates a paradox because apocytochrome activation by HAP2-HAP3 is highly regulated, subject to induction by heme, and subject to further derepression by a shift from glucose medium to one containing a nonfermentable carbon source. We clarify this issue by showing that HEM1 is subject to two additional layers of control that mask regulatory changes. First is a second activation system acting at a site close to the HAP2-HAP3 target sequence that keeps HEM1 turned on under conditions of heme deficiency. Second is a regulated negative control site downstream of the upstream activation site that counteracts derepression in medium containing a nonfermentable carbon source. Thus, transcription of the constitutive gene is actually a composite of opposing regulatory sites. This complex regulatory arrangement may exist to allow HEM1 to be coordinated transiently with apocytochromes for transition to respiratory growth. Conversely, it may reflect the alteration of HEM1 from a regulated to a constitutive gene over evolution.
Alternative to the steady-state method: derivation of reaction rates from first-passage times and pathway probabilities.
Authors: Ninio J
Published: May 2006

An alternative method for deriving rate equations in enzyme kinetics is presented. An enzyme is followed as it moves along the various pathways allowed by the reaction scheme. The times spent in various sections of the scheme and the pathway probabilities are computed, using simple rules. The rate equation obtains as a function of times and probabilities. The results are equivalent to those provided by the steady-state formalism. While the latter applies uniformly to all schemes, the formalism presented here requires adaptation to each additional class of schemes. However, it has the merit of allowing one to leave unspecified many details of the scheme, including topological ones. Furthermore, it allows one to decompose a scheme into subschemes, analyze the parts separately, and use the intermediate results to derive the rate equation of the complete scheme. The method is applied here to derive general equations for one- and two-entry site enzymes.
Hepatocarcinogenicity of the woodchuck hepatitis virus.
Authors: Popper H , Roth L , Purcell R H , Tennant B C , Gerin J L
Published: May 2006

During investigations of the evolution of experimental laboratory infections of woodchucks (Marmota monax) with the woodchuck hepatitis virus (WHV), eight hepatocellular carcinomas (HCC) were observed, six in newborns and two in young adult animals, all within 17-36 months after infection. The absence of an external cocarcinogenic effect in the well-monitored woodchucks indicates the carcinogenicity of WHV and suggests the same for the genetically and biologically similar human hepatitis B virus (HBV). Laboratory infections of woodchucks with two strains of WHV, not reported here in detail, resembled human and chimpanzee HBV infections histologically and serologically. In these studies, eight woodchucks became carriers of surface antigen of WHV for greater than 1 year. All eight woodchucks developed HCC, indicating a 100% risk of HCC in experimentally infected chronic WHV antigen carriers, which is analogous to the high risk of HCC in human hepatitis B surface antigen carriers. Histologically, the absence of cirrhosis in the examined pericarcinomatous tissue permits recognition of gradual transition from normal parenchyma to neoplastic nodules to HCC of rising anaplasia, indicating a continuum of increasingly more malignant neoplastic stages, as known for chemical carcinogenesis. The HCC developed in carrier woodchucks infected as newborns with only minor, if any, hepatitic changes but is associated with antigen-carrying hepatocytes and sometimes with hyperplastic nodules. This stage was preceded in infected adults by an early, acute, weeks-long hepatitis coinciding with the appearance of surface antigen. These findings are also analogous to typical HBV infection in human newborns and young adults, respectively. At the time of HCC development in all animals with adequate histologic material, an acute recent necroinflammation appeared around the tumor, associated with abnormal hematopoietic cells around and within the tumor. A promoting role in carcinogenesis of this necroinflammation of yet unestablished pathogenesis is being postulated, to be confirmed by determination of the status of the WHV DNA in the HCC and by prospective histologic study of the inflammatory reaction.
Prevention of tumorigenesis of oncogene-transformed rat fibroblasts with DNA site inhibitors of poly(ADP ribose) polymerase.
Authors: Tseng A , Lee W M , Jakobovits E B , Kirsten E , Hakam A , McLick J , Buki K , Kun E
Published: May 2006

The EJ-ras gene was placed under the transcriptional control of the steroid-inducible mouse mammary tumor virus promoter/enhancer and introduced into Rat-1 fibroblasts, yielding the 14C cell line. When these cells were exposed to dexamethasone in vitro, EJ-ras mRNA was induced 15- to 20-fold, the cells grew in agar, and, after injection of cells into syngenic Fischer 344 rats, they produced lethal fibrosarcomas. Inhibitors of poly(ADP ribose) polymerase, which prevent the activation of the purified enzyme by a synthetic octadeoxyribonucleotide duplex, inhibited both in vivo tumorigenicity and in vitro growth in soft agar. The enzyme inhibitor 1,2-benzopyrone, which was studied in detail, and other polymerase inhibitors had no effect on EJ-ras mRNA or p21 protein expression. Poly(ADP ribose) polymerase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosyltransferase, EC 2.4.2.30] was inhibited by the drug in both untreated and dexamethasone-treated cells both in vitro and in vivo to the same extent, but biological consequences of enzyme inhibition were manifest only when the cells were in the transformed tumorigenic state.
Images of a lipid bilayer at molecular resolution by scanning tunneling microscopy.
Authors: Smith D P , Bryant A , Quate C F , Rabe J P , Gerber C , Swalen J D
Published: May 2006

The molecular structure of a fatty acid bilayer has been recorded with a scanning tunneling microscope operating in air. The molecular film, a bilayer of cadmium icosanoate (arachidate), was deposited onto a graphite substrate by the Langmuir-Blodgett technique. The packing of the lipid film was found to be partially ordered. Along one axis of the triclinic unit cell the intermolecular distance varied randomly around a mean of 5.84 A with a SD of 0.24 A. Along the other axis the mean distance was 4.1 A and appeared to vary monotonically over several intermolecular distances, indicating that a superstructure of longer range may exist. The molecular density was one molecular per 19.4 A2. The surprising ability of the scanning tunneling microscope to image the individual molecular chains demonstrates that electrons from the graphite can be transferred along the molecular chains for a distance of 50 A.
Abundant expression of homeobox genes in mouse embryonal carcinoma cells correlates with chemically induced differentiation.
Authors: Deschamps J , de Laaf R , Joosen L , Meijlink F , Destrée O
Published: May 2006

Mammalian homeobox-containing genes might play a role in embryonal pattern formation. In favor of this view is the recently reported expression of such genes during mouse embryogenesis [Manley, J. L. & Levine, M. S. (1985) Cell 43, 1-2]. The embryo-derived stem cells and in particular the pluripotent embryonal carcinoma (EC) cell lines are generally considered as a valid model of early mouse development. Homeobox-containing genes were shown to be expressed in differentiating EC cells. We have analyzed the expression of several of these genes in three EC cell lines triggered to differentiate by alternative treatments in the presence or in the absence of retinoic acid. In both types of conditions, C17S1 (clone 1003) and PCC7.S Aza R1 EC cells were induced to differentiate into mainly neurones, and PSA-1 EC cells were induced to differentiate into a large spectrum of tissue derivatives. Induction to high levels of expression of several homeobox-containing genes during differentiation occurs only in the presence of retinoic acid. Nonchemical treatment triggering differentiation does not lead to detectable expression of these genes. Accumulation to high amounts of homeobox-containing gene transcripts in these experiments seems to correlate with retinoic acid-induced EC cell differentiation rather than with EC cell differentiation as such.
Fusion as a mediator of cytolysis in mixtures of uninfected CD4+ lymphocytes and cells infected by human immunodeficiency virus.
Authors: Yoffe B , Lewis D E , Petrie B L , Noonan C A , Melnick J L , Hollinger F B
Published: May 2006

We describe an unusual type of cytopathology in which uninfected CD4+ (helper/inducer) cells (cells expressing the human leukocyte antigen CD4) interact with cells persistently infected with the human immunodeficiency virus (HIV). Prior antigenic stimulation was not required, since CD4+ cells taken either from healthy persons without anti-HIV antibodies or from individuals with anti-HIV antibodies were capable of inducing cytolysis. Neither CD8+ (suppressor/cytotoxic) nor CD16+ (natural killer) cells mediated the reaction. Light microscopic and autoradiographic studies revealed that, prior to cytolysis, multinucleated giant cells were formed from fusions between HIV-infected cells and large numbers of uninfected CD4+ lymphocytes. These data may explain the paradox that exists in vivo in which a dramatic depletion of CD4+ lymphocytes occurs in the presence of a small number of HIV-infected CD4+ cells. These new insights into the pathogenesis of acquired immunodeficiency syndrome (AIDS) may lead to future therapeutic strategies.
B lymphocyte-specific protein binding near an immunoglobulin kappa-chain gene J segment.
Authors: Weaver D , Baltimore D
Published: May 2006

Nuclear extracts from pre-B and B cell lines contain a nuclear DNA binding protein (kappa locus protein, KLP) that specifically recognizes a DNA sequence in the immunoglobulin kappa light chain joining (J) segment gene region. KLP is not observed in mature B cells, T cells, or nonlymphoid cell types. Two tandem binding sites for KLP designated KI and KII have been identified by methylation interference analysis to be immediately proximal to the J kappa 1 nonamer-heptamer recognition sequences and separated by 38 base pairs from each other. Fragments of DNA containing KI and KII sites compete for binding to KLP, and both protein-DNA complexes have the same electrophoretic mobility. Other flanking sequences of immunoglobulin gene fragments do not bind to KLP. The position of KLP-DNA binding and its tissue-specific expression suggest that it may be involved in the regulation of lymphoid gene DNA rearrangements by targeting recombinase to the kappa-chain gene region.
Repair of single-stranded loops in heteroduplex DNA transfected into mammalian cells.
Authors: Weiss U , Wilson J H
Published: May 2006

Repair of heteroduplex DNA, generated between two interacting DNA molecules during homologous recombination, has been implicated as a contributing factor in the process of gene conversion. To assess patterns of heteroduplex repair in mammalian cells, we constructed 13 different heteroduplexes from simian virus 40 wild-type and deletion mutant DNAs. Each heteroduplex contained one or multiple single-stranded loops in the intron of the gene for large tumor antigen, which is not essential during lytic infection. After transfection into cultured monkey cells, cellular repair was evaluated by restriction analysis of the amplified viral progeny from 1123 individual plaques, each representing the clonal expansion of a single repair event. Single-stranded loops were corrected prior to replication with an overall efficiency of 90%. At the position of a loop, one of the two heteroduplex strands served as a template for accurate repair 98% of the time. Repair of single-stranded loops was biased nearly 2 to 1 in favor of the strand without the loop. The efficiency, accuracy, and strand bias of repair were unaffected by loop size within the tested range, which was 25-247 nucleotides. The excision tract associated with repair of single-stranded loops rarely exceeds 200-400 nucleotides in length.
Induction of aldose reductase and sorbitol in renal inner medullary cells by elevated extracellular NaCl.
Authors: Bagnasco S M , Uchida S , Balaban R S , Kador P F , Burg M B
Published: May 2006

Aldose reductase [aldehyde reductase 2; alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21] catalyzes conversion of glucose to sorbitol. Although its activity is implicated in the progression of ocular and neurological complications of diabetes, the normal function of the enzyme in most cells is unknown. Both aldose reductase activity and substantial levels of sorbitol were previously reported in renal inner medullary cells. In this tissue, the extracellular NaCl concentration normally is high and varies considerably depending on the urine concentration. We report here on a line of renal medullary cells in which medium that is high in NaCl greatly increases both aldose reductase activity and intracellular sorbitol. In these tissue culture cells (and presumably also in the renal inner medulla), the intracellular sorbitol helps balance the osmotic pressure of elevated extracellular NaCl and thus prevents cellular dehydration.
Continuing rearrangement of immunoglobulin and T-cell receptor genes in a Ha-ras-transformed lymphoid progenitor cell line.
Authors: Alessandrini A , Pierce J H , Baltimore D , Desiderio S V
Published: May 2006

The arrangement of immunoglobulin genes has been examined in a series of lymphoid cell lines transformed with the Harvey murine sarcoma virus. One cell line, HAFTL-1, expresses antigenic markers characteristic of B-lymphoid cells and undergoes frequent rearrangement at the JH locus (where J = joining and H = heavy chain) during propagation in culture. By molecular cloning and nucleotide sequence determination, these rearrangements were found to represent the earliest postulated step in heavy chain gene assembly: the joining of a diversity (D) segment to a JH segment. The HAFTL-1 cell line also undergoes infrequent D beta-to-J beta joining at the T-cell receptor beta locus in culture. The observations presented here suggest that the HAFTL-1 cell line represents the early stage of B-cell differentiation at which immunoglobulin gene rearrangement is initiated.
pen repeat sequences are GGN clusters and encode a glycine-rich domain in a Drosophila cDNA homologous to the rat helix destabilizing protein.
Authors: Haynes S R , Rebbert M L , Mozer B A , Forquignon F , Dawid I B
Published: May 2006

Several cDNA clones that contain the pen repeat have been isolated and sequenced; pen consists of clusters of GGN triplets, where N can be any nucleotide. Some of the pen repeat sequences are found within long open reading frames in which they encode oligoglycine stretches. For one of the clones, the deduced amino acid sequence of the entire open reading frame, especially in the region preceding the glycine-rich domain, shows strong homology to the rat helix destabilizing protein [Cobianchi, F., SenGupta, D. N., Zmudzka, B. Z. & Wilson, S. H. (1986) J. Biol. Chem. 261, 3536-3543]. The rat protein and homologs in other organisms are single-stranded nucleic acid binding proteins, some of which are major components of heterogeneous nuclear ribonucleoprotein particles. We suggest that we have cloned a cDNA encoding a Drosophila single-stranded nucleic acid binding protein.
Isolation and specificity of a Mr 74,000 cholesteryl ester transfer protein from human plasma.
Authors: Jarnagin A S , Kohr W , Fielding C
Published: May 2006

A cholesteryl ester transfer protein was isolated from human plasma whose ligand specificity, molecular weight, and amino acid composition are significantly different from those of proteins previously reported to have this activity. The protein, purified about 100,000-fold from plasma, is rich in hydrophobic amino acids, especially leucine. It has a molecular weight of 74,000 and an isoelectric point of 5.2. The protein's transfer rate for cholesteryl ester between each of the major plasma lipoprotein classes is similar, and rapid compared to the transfer of triacyglycerol, regardless of the overall lipid composition of the donor and acceptor lipoprotein. These data suggest that this protein functions primarily in the transfer of cholesteryl esters between plasma lipoproteins.
Increased biosynthesis and processing of fibronectin in fibroblasts from diabetic mice.
Authors: Phan-Thanh L , Robert L , Derouette J C , Labat-Robert J
Published: May 2006

Diabetic connective tissues exhibit a deranged regulation of extracellular matrix biosynthesis. Fibronectin is shown to be increased in human dermal connective tissue by immunofluorescence, mainly at the dermoepidermal and capillary basement membranes. The rate of fibronectin biosynthesis, excretion, and incorporation in a pericellular polymeric form was investigated using genetically diabetic KK mouse skin and fibroblasts as compared to Swiss and C57BL mouse skin and fibroblasts. The rate of incorporation of [35S]methionine into proteins recovered in the culture medium or in deoxycholate and NaDodSO4 or urea extracts was investigated. The rate of incorporation in the medium and deoxycholate extracts was comparable. However, the relative rate of incorporation of the tracer in the NaDodSO4-extractable, pericellular polymeric form was increased in the diabetic KK fibroblasts both for total proteins and for fibronectin. In pulse-chase experiments, the deoxycholate-soluble and NaDodSO4-soluble fractions exhibited a precursor-product relationship. The rate of passage of fibronectin from the deoxycholate-soluble (cellular compartment) form to the NaDodSO4-soluble (pericellular polymeric) form was strongly accelerated in the diabetic fibroblast cultures. These results confirm the increased rate of synthesis of fibronectin in diabetic fibroblasts as well as its processing from the cellular compartment to the polymeric pericellular form. The increase of fibronectin in diabetic connective tissues, in the matrix as well as in the basement membranes, may play a role in the mechanism of micro- and macroangiopathies and in the perturbed permeability characteristics of the diabetic capillaries, and as a glycoprotein it may contribute to the increased periodic acid/Schiff reagent staining of diabetic capillary basement membranes.
Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules.
Authors: Swanson J , Bushnell A , Silverstein S C
Published: May 2006

Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate (10 ng/ml). When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of approximately 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4 degrees C or in medium containing 5 microM colchicine or nocodazole at 37 degrees C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37 degrees C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. We conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures.
Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum.
Authors: Winograd E , Greenan J R , Sherman I W
Published: May 2006

Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of 125I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of 125I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes. By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen.
Interleukin 2 binding molecule distinct from the Tac protein: analysis of its role in formation of high-affinity receptors.
Authors: Robb R J , Rusk C M , Yodoi J , Greene W C
Published: May 2006

Interleukin 2 (IL-2) receptors on activated T cells exist in high- and low-affinity configurations, both of which share a ligand-binding component known as the Tac protein. Although almost all binding of IL-2 to such cells was inhibited by an antibody to Tac, the predominant component of binding on the natural killer (NK)-like cell line YT was resistant to this reagent. The ligand-binding component on YT cells also differed from Tac in its affinity constant (Kd approximately 8.2 X 10(-10) M vs. Kd approximately equal to 1.1 X 10(-8) M for low-affinity Tac sites) and in its susceptibility to inhibition by certain antibodies to IL-2. When the YT cells were stimulated in a manner that induced expression of the Tac protein, the IL-2 binding sites were converted to a high-affinity configuration (Kd approximately 1.8 X 10(-11) M). Thus, the original binding component on unstimulated YT cells appeared to combine with Tac and IL-2 to produce a high-affinity receptor complex. Use of bifunctional crosslinking agents following ligand binding to unstimulated YT cells yielded covalent IL-2-receptor complexes of 83 and 90 kDa. These complexes were similar in size to those derived from high-affinity receptors on activated T cells and shared a similar fragmentation pattern upon proteolysis. These results demonstrate the existence of a second IL-2 binding component in addition to the Tac protein and suggest that this component combines with Tac and IL-2 to form high-affinity receptor sites.
Synthetic peptide with inhibin-like activity preferentially inhibits follitropin secretion in comparison with lutropin-releasing hormone antagonists.
Authors: Sairam M R , Ramasharma K , Li C H
Published: May 2006

Biological activity of a synthetic peptide with inhibin-like activity under in vitro and in vivo conditions was compared with three highly potent synthetic lutropin-releasing hormone antagonists. Unlike the synthetic lutropin-releasing hormone antagonists, which effectively inhibited both lutropin and follitropin secretion from the pituitary, the inhibin-like peptide showed a preferential effect by inhibiting follitropin release both in vitro and in vivo. Thus, small peptides such as inhibin-like peptide with a sequence unrelated to lutropin-releasing hormone may provide a basis for design of selective inhibitors of gonadotropin release.
Chloroplast DNA polymorphisms in lodgepole and jack pines and their hybrids.
Authors: Wagner D B , Furnier G R , Saghai-Maroof M A , Williams S M , Dancik B P , Allard R W
Published: May 2006

Samples taken from throughout the ranges of distribution of lodgepole pine (Pinus contorta Dougl. ex. Loud.) and jack pine (Pinus banksiana Lamb.) were assayed for Sal I and Sst I chloroplast DNA restriction fragment variation. Although the chloroplast genome is often regarded as highly conserved, at least 2 distinct Sal I and 13 distinct Sst I restriction fragment banding patterns occur in these closely related species. None of the chloroplast DNA restriction fragment banding patterns observed in allopatric lodgepole pine was observed in allopatric populations of jack pine, and vice versa, even though the two species share an extensive zone of sympatry, and gene flow between the species has been reported for nuclear genes. However, several atypical Sst I restriction fragment banding patterns occur only in or near the zone of sympatry. Chloroplasts have been reported to be inherited maternally in the great majority of species studied; however, restriction fragment analyses indicated that chloroplasts are inherited paternally in controlled matings between lodgepole pine (female) and jack pine (male).
Endothelial cell-derived basic fibroblast growth factor: synthesis and deposition into subendothelial extracellular matrix.
Authors: Vlodavsky I , Folkman J , Sullivan R , Fridman R , Ishai-Michaeli R , Sasse J , Klagsbrun M
Published: May 2006

Bovine aortic and corneal endothelial cells synthesize a growth factor that remains mostly cell-associated but can also be extracted from the subendothelial extracellular matrix (ECM) deposited by these cells. The endothelial cell-derived growth factors extracted from cell lysates and from the extracellular matrix appear to be structurally related to basic fibroblast growth factor by the criteria that they bind to heparin-Sepharose and are eluted at 1.4-1.6 M NaCl, have a molecular weight of about 18,400, cross-react with anti-basic fibroblast growth factor antibodies when analyzed by electrophoretic blotting and immunoprecipitation, and are potent mitogens for bovine aortic and capillary endothelial cells. It is suggested that endothelium can store growth factors capable of autocrine growth promotion in two ways: by sequestering growth factor within the cell and by incorporating it into the underlying extracellular matrix.
Broad spectrum antiretroviral activity of 2',3'-dideoxynucleosides.
Authors: Dahlberg J E , Mitsuya H , Blam S B , Broder S , Aaronson S A
Published: May 2006

Certain dideoxynucleosides have been shown to markedly inhibit the infectivity of human T-lymphotropic virus type III/lymphadenopathy-associated virus, the causative agent of acquired immunodeficiency syndrome (AIDS). Our present studies demonstrate that these drugs are broad spectrum antiretroviral agents capable of inhibiting the infectivity of evolutionarily divergent mammalian type C and animal lentiviruses. Under some conditions, virus infectivity could be inhibited by more than six orders of magnitude. However, the potency of these agents was shown to be greatly influenced by cell-specified determinants. Drug exposure during the initial 24 hr was almost as effective as prolonged treatment on the inhibition of a single cycle of virus infection and expression. Moreover, virus infection was shown directly to be inhibited at the level of proviral DNA synthesis. Thus the time period during which reverse transcription and provirus integration occur is the critical period required for drug action. Our findings have implications concerning strategies to be considered in attempts to utilize 2',3'-dideoxynucleosides in control and treatment of retrovirus-induced diseases of animals and humans.
Complete nucleotide sequence of an attenuated hepatitis A virus: comparison with wild-type virus.
Authors: Cohen J I , Rosenblum B , Ticehurst J R , Daemer R J , Feinstone S M , Purcell R H
Published: May 2006

The complete nucleotide sequence of an attenuated hepatitis A virus, HAV HM-175/7 MK-5, was determined from cloned cDNA. This virus was derived from wild-type HAV HM-175 after 32 passages in African green monkey kidney cells. The resultant cell culture-adapted virus is attenuated for chimpanzees. This virus was passaged an additional three times in monkey kidney cells to obtain sufficient virus for molecular cloning and was designated HM-175/7 MK-5. Three overlapping cDNA clones were obtained that together spanned the entire genome. Comparison of the nucleotide sequence of cDNA from wild-type virus (propagated in marmoset liver in vivo) with attenuated virus (grown in cell culture) showed 24 nucleotide changes distributed throughout the genome. Five base deletions occurred in the 5' noncoding region, and 12 of the 16 base substitutions in the coding region resulted in amino acid changes. Amino acid changes occurred in viral capsid proteins VP1 and VP2 and several of the nonstructural proteins. Thus, a small number of nucleotide changes are responsible for adaptation to cell culture and attenuation of HAV strain HM-175.