bluuebunny/crossref_metadata_2025 huggingface dataset:
The modulatory action of loreclezole at the gamma-aminobutyric acid type A receptor is determined by a single amino acid in the beta 2 and beta 3 subunit.
Authors: Wingrove P B , Wafford K A , Bain C , Whiting P J
Published: May 2006

Type A gamma-aminobutyric acid (GABAA) receptors of the mammalian nervous system are a family of ligand-gated ion channels probably formed from the coassembly of different subunits (alpha 1-6, beta 1-3, gamma 1-3, delta) in the arrangement alpha beta gamma or alpha beta delta. The activation of these receptors by GABA can be modulated by a range of compounds acting at distinct allosteric sites. One such compound is the broad-spectrum anticonvulsant loreclezole, which we have recently shown to act via a specific modulatory site on the beta subunit of the GABAA receptor. The action of loreclezole depends on the type of beta subunit present in the receptor complex; receptors containing beta 2 or beta 3 subunits have > 300-fold higher affinity for loreclezole than receptors containing a beta 1 subunit. We have used this property to identify the amino acid residue in the beta subunit that determines the subunit selectivity of loreclezole. Chimeric beta 1/beta 2 human GABAA receptor subunits were constructed and coexpressed in Xenopus oocytes with human alpha 1 and gamma 2s subunits. The chimera beta 1/beta 2Lys237-Gly334 conferred sensitivity to 1 microM loreclezole. Within this region there are four amino acids that are conserved in beta 2 and beta 3 but differ in beta 1. By mutating single amino acids of the beta 1 subunit to the beta 2/beta 3 equivalent, only the beta 1 mutation of Ser-290-->Asn conferred potentiation by loreclezole. Similarly, mutation of the homologous residue in the beta 2 and beta 3 subunits to the beta 1 equivalent (Asn-->Ser) resulted in loss of sensitivity to loreclezole. The affinity for GABA and the potentiation by flunitrazepam were unchanged in receptors containing the mutated beta subunits. Thus, a single amino acid, beta 2 Asn-289 (beta 3 Asn-290), located at the carboxyl-terminal end of the putative channel-lining domain TM2, confers sensitivity to the modulatory effects of loreclezole.
Adipocyte differentiation selectively represses the serum inducibility of c-jun and junB by reversible transcription-dependent mechanisms.
Authors: Wang H , Scott R E
Published: May 2006

Nonterminally differentiated 3T3 T adipocytes are resistant to growth stimulation by 10% (vol/vol) fetal bovine serum even though they can be induced to proliferate with extremely high serum concentrations. We now report that in adipocytes 10% fetal bovine serum also fails to typically induce c-jun or junB. Rather, after 10% fetal bovine serum treatment, c-jun and junB expression is markedly repressed after a brief initial slight induction. Gel mobility shift studies confirm that AP-1 DNA binding activity is inhibited in adipocytes. Repression in c-jun and junB inducibility in adipocytes results from transcriptional mechanisms, can be reversed by treatment with protein synthesis inhibitors or higher serum concentrations, and does not affect c-fos or c-myc expression. These data suggest that adipocyte differentiation selectively and transcriptionally represses the inducibility of c-jun and junB so as to decrease the cell's ability to proliferate in response to 10% fetal bovine serum.
The expression of nifA in Azorhizobium caulinodans requires a gene product homologous to Escherichia coli HF-I, an RNA-binding protein involved in the replication of phage Q beta RNA.
Authors: Kaminski P A , Desnoues N , Elmerich C
Published: May 2006

We report the characterization of a mutant of Azorhizobium caulinodans, isolated after ethyl methanesulfonate mutagenesis. This Nod+ Nif- Fix- mutant is unable to synthesize 10 of 15 polypeptides normally induced under conditions of nitrogen fixation. By using lacZ fusions it was shown that nifA and nifA-regulated genes were not expressed in this strain. The mutation was complemented by a constitutively expressed nifA gene or by a 1.1-kb DNA fragment from the wild-type strain, whose nucleotide sequence revealed a single open reading frame of 255 bp coding for an 85-amino acid polypeptide. The deduced amino acid sequence is similar to that of HF-I, an RNA-binding protein of Escherichia coli, which is required for replication of bacteriophage Q beta RNA. The similarity can be extended to the function since hfq, the structural gene for HF-I, complemented the A. caulinodans mutant. The corresponding gene in A. caulinodans was termed nrfA (for nif regulatory factor). Inactivation of nrfA in the mutant was due to a missense mutation resulting in the replacement of a cysteine residue by arginine. A null mutant, constructed by disruption of nrfA, exhibited the same phenotype as the missense mutant. Thus, an additional factor can be added to the already complex system of nifA regulation in A. caulinodans.
Involvement of human muscle acetylcholine receptor alpha-subunit gene (CHRNA) in susceptibility to myasthenia gravis.
Authors: Garchon H J , Djabiri F , Viard J P , Gajdos P , Bach J F
Published: May 2006

The muscle acetylcholine receptor is the major target of the autoimmune response in generalized myasthenia gravis. To investigate the role of the gene encoding the alpha subunit of the receptor (CHRNA), two stable polymorphic d[(GT).(CA)]dinucleotide repeats, designated HB and BB, were characterized within the first intron of CHRNA. The HB*14 allele conferred a relative risk for myasthenia gravis of 2.5 in 81 unrelated patients compared with 100 control subjects. Very significantly, family analysis based on haplotype segregation data indicated that parental haplotypes associated with HB*14 always segregated to the child with myasthenia gravis (P < 0.0002 for the comparison with the transmission of haplotypes not bearing HB*14), whereas their transmission to unaffected siblings was equilibrated. Myasthenia gravis patients also showed a high frequency of microsatellite variants unseen in controls. These findings implicate the CHRNA in susceptibility to myasthenia gravis.
Mobile Minos elements from Drosophila hydei encode a two-exon transposase with similarity to the paired DNA-binding domain.
Authors: Franz G , Loukeris T G , Dialektaki G , Thompson C R , Savakis C
Published: May 2006

Elements related to the Tc1-like Minos mobile element have been cloned from Drosophila hydei and sequenced. Southern blot and sequence analyses show that (i) the elements are actively transposing in the Drosophila hydei germ line, (ii) they are characterized by a striking degree of sequence and size homogeneity, and (iii) like Tc1, they insert at a TA dinucleotide that is probably duplicated during the process. The nucleotide sequences of two elements, Minos-2 and Minos-3, differ at only one position from each other and contain two nonoverlapping open reading frames that are separated by a putative 60-nucleotide intron. The amino-terminal part of the Minos putative transposase shows sequence similarity to the paired DNA-binding domain. Forced transcription of a modified Minos element that was introduced into the Drosophila melanogaster germ line by P element-mediated transformation resulted in the production of accurately spliced polyadenylylated RNA molecules. It is proposed that Minos-2 and/or Minos-3 may encode an active transposase containing an amino-terminal DNA-binding domain that is distantly related to the paired DNA-binding domain.
Molecular basis of dark-eyed albinism in the mouse.
Authors: Schmidt A , Beermann F
Published: May 2006

Dark-eyed albino (C44H) is a recessive allele at the mouse albino (c) locus, which encodes tyrosinase (monophenol,L-dopa:oxygen oxidoreductase, EC 1.14.18.1), the key enzyme in melanin synthesis. Similar to type IB oculocutaneous albinism in humans, overall production of pigment is greatly reduced in dark-eyed albino mice and obvious only in the eyes. We have studied the molecular basis of the c44H mutation and show that expression of the tyrosinase gene is not affected. After sequencing tyrosinase cDNA isolated from c44H/c44H homozygotes, we uncovered a single base alteration from wild type leading to a serine-to-isoleucine exchange. The importance of this mutation was demonstrated by generating transgenic mice containing a mutated tyrosinase minigene. This showed that the single base change was sufficient to severely depress pigment production in transgenic mice. We therefore conclude that the point mutation is responsible and sufficient to generate the dark-eyed albino phenotype.
Mitogen-stimulated phosphorylation of histone H3 is targeted to a small hyperacetylation-sensitive fraction.
Authors: Barratt M J , Hazzalin C A , Cano E , Mahadevan L C
Published: May 2006

Diverse agents, including growth factors and phorbol esters, induce rapid transcriptional activation of a subset of immediate-early (IE) genes that include the protooncogenes c-fos and c-jun. Among the earliest nuclear signaling events concomitant with IE gene activation is the phosphorylation of nucleosomal histone H3 in its basically charged N-terminal tail. This highly conserved domain is also subject to reversible posttranslational acetylation at specific lysine residues, a process implicated in transcriptional regulation. We show here that H3 phosphorylation associated with G0-G1 transition affects only a small fraction of this histone in the nucleus. Moreover, this fraction is biochemically distinct from bulk H3 in being extremely sensitive to sodium butyrate-induced hyperacetylation. However, acetylation itself does not predispose H3 to phosphorylation, nor does phosphorylation predispose H3 to enhanced acetylation. Further, selectivity is not based on preferential modification of particular histone H3 subtypes. Thus, the mitogen-regulated kinase that phosphorylates histone H3 is restricted to a small subset of nucleosomes that is especially susceptible to hyperacetylation.
A lymphokine, provisionally designated interleukin T and produced by a human adult T-cell leukemia line, stimulates T-cell proliferation and the induction of lymphokine-activated killer cells.
Authors: Burton J D , Bamford R N , Peters C , Grant A J , Kurys G , Goldman C K , Brennan J , Roessler E , Waldmann T A
Published: May 2006

In early phases of human T-cell lymphotrophic virus I-induced adult T-cell leukemia (ATL), the malignant cell proliferation is associated with an autocrine process involving coordinate expression of interleukin (IL) 2 and its receptor. However, during late-phase ATL, leukemic cells no longer produce IL-2 yet continue to express high-affinity IL-2 receptors. During studies to define pathogenic mechanisms that underlie this IL-2-independent proliferation, we demonstrated that the ATL cell line HuT-102 secretes a lymphokine, provisionally designated IL-T, that stimulates T-cell proliferation and the induction of lymphokine-activated killer cells. Conditioned medium from HuT-102, when added to the IL-2-dependent CTLL-2 line, yielded a stimulation index of 230. Since CTLL-2 was purported to be IL-2-specific, we performed a number of studies to exclude IL-2 production by HuT-102. Stimulation of CTLL-2 cells by HuT-102-conditioned medium was not meaningfully inhibited by addition of an antiserum to IL-2. Furthermore, uninduced HuT-102 cells did not express mRNA encoding IL-2 as assessed by Northern blot analysis. No biological activity on CTLL-2 cells was mediated by purified IL-1, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, or granulocyte/macrophage colony-stimulating factor, thus differentiating these factors from IL-T. Based on preliminary biochemical data, IL-T is a protein with a pI value of 4.5 and a molecular mass in SDS gels of 14 kDa. In addition to its action on CTLL-2 cells, 3200-fold-purified IL-T stimulated proliferation of the human cytokine-dependent T-cell line Kit-225. Furthermore, addition of IL-T enhanced cytotoxic activity of large granular lymphocytes (i.e., induced lymphokine-activated killer cells). Thus, IL-T is a lymphokine that plays a role in T-cell proliferation and induction of lymphokine-activated killer cells. Furthermore, IL-T may contribute to IL-2-independent proliferation of select ATL cells and lines.
Protein-tyrosine-phosphatase 2C is phosphorylated and inhibited by 44-kDa mitogen-activated protein kinase.
Authors: Peraldi P , Zhao Z , Filloux C , Fischer E H , Van Obberghen E
Published: May 2006

Protein-tyrosine-phosphatase 2C (PTP2C, also named SHPTP2, SHPTP3, or PTP1D) is a cytosolic enzyme with two Src homology 2 domains. We have investigated its regulation by phosphorylation in PC12 rat pheochromocytoma cells. In untreated cells, PTP2C was phosphorylated predominantly on serine residues. A 5-min treatment with epidermal growth factor (EGF) induced an increase in phosphorylation on threonine and, to a lesser degree, on serine. After 45 min of exposure to EGF, PTP2C phosphorylation returned to basal levels. Using an in vitro kinase assay, we found that the 44-kDa mitogen-activated protein kinase, p44mapk, phosphorylated PTP2C on serine and threonine residues. This phosphorylation resulted in a pronounced inhibition of PTP2C enzyme activity measured with phosphorylated EGF receptors as substrate. Moreover, in intact PC12 cells, PTP2C was also inhibited following a short EGF treatment, but its activity returned to normal when the exposure to EGF was maintained for 45 min. The profile of this response to EGF can be inversely correlated to that of the stimulatory action of EGF on p44mapk. These data suggest that the EGF-induced regulation of PTP2C activity is mediated by p44mapk. These findings provide evidence for an additional role of the mitogen-activated protein kinase cascade--namely, the regulation of a PTP.
Nuclear transformation of Volvox carteri.
Authors: Schiedlmeier B , Schmitt R , Müller W , Kirk M M , Gruber H , Mages W , Kirk D L
Published: May 2006

Stable nuclear transformation of Volvox carteri was achieved using the cloned V. carteri nitA+ gene (which encodes nitrate reductase) to complement a nitA- mutation. Following bombardment of mutant cells with plasmid-coated gold particles, putative transformants able to utilize nitrate as a nitrogen source were recovered with an efficiency of approximately 2.5 x 10(5). DNA analysis indicated that the plasmid integrated into the genome, often in multiple copies, at sites other than the nitA locus. Cotransformants were recovered with a frequency of 40-80% when cells were cobombarded with a selected and an unselected marker. Thus, V. carteri becomes one of the simplest multicellular organisms that is accessible to detailed molecular studies of genes regulating cellular differentiation and morphogenesis.
Prevention of autoimmune lysis by T cells with specificity for a heat shock protein by antisense oligonucleotide treatment.
Authors: Steinhoff U , Zügel U , Wand-Württenberger A , Hengel H , Rösch R , Munk M E , Kaufmann S H
Published: May 2006

T lymphocytes with specificity for the bacterial heat shock protein (hsp) 60 recognize stressed host cells, thus possibly promoting pathogenesis of certain infectious and autoimmune diseases. Here, we show that autoimmune destruction of stressed Schwann cells and macrophages by cytotoxic T lymphocytes raised against mycobacterial hsp60 can be inhibited by the use of hsp60-specific antisense oligodeoxynucleotides (A-ODNs). The inhibitory effect of hsp60 A-ODNs was specific because lysis of murine cytomegalovirus-infected host cells by virus-specific cytotoxic lymphocytes was not affected. Immunoblot analysis and immunoprecipitation studies suggest that different forms of stress increase hsp60 synthesis in Schwann cells and that this neosynthesis is reduced by hsp60 A-ODNs. These findings (i) provide evidence for participation of endogenous hsp60 in the recognition of stressed host cells by mycobacterial hsp60-crossreactive T cells and (ii) suggest the feasibility of inhibiting autoimmune reactions by target-cell treatment with specific A-ODNs.
Fast photoinduced electron transfer through DNA intercalation.
Authors: Murphy C J , Arkin M R , Ghatlia N D , Bossmann S , Turro N J , Barton J K
Published: May 2006

We report evidence for fast photoinduced electron transfer mediated by the DNA helix that requires metal complexes that are avid intercalators of DNA. Here the donor bis(phenanthroline)(dipyridophenazine)ruthenium(II) [Ru(phen)2dppz2+] and acceptor bis(9,10-phenanthrenequinone diimine)(phenanthroline)rhodium(III) [Rh(phi)2phen3+] intercalate into DNA with Kb > 10(6) M-1. Luminescence quenching experiments in the presence of two different lengths of DNA yield upward-curving Stern-Volmer plots and the loss of luminescence intensity far exceeds the change in emission lifetimes. In the presence of a nonintercalative electron acceptor, Ru(NH3)3+(6), Ru(phen)2dppz2+ luminescence is quenched much less efficiently compared to that found for the intercalative Rh(phi)2phen3+ quencher and follows linear Stern-Volmer kinetics; steady-state and time-resolved Stern-Volmer plots are comparable in scale. These experiments are consistent with a model involving fast long-range electron transfer between intercalators through the DNA helix.
Methane production in terrestrial arthropods.
Authors: Hackstein J H , Stumm C K
Published: May 2006

We have screened more than 110 representatives of the different taxa of terrestrial arthropods for methane production in order to obtain additional information about the origins of biogenic methane. Methanogenic bacteria occur in the hindguts of nearly all tropical representatives of millipedes (Diplopoda), cockroaches (Blattaria), termites (Isoptera), and scarab beetles (Scarabaeidae), while such methanogens are absent from 66 other arthropod species investigated. Three types of symbiosis were found: in the first type, the arthropod's hindgut is colonized by free methanogenic bacteria; in the second type, methanogens are closely associated with chitinous structures formed by the host's hindgut; the third type is mediated by intestinal anaerobic protists with intracellular methanogens. Such symbiotic associations are likely to be a characteristic property of the particular taxon. Since these taxa represent many families with thousands of species, the world populations of methane-producing arthropods constitute an enormous biomass. We show that arthropod symbionts can contribute substantially to atmospheric methane.
Effective amplification of long targets from cloned inserts and human genomic DNA.
Authors: Cheng S , Fockler C , Barnes W M , Higuchi R
Published: May 2006

We have used the polymerase chain reaction (PCR) to amplify up to 22 kb of the beta-globin gene cluster from human genomic DNA and up to 42 kb from phaga lambda DNA. We have also amplified 91 human genomic inserts of 9-23 kb directly from recombinant lambda plaques. To do this, we increased pH, added glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and used a secondary thermostable DNA polymerase that possesses a 3'-to 5'-exonuclease, or "proofreading," activity. Our "long PCR" protocols maintain the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing. The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics.
A role for molecular genetics in biological conservation.
Authors: O'Brien S J
Published: May 2006

The recognition of recent accelerated depletion of species as a consequence of human industrial development has spawned a wide interest in identifying threats to endangered species. In addition to ecological and demographic perils, it has become clear that small populations that narrowly survive demographic contraction may undergo close inbreeding, genetic drift, and loss of overall genomic variation due to allelic loss or reduction to homozygosity. I review here the consequences of such genetic depletion revealed by applying molecular population genetic analysis to four endangered mammals: African cheetah, lion, Florida panther, and humpback whale. The accumulated genetic results, combined with physiological, ecological, and ethological data, provide a multifaceted perspective of the process of species diminution. An emerging role of population genetics, phylogenetics, and phylogeography as indicators of a population's natural history and its future prognosis provides valuable data of use in the development of conservation management plans for endangered species.
Genetic and physiological association of diabetes susceptibility with raised Na+/H+ exchange activity.
Authors: Morahan G , McClive P , Huang D , Little P , Baxter A
Published: May 2006

Insulin-dependent diabetes mellitus is a multigenic autoimmune disease, for which one of the best animal models is the nonobese diabetic (NOD) mouse strain. In both humans and NOD mice, major histocompatibility complex genes are implicated as risk factors in the disease process. Other susceptibility genes are also involved, and a number have been mapped in the mouse to specific chromosomal locations. To identify further susceptibility genes, diabetic backcross mice, produced after crossing NOD/Lt to the nondiabetic strains SJL and C57BL/6 (B6), were examined for markers not previously associated with disease susceptibility. Linkage was found to loci on chromosomes 4 and 14. Of the candidate loci on chromosome 4, the gene encoding the Na+/H+ exchanger-1, Nhe-1, was the most likely, since the NOD allele was different from that of both nondiabetic strains. NOD lymphocytes were found to have a higher level of Na+/H+ exchange activity than lymphocytes from either B6 or SJL mice. Since the chromosome 4 susceptibility gene is recessive, the B6 allele should prevent diabetes. This prediction was tested in fourth-generation backcross mice, selected for retention of the B6 allele at Nhe-1. Mice homozygous for Nhe-1 developed diabetes after cyclophosphamide treatment, but heterozygotes were largely protected from disease. These results implicate the Na+/H+ exchanger (antiporter) in the development of type 1 diabetes and may provide a screening test for at-risk individuals as well as offering prospects for disease prevention.
Inhibition of thrombosis and intimal thickening by in situ photopolymerization of thin hydrogel barriers.
Authors: Hill-West J L , Chowdhury S M , Slepian M J , Hubbell J A
Published: May 2006

Thin hydrogel barriers formed on the inner surface of injured arteries by interfacial photopolymerization dramatically reduced thrombosis and intimal thickening in rat and rabbit models of vascular injury. This polymerization technique allowed the synthesis of a thin hydrogel barrier that conformed to the vessel wall, directly blocking contact between blood and the damaged vessel. The illumination conditions could be varied to control the thickness of the barrier from 10 microns to > 50 microns. The hydrogel was designed to degrade by nonenzymatic hydrolysis. In rats in which the carotid artery had been severely injured by crushing, treatment with the hydrogel barrier completely eliminated thrombosis (P < 0.01) and preserved long-term patency (P < 0.01). Treatment in a rabbit model of balloon injury inhibited thrombosis (P < 0.02) and reduced long-term intimal thickening by approximately 80% (P < 0.003). These results suggest that blood-borne signals acting in the early phases of healing play an important role in stimulating thickening of the intima.
Heterologous expression of plant vacuolar pyrophosphatase in yeast demonstrates sufficiency of the substrate-binding subunit for proton transport.
Authors: Kim E J , Zhen R G , Rea P A
Published: May 2006

The membrane bounding the vacuole of plant cells contains two electrogenic proton pumps. These are the vacuolar H(+)-ATPase (EC 3.6.1.3), an enzyme common to all eukaryotes, and a vacuolar H(+)-translocating pyrophosphatase (EC 3.6.1.1), which is ubiquitous in plants but otherwise known in only a few phototrophic bacteria. Although the substrate-binding subunit of the vacuolar H(+)-pyrophosphatase has been identified and purified and cDNAs encoding it have been isolated and characterized, the minimal unit competent in pyrophosphate (PPi)-energized H+ translocation is not known. Here we address this question and show that heterologous expression of the cDNA (AVP) encoding the substrate-binding subunit of the vacuolar H(+)-pyrophosphatase from the vascular plant Arabidopsis thaliana in the yeast Saccharomyces cerevisiae results in the production of vacuolarly localized functional enzyme active in PPi-dependent H+ translocation. Since the heterologously expressed pump is indistinguishable from the native plant enzyme with respect to PPi hydrolysis, H+ translocation, activation by potassium, and selective inhibition by calcium and 1,1-diphosphonates, it is concluded that all of the known catalytic functions of the enzyme map to the one subunit encoded by AVP.
Evidence that most human Alu sequences were inserted in a process that ceased about 30 million years ago.
Authors: Britten R J
Published: May 2006

The primate Alu interspersed repeats can be subdivided into classes on the basis of shared nucleotides at a set of diagnostic positions. Each of the classes of Alu sequences is apparently the result of past retrotransposition of transcripts of highly conserved class-specific source genes that differed from each other at the diagnostic positions. The nucleotides at the majority of positions are identical among the source genes and therefore were identical among all of the Alu sequences at the time of their insertion. These CONSBI (conserved before insertion) positions are useful because the changes that have occurred after insertion are recognizable and the divergence resulting from nucleotide substitutions, insertions, and deletions is informative. The divergence of Alu sequences at the CONSBI positions is a measure of the time since a class was inserted. The greatest majority of Alu sequences are in one class (identified as class II), and it is particularly suitable for such examination, since nearly full-length sequences are now known for nearly a thousand members of this class. The average divergence of class II members indicates that the class has an average age of about 40 million years. The distribution in divergence of class II accurately fits a sum of two Poisson distributions. The implication is that class II Alu sequences were derived from two massive past events of insertion of many Alu sequences. In this model the younger subset of class II sequences (corresponding to about 300,000 copies in the genome) has an average divergence of 5% at CONSBI positions. The older set of class II sequences (corresponding to about 150,000 genomic copies) has a 9% average divergence. Based on the drift rate of primate DNA sequences, the events of insertion probably occurred 30-50 million years ago. The goodness of the fit to the Poisson distribution indicates that no significant number of members of class II have been inserted since 30 million years ago.
Polarized Na+/H+ exchange function is pliable in response to transepithelial gradients of propionate.
Authors: Rowe W A , Lesho M J , Montrose M H
Published: May 2006

Short-chain fatty acids are produced at high concentration in the colonic lumen and stimulate electroneutral Na+ absorption by activating apical Na+/H+ exchange in colonocytes. We used an epithelial cell line derived from a human colon carcinoma (HT29-18-C1) to study activation of apical and basolateral Na+/H+ exchange by a short-chain fatty acid, propionate. Confluent cell monolayers on membrane filters were loaded with 2',7'-bis(2-carboxyethyl)-5 (and 6)-carboxyfluorescein (a fluorescent pH indicator) and intracellular pH was monitored with a digital fluorescence imaging microscope. Cells acidified by transient exposure to NH4Cl demonstrated both apical and basolateral Na+/H+ exchange. In this condition, apical Na+/H+ exchange was 50% of the total Na+/H+ exchange activity. Similar results were obtained when cells were bilaterally perfused with apical and basolateral propionate in an isosmotic medium (130 mM propionate at each membrane surface). However, apical Na+/H+ exchange was a significantly larger fraction (76%) of the total Na+/H+ exchange activity when cells were acidified by exposure to apical propionate alone. Conversely, in cells acidified by basolateral propionate alone, apical Na+/H+ exchange was 21% of the total Na+/H+ exchange activity. The change in relative activity was observed in individual cells which expressed both apical and basolateral Na+/H+ exchange and occurred rapidly (within 7 min). In the presence of transepithelial propionate gradients, all Na(+)-dependent alkalinization was sensitive to 3 microM 5-(N-ethyl-N-isopropyl)amiloride, a potent Na+/H+ exchange inhibitor. These results suggest that transepithelial gradients of short-chain fatty acids, which occur in vivo, can cause preferential activation of apical Na+/H+ exchange.
A hyphal-specific chitin synthase gene (CHS2) is not essential for growth, dimorphism, or virulence of Candida albicans.
Authors: Gow N A , Robbins P W , Lester J W , Brown A J , Fonzi W A , Chapman T , Kinsman O S
Published: May 2006

In the dimorphic fungus Candida albicans, the CHS2 gene encodes a chitin synthase that is expressed preferentially in the hyphal form. Gene disruption of CHS2 in this diploid asexual fungus was achieved by the "ura-blaster" protocol described for Saccharomyces [Alani, E., Cao, L. & Kleckner, N. (1987) Genetics 116, 541-545]. This involves the sequential disruption of multiple alleles by integrative transformation with URA3 as a single selectable marker. After disrupting the first CHS2 allele, the Ura- phenotype was recovered through cis recombination between repeated hisG sequences that flanked the URA3 marker in the disruption cassette, which was then used again to disrupt further CHS2 alleles. This method of gene disruption is well suited to the mutational analysis of this genetically recalcitrant human pathogen. Three rounds of disruption were required, suggesting that the strain SGY243 is triploid for the CHS2 locus. The resulting homozygous delta chs2::hisG null mutants were viable and made germ tubes with a normal morphology. The germ tubes were formed more slowly than parental strains in serum-containing medium and the germinating cells had a 40% reduction in their chitin content compared to germ tubes of the parent strain. The chitin content of the yeast form was not affected. A prototrophic strain of the chs2 null mutant was not attenuated significantly in its virulence when tested in normal and immunosuppressed mice.
ALL-1 partial duplication in acute leukemia.
Authors: Schichman S A , Caligiuri M A , Gu Y , Strout M P , Canaani E , Bloomfield C D , Croce C M
Published: May 2006

The ALL-1 gene, located on chromosome band 11q23, is fused to a variety of other genes by reciprocal chromosomal translocations present in 5-10% of human acute leukemias. We have recently reported the detection by Southern blot of ALL-1 gene rearrangements in adult patients with acute myeloid leukemia lacking cytogenetic evidence of 11q23 translocations. These include 2 of 19 patients with normal karyotypes as well as 3 of 4 patients with trisomy 11. To characterize the abnormal ALL-1 genes, we cloned the ALL-1 rearrangements from two patients with trisomy 11. Characterization of the clones, together with Southern blot analysis, indicates that the ALL-1 rearrangement in both patients is the result of a direct tandem duplication of a portion of the ALL-1 gene spanning exons 2-6. The partial ALL-1 duplication is also detected by Southern blot analysis in a patient with a normal karyotype. RNA PCR and DNA sequence analysis show that the partially duplicated ALL-1 gene is transcribed into mRNA capable of encoding a partially duplicated protein. Partial duplication of ALL-1, in which a portion of a putative protooncogene is fused with itself, represents an additional genetic mechanism for leukemogenesis. Our findings suggest that the presence of trisomy in malignancy may sometimes indicate the partial duplication of a cellular protooncogene.
Infection and immunoregulation of T lymphocytes by parainfluenza virus type 3.
Authors: Sieg S , Muro-Cacho C , Robertson S , Huang Y , Kaplan D
Published: May 2006

Human parainfluenza virus type 3 (HPIV3) is a major cause of disease in newborns and infants. It also has a striking potential to reinfect individuals throughout their lives, suggesting that HPIV3 does not induce lifelong immunity; however, the operative mechanism for the failure to prevent reinfection is not known. We have assessed the potential of the virus to infect nontransformed human T lymphocytes and have found that T cells are readily infected by the virus. Productive infection requires activation of the T cells and results in a marked inhibition of proliferation. Furthermore, our results indicate that exposure to the virus, even without overt expression of viral proteins as detected by immunohistology, profoundly alters the functional capacity of the T cells. The capacity of the virus to regulate T-lymphocyte function may play an important role in the failure of the virus to induce lifelong immunity.
LIGA20, a lyso derivative of ganglioside GM1, given orally after cortical thrombosis reduces infarct size and associated cognition deficit.
Authors: Kharlamov A , Zivkovic I , Polo A , Armstrong D M , Costa E , Guidotti A
Published: May 2006

A bilateral photochemically induced thrombotic lesion of rat sensorimotor cortex (approximately 3 mm in diameter and 25 mm3 in volume) is associated with a persistent cognition (learning and memory) deficit, which was evaluated with water maze tasks. The N-dichloroacetylsphingosine derivative of lysoGM1 (LIGA20) administered after the lesion either i.v. or per or reduces the infarct size by 30-40% and attenuates the associated cognition deficits, presumably by limiting the extent of damage of neurons at risk located in the surroundings of the infarcted core (i.e., area penumbra). The LIGA20 protection is dose and time dependent. Maximal protection is afforded by a single dose of LIGA20 of 34 mumol/kg i.v. 1 hr after lesion or by a dose of 270 mumol/kg per os when administered 1 hr and 24 hr after the lesion. The protective effect of LIGA20 can be observed when the drug is administered i.v. up to 6 hr after the lesion. The protective efficacy of the oral administration of LIGA20 is related to its physiochemical properties, which, unlike those of GM1, allow absorption from the gastrointestinal tract. LIGA20 given orally reaches the brain promptly and rapidly inserts into the neuronal membranes. Here, by an unknown molecular mechanism, LIGA20 selectively reduces the pathological amplification of Ca2+ signaling elicited by persistent stimulation of ionotropic glutamate receptors in the area penumbra.
Domain structure of a mammalian myosin I beta.
Authors: Reizes O , Barylko B , Li C , Südhof T C , Albanesi J P
Published: May 2006

We have determined the primary structure of a myosin I (called mammalian myosin I beta, MMI beta) from bovine brain and identified its functional domains. The protein was previously purified from brain and adrenal gland. Several constructs were generated and expressed in Escherichia coli as glutathione S-transferase fusion proteins and the recombinant proteins were recognized by monoclonal antibodies that recognize either "head" or "tail" domains of native myosin I. A gel overlay method was used to confirm that calmodulin binds to the consensus calmodulin-binding sequence in MMI beta. Binding assays were used to detect interaction with anionic phospholipid vesicles. We conclude that MMI beta consists of an amino-terminal 80.5-kDa domain that contains the ATP- and actin-binding sites, followed by an 8.5-kDa domain with three calmodulin-binding sequences and a basic 30-kDa carboxyl-terminal tail segment that binds to anionic phospholipids and membranes.
A role for a eukaryotic GrpE-related protein, Mge1p, in protein translocation.
Authors: Laloraya S , Gambill B D , Craig E A
Published: May 2006

The 70-kDa heat shock proteins (hsp70s) function as molecular chaperones in a wide variety of cellular processes through cycles of binding and release from substrate proteins coupled to cycles of ATP hydrolysis. In the prokaryote Escherichia coli, the hsp70 DnaK functions with two other proteins, DnaJ and GrpE, which modulate the activity of DnaK. While numerous hsp70s and DnaJ-related proteins have been identified in eukaryotes, to our knowledge no GrpE-related proteins have been reported. We report the isolation and characterization of a eukaryotic grpE-related gene, MGE1. MGE1, an essential nuclear gene of the yeast Saccharomyces cerevisiae, encodes a soluble protein of the mitochondrial matrix. Cells with reduced expression of Mge1p accumulate the precursor form of a mitochondrial protein. Since mitochondrial hsp70 is required for translocation of precursors of mitochondrial proteins from the cytosol into the matrix of mitochondria, these data suggest that Mge1p acts in concert with mitochondrial hsp70 in protein translocation.
Timing of fungal invasion using host's ripening hormone as a signal.
Authors: Flaishman M A , Kolattukudy P E
Published: May 2006

In many postharvest fruit diseases, fungi remain latent until the fruit ripens. How the fungus times its infection at ripening of the host is not known. We have found that the volatiles produced by the climacteric tomato, avocado, and banana fruits induce germination and appressorium formation in Colletotrichum gloeosporioides and Colletotrichum musae. Exposure of the spores of these fungi to ethylene, the host's ripening hormone, at </=1 microl/liter, caused germination, branching of the germ tube, and formation of up to six appressoria from a single spore. Propylene, an ethylene analog, but not the hydrocarbon gas methane was able to induce spore germination and multiple appressorium formation. The ethylene effect on the fungi appears to be a plant-like response as it was inhibited by silver ion and 2,5-norbornadiene; the inhibition by the latter could be reversed by higher ethylene concentrations. Ethylene induced germination and appressorium formation in the Colletotrichum sp. penetrating climacteric fruit but not in other Colletotrichum strains. That the ethylene induction of multiple appressorium formation could be relevant to postharvest infection was indicated by the observation that C. gloeosporioides spores formed multiple appressoria on normally ripening tomato that produces ethylene, whereas on transgenic tomato and orange, fruits incapable of producing ethylene, exogenous ethylene was required to induce multiple appressorium formation and lesion formation. These results strongly suggest that these fungi must have coevolved to develop a mechanism to use the host's ripening hormone as a signal to differentiate into multiple infection structure and thus time the infection process.
Delineation of a T-cell activation motif required for binding of protein tyrosine kinases containing tandem SH2 domains.
Authors: Koyasu S , Tse A G , Moingeon P , Hussey R E , Mildonian A , Hannisian J , Clayton L K , Reinherz E L
Published: May 2006

To define the T-cell receptor signal transduction motif, we have transfected human and murine T-cell lines with a chimeric receptor consisting of the extracellular and transmembrane domains of human CD8 alpha and the membrane-proximal portion of CD3 zeta containing at its C terminus either an 18-amino acid segment (NQLYNELNLGRREEYDVL) or alanine-scanning point mutant derivatives. Crosslinking of the extracellular domain of the chimera is sufficient to initiate Ca2+ flux, interleukin 2 production, and tyrosine phosphorylation of cellular proteins including the chimera. Subsequently, the chimera becomes associated with several tyrosine-phosphorylated proteins, among them the 70-kDa protein tyrosine kinase ZAP70. Mutational data identify the T-cell activation motif as Y(X)2L(X)7Y(X)2L and show that each of the four designated residues is necessary for the above activation events. Recombinant protein containing the two tandem SH2 domains derived from ZAP70 binds to a synthetic peptide corresponding to the above 18-amino acid motif but only when both tyrosines are phosphorylated; in contrast, little or no binding is observed to monophosphorylated or nonphosphorylated analogues. These results imply that after receptor crosslinking in T cells, and by inference also in B cells and mast cells, the motif is phosphorylated on both tyrosine residues, thereafter serving as a docking site for protein tyrosine kinases containing tandem SH2 domains.
Molecular genetics of speciation and human origins.
Authors: Ayala F J , Escalante A , O'Huigin C , Klein J
Published: May 2006

The major histocompatibility complex (MHC) plays a cardinal role in the defense of vertebrates against parasites and other pathogens. In some genes there are extensive and ancient polymorphisms that have passed from ancestral to descendant species and are shared among contemporary species. The polymorphism at the DRB1 locus, represented by 58 known alleles in humans, has existed for at least 30 million years and is shared by humans, apes, and other primates. The coalescence theory of populations genetics leads to the conclusion that the DRB1 polymorphism requires that the population ancestral to modern humans has maintained a mean effective size of 100,000 individuals over the 30-million-year persistence of this polymorphism. We explore the possibility of occasional population bottlenecks and conclude that the ancestral population could not have at any time consisted of fewer than several thousand individuals. The MHC polymorphisms exclude the theory claiming, on the basis of mitochondrial DNA polymorphisms, that a constriction down to one or few women occurred in Africa, at the transition from archaic to anatomically modern humans, some 200,000 years ago. The data are consistent with, but do not provide specific support for, the claim that human populations throughout the World were at that time replaced by populations migrating from Africa. The MHC and other molecular polymorphisms are consistent with a "multiregional" theory of Pleistocene human evolution that proposes regional continuity of human populations since the time of migrations of Homo erectus to the present, with distinctive regional selective pressures and occasional migrations between populations.
Error-prone replication of repeated DNA sequences by T7 DNA polymerase in the absence of its processivity subunit.
Authors: Kunkel T A , Patel S S , Johnson K A
Published: May 2006

We have examined the effect of thioredoxin, an accessory protein that confers high processivity to bacteriophage T7 DNA polymerase, on the fidelity of DNA synthesis. In the presence of thioredoxin, exonuclease-proficient T7 DNA polymerase is highly accurate. In fidelity assays that score errors that revert M13mp2 lacZ alpha-complementation mutants, error rates are < or = 2.2 x 10(-6) for base substitution and < or = 3.7 x 10(-7) and < or = 4.5 x 10(-7) for frameshifts that revert mutations in the +1 and -1 reading frames, respectively. Rates are more than 10-fold higher during synthesis by polymerase.thioredoxin complex lacking 3'-->5' exonuclease activity, demonstrating that frameshift as well as substitution errors are subject to proofreading. The contribution of thioredoxin to accuracy has been examined by comparing the fidelity of the exonuclease-deficient polymerase in the presence or absence of the accessory protein. Thioredoxin either enhances or reduces fidelity, depending on the type of error considered. In the absence of thioredoxin, T7 DNA polymerase is 3-fold more accurate for base substitutions and > or = 27-fold and 9-fold more accurate, respectively, for 1- and 2-nt deletion errors at nonreiterated nucleotide sequences. Higher fidelity for all three errors may reflect the inability of the polymerase to continue synthesis from the premutational intermediates in the absence of the accessory protein. In marked contrast, the rate for frameshift errors wherein one or more nucleotides has been added to a repeated DNA sequence increases 46-fold when thioredoxin is absent from the polymerization reaction. The error rate increases as the length of the repeated sequence increases, consistent with a model where strand slippage creates misaligned template-primers. Thus, replicative expansion of repetitive sequences occurs in the absence of a replication accessory protein.
Rearrangement of the histone H2A C-terminal domain in the nucleosome.
Authors: Usachenko S I , Bavykin S G , Gavin I M , Bradbury E M
Published: May 2006

Using zero-length covalent protein-DNA crosslinking, we have mapped the histone-DNA contacts in nucleosome core particles from which the C- and N-terminal domains of histone H2A were selectively trimmed by trypsin or clostripain. We found that the flexible trypsin-sensitive C-terminal domain of histone H2A contacts the dyad axis, whereas its globular domain contacts the end of DNA in the nucleosome core particle. The appearance of the histone H2A contact at the dyad axis occurs only in the absence of linker DNA and does not depend on the absence of linker histones. Our results show the ability of the histone H2A C-terminal domain to rearrange. This rearrangement might play a biological role in nucleosome disassembly and reassembly and the retention of the H2A-H2B dimer (or the whole octamer) during the passing of polymerases through the nucleosome.
A carbonic anhydrase from the archaeon Methanosarcina thermophila.
Authors: Alber B E , Ferry J G
Published: May 2006

Carbonic anhydrase (CA) from acetate-grown Methanosarcina thermophila was purified > 10,000-fold (22% recovery) to apparent homogeneity with a specific activity of 4872 units/mg. The estimated native molecular mass of the enzyme is 84 kDa based on gel filtration chromatography. SDS/PAGE revealed one protein band with an apparent molecular mass of 40 kDa. The M. thermophila CA is less sensitive than human CA isozyme II toward inhibition by sulfonamides and monovalent ions. The gene encoding this CA was cloned into pUC18 and sequenced. Escherichia coli harboring the recombinant plasmid expresses CA activity (2.3 units/mg of cell extract protein). Comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein shows that the gene encodes an additional 34 N-terminal residues with properties characteristic of signal peptides in secretory proteins. The calculated molecular mass (22.9 kDa) and pI (4.0) suggest that SDS/PAGE overestimates the subunit size and that the native enzyme is a tetramer. To our knowledge, the deduced amino acid sequence has no significant identity to any known CA but has 35% sequence identity to the first 197 deduced N-terminal amino acids of a proposed CO2-concentrating-mechanism protein from Synechococcus PCC7942 and 28% sequence identity to the deduced sequence of ferripyochelin binding protein from Pseudomonas aeruginosa. Thus, our results indicate that this archaeal CA represents a distinct class of CAs and provide a basis to determine physiological roles for CA in acetotrophic anaerobes.
The A1 and A1B proteins of heterogeneous nuclear ribonucleoparticles modulate 5' splice site selection in vivo.
Authors: Yang X , Bani M R , Lu S J , Rowan S , Ben-David Y , Chabot B
Published: May 2006

Recent in vitro results suggest that the heterogeneous nuclear ribonucleoparticle (hnRNP) A1 protein modulates alternative splicing by favoring distal 5' splice site (5'SS) selection and exon skipping. We used a mouse erythroleukemia (MEL) cell line (CB3C7) deficient in the expression of hnRNP A1 to test whether variations in hnRNP A1 and AlB protein levels affected alternative splicing in vivo. In contrast to A1-expressing MEL cell lines, CB3C7 cells preferentially selected the proximal 13S and 12S 5'SS on the adenovirus E1A pre-mRNA. Transiently expressing the A1 or A1B cDNA in CB3C7 cells shifted 5'SS selection toward the more distal 9S donor site. A1 protein synthesis was required for this effect since the expression of a mutated A1 cDNA did not affect 5'SS selection. These results demonstrate that in vivo variations in hnRNP A1 protein levels can influence 5'SS selection.