bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple integrins mediate cell attachment to cytotactin/tenascin.
Authors: Prieto A L , Edelman G M , Crossin K L
Published: May 2006

To identify potential cell surface receptors for chicken cytotactin (CT), we have characterized the ability of recombinant fusion proteins spanning the proximal fibronectin (FN) type III repeats of the molecule to support attachment of glioma and carcinoma cell lines. The third FN type III repeat, which contains the RGD tripeptide, supported cell attachment and cell spreading; however, mutation of RGD to RAD did not result in significant loss of either activity. In addition, the same repeat of mouse CT, which contains a natural mutant, RVD, also supported cell attachment and spreading, although at a lower level; both activities were increased by mutation of the RVD sequence to RGD. Studies utilizing RGD-containing peptides and well-characterized antibodies to integrins indicated that cell attachment to the third FN type III repeat was mediated by at least two different integrin receptors of the alpha v subtype. Additional cellular receptors may also be involved in cell attachment to CT. For example, an antibody to the beta 1 subfamily of integrins partially inhibited binding of cells to intact CT but did not inhibit cell binding to the third FN type III repeat. These findings suggest that the RGD site in CT is able to mediate cell attachment to integrins and thus is not a cryptic adhesion site. They also open the possibility that the functions of CT in processes such as counteradhesion, cell migration, cell proliferation, and cell differentiation may be mediated in part by interaction with multiple integrins.
Transcription factors NF-IL6 and NF-kappa B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8.
Authors: Matsusaka T , Fujikawa K , Nishio Y , Mukaida N , Matsushima K , Kishimoto T , Akira S
Published: May 2006

Single binding sites for transcription factors NF-IL6 and NF-kappa B are present in the promoter of the interleukin (IL) 6 gene. Previous studies of internally deleted promoter mutants demonstrated that these two sites are important for the transcriptional regulation of this gene. In this report, we describe the synergistic activation of the IL-6 promoter by transcription factors NF-IL6 and NF-kappa B. Cotransfection of NF-IL6 with the NF-kappa B p65 subunit resulted in strong synergistic activation of an IL-6 promoter-reporter construct. Both the NF-IL6 and NF-kappa B binding sites in the IL-6 promoter were required for synergistic activation. Similar synergistic activation was observed in the IL-8 promoter, which also contains both NF-IL6 and NF-kappa B binding sites. Furthermore, we demonstrated that NF-IL6 and the NF-kappa B p65 subunit directly associated via the basic leucine-zipper domain of NF-IL6 and the Rel homology domain of p65. Since the promoters of many other genes involved in the inflammatory and acute-phase responses also contain binding sites for NF-IL6 and NF-kappa B, the cooperation between these two factors may have an important role in these responses. We also discuss the possible interplay between various viral gene products and these two factors in the process of viral infection and constitutive cytokine production.
Sex pheromone for the brownbanded cockroach is an unusual dialkyl-substituted alpha-pyrone.
Authors: Charlton R E , Webster F X , Zhang A , Schal C , Liang D , Sreng I , Roelofs W L
Published: May 2006

Female brownbanded cockroaches, Supella longipalpa, emit a sex pheromone that attracts males from a distance. This pheromone was isolated and identified as 5-(2,4-dimethylheptanyl)-3-methyl-2H-pyran-2-one (which we refer to as supellapyrone), and its structure was confirmed by synthesis. A racemic blend of the synthetic compound elicited behavioral and electrophysiological responses comparable to the natural pheromone across a range of doses. This compound is not only a very different type of cockroach pheromone but also makes up an additional class of natural products--namely, 3,5-dialkyl-substituted alpha-pyrones.
Mutant woodchuck hepatitis virus genomes from virions resemble rearranged hepadnaviral integrants in hepatocellular carcinoma.
Authors: Kew M C , Miller R H , Chen H S , Tennant B C , Purcell R H
Published: May 2006

Although hepadnaviruses are implicated in the etiology of hepatocellular carcinoma, the pathogenic mechanisms involved remain uncertain. Clonally propagated integrations of hepadnaviral DNA into cellular DNA can be demonstrated in most virally induced hepatocellular carcinomas. Integration occurs at random sites in cellular DNA, but the highly preferred sites in viral DNA are adjacent to the directly repeated sequence DR1, less often DR2, or in the cohesive overlap region. Integrants invariably contain simple deletions or complex rearrangements that have been thought to occur after integration. We report here the detection of mutant woodchuck hepatitis virus (WHV) genomes cloned from virions in serum that are strikingly similar to the rearranged hepadnaviral genomes found previously as integrated sequences in cellular DNA. Of 102 cloned genomes studied, 2 had large inverted duplications, 1 a 219-nucleotide direct duplication, and 1 a 219-nucleotide deletion. Virus-virus DNA junctions occurred either adjacent to DR1 or DR2 or in the cohesive overlap region at preferred topoisomerase I cleavage sites. Since these sites are located in the single-stranded regions of the genome, cleavage by topoisomerase I would produce linear molecules that would be expected to be highly recombinogenic since this enzyme, possessing nicking and ligating activities, would remain covalently attached. Sucrose density gradient centrifugation coupled with polymerase chain reaction studies confirmed that the mutant WHV DNA forms resided in virions and did not represent free viral DNA released from infected cells or were unlikely to be an artifact of the cloning process. Thus, the finding in virions of mutant WHV DNA similar to WHV DNA integrated into cellular DNA suggests that the processes of mutation and integration are linked in some instances. Furthermore, the mutant genomes that are preferentially integrated into cellular DNA may have an etiologic role in hepatocarcinogenesis.
Increasing cAMP attenuates activation of mitogen-activated protein kinase.
Authors: Sevetson B R , Kong X , Lawrence J C
Published: May 2006

Activation of the mitogen-activated protein kinase (MAP kinase) isoforms ERK1 and ERK2 was investigated in rat adipocytes. Kinase activities were measured by using myelin basic protein as substrate after the isoforms were resolved by Mono Q chromatography or by immunoprecipitation with specific antibodies. Insulin increased the activity of both isoforms by 3- to 4-fold. The beta-adrenergic agonist isoproterenol was without effect in the absence of insulin but markedly reduced the increases in ERK1 and ERK2 activities produced by the hormone. MAP kinase activation was also attenuated by forskolin and glucagon, which increase intracellular cAMP, and by dibutyryl-cAMP, 8-bromo-cAMP, and 8-(4-chlorophenylthio)-cAMP. Thus, increasing cAMP is associated with decreased activation of MAP kinase by insulin. Forskolin also inhibited activation of MAP kinase by several agents (epidermal growth factor, phorbol 12-myristate 13-acetate, and okadaic acid) that act independently of insulin receptors. Moreover, forskolin did not inhibit insulin-stimulated tyrosine phosphorylation of the insulin receptor substrate IRS-1. Therefore, the inhibitory effect on MAP kinase did not result from compromised functioning of the insulin receptor. The inhibitory effect was not confined to adipocytes, as forskolin and dibutyryl-cAMP inhibited the increase in MAP kinase activity by phorbol 12-myristate 13-acetate in wild-type CHO cells. In contrast, these agents did not inhibit MAP kinase activity in mutant CHO cells (line 10248) that express a cAMP-dependent protein kinase resistant to activation by cAMP. Our results suggest that activation of cAMP-dependent protein kinase represents a general counter-regulatory mechanism for opposing MAP kinase activation.
Transforming growth factor beta effects on expression of G1 cyclins and cyclin-dependent protein kinases.
Authors: Geng Y , Weinberg R A
Published: May 2006

Transforming growth factor beta 1 (TGF-beta 1) is a potent growth-inhibitory polypeptide. The mechanism of TGF-beta 1 inhibition has been related to its ability to prevent the hyperphosphorylation of retinoblastoma protein (pRb). Several lines of evidence have suggested that cell cycle-regulated protein kinases are responsible for the hyperphosphorylation of pRb. We demonstrate here that TGF-beta 1 has profound effects on the expression of genes encoding certain G1 cyclins and their associated kinases, which provides one explanation of TGF-beta 1 effects on pRb hyperphosphorylation. These results also suggest that the growth-inhibitory effects of TGF-beta 1 in many cells are attributable to its effects on the cell cycle apparatus involved in programming G1 transit.
The Fas protein is expressed at high levels on CD4+CD8+ thymocytes and activated mature lymphocytes in normal mice but not in the lupus-prone strain, MRL lpr/lpr.
Authors: Drappa J , Brot N , Elkon K B
Published: May 2006

The lymphoproliferation (lpr) mutation in the MRL strain of mice is caused by the insertion of the early transposable element ETn in the Fas gene. The insertion causes a striking decrease in Fas mRNA expression and is associated clinically with marked acceleration of the lupus-like disease. To further explore the role of the Fas protein in T-cell selection in the thymus and tolerance in the peripheral immune system, we produced a monospecific polyclonal anti-murine Fas antibody that binds to a polymorphic region of the protein. Fas protein expression was detected on approximately 90% of BALB/c and MRL +/+ thymocytes, and the expression was highest on CD4+CD8+ thymocytes, the stage at which most thymocytes die by apoptosis. In contrast to the high level of expression of Fas on thymocytes, Fas was detected on < 10% of normal splenic T cells. After activation of splenic T cells with Con A or anti-CD3 and interleukin 2, Fas expression increased approximately 10-fold. Fas expression on splenic B cells was also markedly up-regulated after activation with lipopolysaccharide or anti-mu antibodies. The Fas protein was not detected on resting or activated lymphocytes obtained from MRL lpr/lpr mice. Together, these findings suggest that Fas plays a role in both thymic selection and T-cell survival in the periphery and that the accelerated autoimmunity in MRL lpr/lpr mice results from a defect in both of these pathways.
Brain-specific tropomyosins TMBr-1 and TMBr-3 have distinct patterns of expression during development and in adult brain.
Authors: Stamm S , Casper D , Lees-Miller J P , Helfman D M
Published: May 2006

In this study we report on the developmental and regional expression of two brain-specific isoforms of tropomyosin, TMBr-1 and TMBr-3, that are generated from the rat alpha-tropomyosin gene via the use of alternative promoters and alternative RNA splicing. Western blot analysis using an exon-specific peptide polyclonal antibody revealed that the two isoforms are differentially expressed in development with TMBr-3 appearing in the embryonic brain at 16 days of gestation, followed by the expression of TMBr-1 at 20 days after birth. TMBr-3 was detected in all brain regions examined, whereas TMBr-1 was detected predominantly in brain areas that derived from the prosencephalon. Immunocytochemical studies on mixed primary cultures made from rat embryonic midbrain indicate that expression of the brain-specific epitope is restricted to neurons. The developmental pattern and neuronal localization of these forms of tropomyosin suggest that these isoforms have a specialized role in the development and plasticity of the nervous system.
Analysis of somatic hypermutation in mouse Peyer's patches using immunoglobulin kappa light-chain transgenes.
Authors: González-Fernández A , Milstein C
Published: May 2006

We have exploited mice transgenic for an immunoglobulin kappa light chain in order to show that immunoglobulin genes in the B cells of Peyer's patches in unimmunized mice carry a high level of somatic mutations. Most of the mutations are found in the subpopulation of B cells which, based on peanut agglutinin binding, derive from the germinal centers. The number of mutations per clone and their distribution along the variable gene segment (indicative of untemplated point mutations) are very similar to those found in antigen-specific splenic B cells of normal mice after secondary immunization. The mutations accumulate mainly in complementarity-determining region 1, in particular in some specific codons (Ser-26, Ser-31, and Ser-77) which have been previously recognized as intrinsic hypermutational hotspots. These results suggest that, as in the spleen, somatic mutation occurs in B cells which have migrated to the germinal centers, probably as a consequence of stimulation by antigens present in the gut environment. Transgenic animals are increasingly being used to define the signals involved in hypermutation. However, their subsequent study is very time-consuming because it is based on immunization and analysis of hybridomas or antigen-selected cells. We propose that the use of Peyer's patches of unimmunized adult mice offers a reliable and simple approach to analyze hypermutation of transgenes.
Characterization of composite aminodeoxyisochorismate synthase and aminodeoxyisochorismate lyase activities of anthranilate synthase.
Authors: Morollo A A , Bauerle R
Published: May 2006

Anthranilate synthase [chorismate pyruvatelyase (amino-accepting), E.C.4.1.3.27] catalyzes the formation of anthranilate (o-aminobenzoate) and pyruvic acid from chorismate and glutamine. A mutant form of the enzyme from Salmonella typhimurium accumulates a compound that we had isolated and identified as trans-6-amino-5-[(1-carboxyethenyl)-oxy]-1,3- cyclohexadiene-1-carboxylic acid, commonly called aminodeoxyisochorismate (ADIC). Here we report that ADIC is formed by a reversible, Mg(2+)-dependent ADIC synthase activity of anthranilate synthase that can be functionally uncoupled from a Mg(2+)-dependent ADIC lyase activity of the enzyme by single amino acid substitutions in the TrpE subunit of the anthranilate synthase complex of S. typhimurium. Both of the component activities of the enzyme are sensitive to feedback inhibition by L-tryptophan. Purified ADIC is quantitatively converted to anthranilate and pyruvic acid by the ADIC lyase activity of wild-type anthranilate synthase. ADIC also serves as a substrate for the formation of chorismate by the enzyme in the absence of glutamine and (NH4)2SO4. The rate of ADIC formation by the mutant enzyme and the steady-state parameters for ADIC utilization by the wild-type enzyme are consistent with a role for ADIC as an enzyme-bound intermediate that does not accumulate during the course of the anthranilate synthase reaction. The altered catalytic specificity of mutant anthranilate synthase enzymes suggests a potential role for ADIC in secondary metabolism.
Characterization of functional organization within rat barrel cortex using intrinsic signal optical imaging through a thinned skull.
Authors: Masino S A , Kwon M C , Dory Y , Frostig R D
Published: May 2006

We used optical imaging of intrinsic signals to characterize the functional representations of mystacial vibrissae (whiskers) in rat somatosensory cortex. Stimulation of individual whiskers for 2 s at 5 Hz resulted in a discrete area of functional activity in the cortex. Images of whisker representations were collected both through the dura and through a thinned skull. We characterized the functional representation of a whisker both spatially and temporally with two-dimensional images and three-dimensional surface plots of intrinsic signal development in the cortex in response to whisker stimulation. Single unit recordings verified that the representation of the whisker obtained with optical imaging corresponded with the electrophysiological response area of that whisker in the cortex. Lesions in the center of the functional activity were found to be in the center of the dense cytochrome oxidase patch for the corresponding whisker. In addition, a 3 x 3 matrix of whiskers was stimulated and the distances between the centers of the imaged representations and the distances between the centers of the layer IV cytochrome oxidase staining of the nine whiskers were found to be highly correlated (r = 0.98). This study shows a striking correspondence among imaging, physiology, and anatomy in the rat somatosensory cortex. Furthermore, the ability to use optical imaging through a thinned skull should allow investigations into the long-term changes in a sensory representation within a single animal.
Human immunodeficiency virus infection of human brain capillary endothelial cells occurs via a CD4/galactosylceramide-independent mechanism.
Authors: Moses A V , Bloom F E , Pauza C D , Nelson J A
Published: May 2006

Neuropathologic studies of AIDS patients have shown that brain capillary endothelial cells are a cellular target for human immunodeficiency virus (HIV) in vivo. We have established in vitro cultures of primary human brain capillary endothelial (HBCE) cells. Using this model system, we have shown a significant HIV infection of HBCE cells that is productive yet noncytopathic. The infection is mediated by a cellular interaction with gp120 that does not involve CD4 or galactosylceramide. HIV infection of HBCE cells may contribute to AIDS-associated neuropathology by disturbing the physiology of the endothelium and directly or indirectly facilitating dissemination of virus to the central nervous system.
The lysosomal proenzyme receptor that binds procathepsin L to microsomal membranes at pH 5 is a 43-kDa integral membrane protein.
Authors: McIntyre G F , Erickson A H
Published: May 2006

Two lysosomal proenzymes, procathepsins L and D, bind to mouse fibroblast microsomal membranes at acidic pH. This membrane association is independent of the mannose-6-phosphate receptors and requires the presence of the N-terminal propeptides of the enzymes. We have identified the protein that specifically binds procathepsin L at pH 5. A 43-kDa membrane protein coimmunoprecipitated with procathepsin L at pH 5 but not at pH 7 when cells were denatured with detergents. Similarly, a 43-kDa integral membrane protein bound procathepsin L in three kinds of ligand blots at pH 5 but not at pH 7. A synthetic peptide containing the 24 N-terminal residues of mouse procathepsin L blocked the binding of procathepsin L to this integral membrane protein on ligand blots. These results indicate that the 43-kDa integral membrane protein is a lysosomal proenzyme receptor that specifically binds the procathepsin L activation peptide at acidic pH.
Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus.
Authors: Hijikata M , Mizushima H , Tanji Y , Komoda Y , Hirowatari Y , Akagi T , Kato N , Kimura K , Shimotohno K
Published: May 2006

By using a plasmid-based transient protein expression system in cultured cells and an in vitro transcription/translation system, we analyzed the proteolytic processing of the putative nonstructural protein region of the precursor polyprotein from a Japanese type of hepatitis C virus. In addition to the previously reported viral proteins, p21 and p70, we identified products of 4 kDa (p4), 27 kDa (p27), 56 kDa (p56), 58 kDa (p58), and 66 kDa (p66). These products were produced in a viral serine proteinase (proteinase 2)-dependent manner from the region downstream of p70 in the precursor polyprotein and were arranged as NH2-p70-p4-p27-p58(p56)-p66-COOH as determined with region-specific antibodies. We showed that p56 was an N-terminally truncated form of p58, which suggested that a small polypeptide of 2 kDa (p2) was produced from the N-terminal part of p58. Cleavage between p4 and p27 was inefficient in vitro and we saw the 31-kDa precursor polypeptide (p31) accumulate. Furthermore, efficient cleavage at this site in vivo required the presence of p58/p56. Immunoprecipitation analysis in vitro also suggested the mutual interaction of those nonstructural protein products. An especially close association of p4 with p70 may contribute to association of p70 with microsomal membranes.
Acetalins: opioid receptor antagonists determined through the use of synthetic peptide combinatorial libraries.
Authors: Dooley C T , Chung N N , Schiller P W , Houghten R A
Published: May 2006

A synthetic peptide combinatorial library made up of 52,128,400 hexapeptides, each having an acetyl group at the N terminus and an amide group on the C terminus, was screened to find compounds able to displace tritiated [D-Ala2,MePhe4,Gly-ol5]enkephalin from mu opioid receptor binding sites in crude rat brain homogenates. Individual peptides with mu receptor affinity were found using an iterative process for successively determining the most active peptide mixtures. Upon completion of this iterative process, the three peptides with the highest affinity were Ac-RFMWMT-NH2, Ac-RFMWMR-NH2, and Ac-RFMWMK-NH2. These peptides showed high affinity for mu and kappa 3 opioid receptors, somewhat lower affinity for delta receptors, weak affinity for kappa 1 receptors, and no affinity for kappa 2 receptors. They were found to be potent mu receptor antagonists in the guinea pig ileum assay and relatively weak antagonists in the mouse vas deferens assay. These peptides represent a class of opioid receptor ligands that we have termed acetalins (acetyl plus enkephalin).
Times to extinction for small populations of large birds.
Authors: Pimm S L , Diamond J , Reed T M , Russell G J , Verner J
Published: May 2006

A major practical problem in conservation biology is to predict the survival times-"lifetimes"-for small populations under alternative proposed management regimes. Examples in the United States include the 'Alala (Hawaiian Crow; Corvus hawaiiensis) and Northern Spotted Owl (Strix occidentalis caurina). To guide such decisions, we analyze counts of all crow, owl, and hawk species in the most complete available data set: counts of bird breeding pairs on 14 European islands censused for 29-66 consecutive years. The data set yielded 129 records for analysis. We define the population ceiling as the highest number of breeding pairs observed from colonization to extinction, within a consecutive series of counts for a given species on a given island. The resulting distributions of population lifetimes as a function of population size prove to be highly skewed: most small populations disappear quickly, but a few last for a long time. Median (i.e., 50th percentile) lifetimes are calculated as only 1-5 yr for hawk, owl, and crow populations with ceilings of one or two breeding pairs. As expected if demographic accidents are the main cause of extinction for small populations, lifetimes rise by a factor of 3-4 for each additional pair up to three pairs. They rise more slowly thereafter. These observations suggest that lifetimes of the 'Alala (now reduced to about three pairs in the wild), and of populations of Northern Spotted Owl in the smallest forest fragments, will be short unless active management is implemented.
Raf-1 forms a stable complex with Mek1 and activates Mek1 by serine phosphorylation.
Authors: Huang W , Alessandrini A , Crews C M , Erikson R L
Published: May 2006

Recombinant Mek1 and Raf-1 proteins produced in Sf9 cells undergo a tight association both in vivo and in vitro, which apparently does not depend on additional factors or the kinase activity of Mek1 or Raf-1. The complex can be disrupted by two polyclonal antibodies raised against Raf-1 peptides. Coinfection with Raf-1 activates Mek1 > 150-fold, and coinfection with Raf-1 and Mek1 activates Erk1 approximately 90-fold. The activation of Mek1 by Raf-1 involves only serine phosphorylation, which is directly proportional to the extent of Mek1 activation. Phosphopeptide maps suggest a single Raf-1 phosphorylation site on mek1.
Photolysis of a protecting group for the carboxyl function of neurotransmitters within 3 microseconds and with product quantum yield of 0.2.
Authors: Ramesh D , Wieboldt R , Niu L , Carpenter B K , Hess G P
Published: May 2006

The synthesis of a photosensitive blocking group for the carboxyl function of neurotransmitters, in this case glycine, is reported. The compound, 2-methoxy-5-nitrophenyl glycine ester (caged glycine), is photolyzed by a laser pulse at 308 or 337 nm within 3 microseconds and with a product quantum yield of 0.2. The compound is hydrolyzed in water with a time constant tau of 6.1 min at pH 7.1 and 3 hr at pH 4.0. Mouse cerebral cortical neurons containing glycine receptors were used in biological assays. A cell-flow device, in which solutions of caged glycine at pH 4.0 were mixed with buffer to give a final pH of 7.1, was used to equilibrate the compound with receptors on the cell surface. Neither the caged compound nor the 2-methoxy-5-nitrophenol photolysis product affected the glycine receptors or modified their response to glycine. When cells equilibrated with caged glycine are irradiated by a laser pulse at 337 nm, glycine receptor channels are opened, as detected in whole-cell current recordings. The approach described may be used in the synthesis and characterization of photolabile precursors of neurotransmitters and other compounds that contain carboxyl groups and for kinetic investigations of neurotransmitter receptors in central nervous system cells in the microsecond time domain.
Evidence for an additional ligand, distinct from B7, for the CTLA-4 receptor.
Authors: Razi-Wolf Z , Galvin F , Gray G , Reiser H
Published: May 2006

Activation of T lymphocytes requires the recognition of peptide-major histocompatibility complex complexes and costimulatory signals provided by antigen-presenting cells (APCs). The best-characterized costimulatory molecule to date is the B7 antigen, a member of the immunoglobulin family that binds two receptors, CD28 and CTLA-4, expressed on the T-cell surface. Using the anti-mouse B7 (mB7) monoclonal antibody (mAb) 16-10A1, which we recently developed, we found that mB7 is indeed an important costimulatory ligand for the antigen-specific activation of murine T cells by B lymphocytes. Three lines of evidence suggest, however, the existence of at least one additional ligand for the CTLA-4 receptor. First, a soluble fusion protein of human CTLA-4 and the IgG1 Fc region, termed CTLA4Ig, blocks better than the anti-mB7 mAb the allogeneic stimulation of T cells by unfractionated splenic APCs. Second, saturating amounts of anti-mB7 mAb do not significantly block binding of fluorescein isothiocyanate-conjugated CTLA4Ig to activated splenic APCs. Furthermore, CTLA4Ig but not the anti-mB7 mAb reacts with the M12 and M12.C3 cell lines. The identification of an additional ligand for CTLA-4 may have applications to the treatment of autoimmune disease and transplant-associated disorders.
Identifying the catalytic residue of the ATPase reaction of DNA gyrase.
Authors: Jackson A P , Maxwell A
Published: May 2006

We propose a mechanism for the hydrolysis of ATP by the DNA gyrase B protein in which Glu42 acts as a general base and His38 has a role in aligning and polarizing the glutamate residue. We have tested this mechanism by site-directed mutagenesis, converting Glu42 to Ala, Asp, and Gln, and His38 to Ala. In the presence of wild-type A protein, B proteins bearing the mutations Ala42 and Gln42 show no detectable supercoiling or ATPase activities, while Asp42 and Ala38 proteins have reduced activities. In the DNA cleavage and relaxation reactions of gyrase, which do not require ATP hydrolysis, wild-type and mutant proteins have similar activities. When the 43-kDa N-terminal fragment of the gyrase B protein (which hydrolyzes ATP) contained the mutations Ala42 or Gln42, ATP was bound but not hydrolyzed, supporting the idea that Glu42 is involved in hydrolysis but not nucleotide binding.
Cloning and characterization of a transmembrane serine kinase that acts as an activin type I receptor.
Authors: Tsuchida K , Mathews L S , Vale W W
Published: May 2006

Activin type II receptors are transmembrane protein-serine/threonine kinases. By using a reverse-transcription PCR assay to screen for protein kinase sequences, we isolated a cDNA clone, activin X1 receptor, from rat brain that encodes a 55-kDa transmembrane protein-serine kinase which is structurally related to other receptors in this kinase subfamily. The predicted protein consists of 509 amino acids, and the kinase domain shows 40% and 37% identity to the activin and transforming growth factor beta type II receptors, respectively. No activin-binding was observed when activin X1 receptor was expressed alone in COS-M6 cells; however, coexpression with type II activin receptors gave rise to a 68-kDa affinity-labeled complex in addition to the 85-kDa type II receptor complex. The size of this cross-linked band is consistent with the size of the type I activin receptor; furthermore, activin X1 receptor associated with type II receptors, as judged by coimmunoprecipitation with type II receptor antibodies. These data suggest that activin X1 receptor can serve as an activin type I receptor and that the diverse biological effects of activins may be mediated by a complex formed by the interaction of two transmembrane protein-serine kinases.
A receptor for interleukin 10 is related to interferon receptors.
Authors: Ho A S , Liu Y , Khan T A , Hsu D H , Bazan J F , Moore K W
Published: May 2006

We isolated cDNAs encoding a mouse interleukin 10 receptor (mIL-10R) from mouse mast cell and macrophage cell lines. The two cDNAs are substantially identical and express an approximately 110-kDa polypeptide in COS7 cells, which binds mIL-10 specifically. A mouse pro-B-cell line (Ba/F3) expressing transfected recombinant mIL-10R binds IL-10 with high affinity (approximately 70 pM) and proliferates in response to mIL-10. mIL-10R is structurally related to interferon receptors (IFNRs). Since IL-10 inhibits macrophage activation by IFN-gamma, a possible implication of this relationship interaction of IL-10R and IFN-gamma R or their signaling pathways.
Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase.
Authors: Björklund S , Hjortsberg K , Johansson E , Thelander L
Published: May 2006

Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two nonidentical subunits, proteins R1 and R2, both required for enzyme activity. The structure of the genomic mouse ribonucleotide reductase R1 gene was compiled from a number of overlapping lambda clones isolated from a Charon 4A mouse sperm genomic library. The R1-encoding gene covers 26 kb and consists of 19 exons. All exon-intron boundaries were located by dideoxynucleotide sequencing, showing that intron 7 starts with the variant GC instead of GT. About 3.5 kb of DNA from the 5'-flanking region of the R1-encoding gene were cloned and sequenced, and the transcriptional start site was determined by nuclease S1 mapping of RNA. DNase I footprinting assays on the R1 promoter identified two nearly identical 23-bp-long protein-binding regions. Three protein complexes binding to one of the 23-mer regions were resolved and partially identified by using gel-retardation mobility-shift assays and UV crosslinking. One complex most likely contained Sp1, and another complex showed S-phase-specific binding, suggesting a direct role in the cell-cycle-dependent R1 gene expression.
kappa-Opioid receptors also increase potassium conductance.
Authors: Grudt T J , Williams J T
Published: May 2006

Decrease of calcium conductance induced by opioid agonists has been reported by others for mu-, delta-, and kappa-opioid receptors. On the other hand, only mu- and delta-opioid receptors have been reported to increase potassium conductance. Intracellular recordings were made from guinea pig substantia gelatinosa neurons in a brain slice. A subset of cells (29 of 83) were hyperpolarized by the kappa-opioid receptor agonist U69593 with an EC50 of 23 nM. The kappa-opioid receptor antagonist norbinaltorphimine (10 nM) blocked the hyperpolarization by U69593 but had no effect on the mu-opioid hyperpolarization present in these cells. Naloxone (300 nM) shifted the U69593 dose-response curve to the right, giving an estimated Kd for naloxone of 7.5 and 8.1 nM measured in two cells. The hyperpolarization caused by U69593 was mediated by a potassium conductance as determined with voltage clamp experiments. This demonstrates, depending on the cell type, that all three major opioid receptors (mu, delta, and kappa) can increase potassium conductance as well as decrease calcium conductance.
The Arabidopsis endoplasmic reticulum retention receptor functions in yeast.
Authors: Lee H I , Gal S , Newman T C , Raikhel N V
Published: May 2006

Soluble proteins retained in the lumen of the endoplasmic reticulum (ER) contain a carboxyl-terminal tetrapeptide sequence that functions presumably to recycle these proteins from a subsequent compartment. Biochemical and genetic evidence indicate that the ERD2 gene product is the receptor for these ER retention signals. Here we report the identification of a cDNA clone from Arabidopsis thaliana (aERD2) similar in sequence and size to members of the ERD2 gene family. Southern and Northern blot analyses indicate that Arabidopsis contains a single aERD2 gene which is expressed at different levels in various plant tissues. A functional assay demonstrates that the Arabidopsis homologue, unlike the mammalian protein, can complement the lethal phenotype of the erd2 deletion mutant of Saccharomyces cerevisiae, indicating that this protein may have a similar function in plants. As the plant protein may have a binding specificity similar to the human Erd2 protein but can function in yeast, we suggest that the plant homologue is the functional link between yeast and animals.
Microtubules and Src homology 3 domains stimulate the dynamin GTPase via its C-terminal domain.
Authors: Herskovits J S , Shpetner H S , Burgess C C , Vallee R B
Published: May 2006

Dynamin is a 100-kDa GTPase that plays a critical role in the initial stages of endocytosis. Dynamin binds to microtubules, which potently stimulate its GTPase activity. Binding to Src homology 3 (SH3) domains of proteins involved in signal transduction has also recently been reported. In the present study, the protein was digested with a variety of proteases to define its functional domains. Limited digestion with papain split the protein into an approximately 7- to 9-kDa microtubule-binding fragment and a 90-kDa nonbinding fragment. Immunoblotting with an antibody to the C-terminal 20 amino acids of rat dynamin showed the small fragment to derive from the C-terminal end of the polypeptide. Microtubule-activated GTPase activity, but not basal GTPase activity, was abolished by papain digestion, identifying the basic, proline-rich C-terminal region of dynamin as an important regulatory site. Bacterially expressed growth factor receptor-bound protein 2 (GRB2) and the SH3 domain of c-Src were also found to stimulate GTPase activity, although to a lesser extent than microtubules. Stimulation of GTPase activity by the recombinant proteins was similarly abolished by papain digestion. These results identify the basic, proline-rich C-terminal region of dynamin as the binding site for both microtubules and SH3 domains and demonstrate an allosteric interaction between this region of the molecule and the N-terminal GTPase domain.
Localization of the colchicine-binding site of tubulin.
Authors: Uppuluri S , Knipling L , Sackett D L , Wolff J
Published: May 2006

We have previously shown that rat brain tubulin, a heterodimer consisting of an alpha and beta monomer, can be covalently labeled with [3H]colchicine by near UV irradiation. Most of the label appears in beta-tubulin. We show here that beta-tubulin can be separated and purified from SDS preparative gels and analyzed by proteolysis. Chymotrypsin yielded a labeled approximately 4-kDa band that contained two peptides. Tryptic digestion also yielded an approximately 4-kDa band containing two peptides. Sequence analysis revealed a peptide of residues 1-36 and 213-242 for chymotrypsin and a peptide of residues 1-46 and 214-241 for trypsin. To identify which peptide carried the label, limited hydrolysis of beta-tubulin was done with trypsin; this procedure yielded a labeled 16-kDa N-terminal peptide and a 35-kDa C-terminal peptide, as identified by antibodies. Isolation of these peptides and extensive digestion with trypsin yielded two labeled peptides corresponding to residues 1-46 from the 16-kDa N-terminal fragment and residues 214-241 from the 35-kDa C-terminal fragment. These results show that at least two regions in beta-tubulin are specifically involved in colchicine binding and that the span of the colchicine molecule, < or = 11 A, bridges these two regions in the native beta monomer.
Evidence that facilitative glucose transporters may fold as beta-barrels.
Authors: Fischbarg J , Cheung M , Czegledy F , Li J , Iserovich P , Kuang K , Hubbard J , Garner M , Rosen O M , Golde D W
Published: May 2006

A widely accepted model for the structure of the facilitative glucose transporters (GLUTs) predicts that they form 12 transmembrane alpha-helices and that the highly conserved sequence Ile-386-Ala-405 in GLUT1 is intracellular. We raised a polyclonal antibody against a synthetic peptide encompassing this conserved sequence and found that antibody treatment increased 2-deoxy-D-glucose (DOG) uptake in Xe-nopus oocytes expressing GLUT1, GLUT2, or GLUT4 only when applied to the extracellular side. This effect was dose dependent and was specifically blocked by competition with the peptide Ile-386-Ala-405; it was due to a decrease in the Km for the transport of DOG. To ascertain GLUT orientation, we raised anti-peptide antibodies against the last 21 and 25 C-terminal amino acids of GLUT1 and GLUT4, respectively, which were previously shown to be intracellular. These antibodies increased DOG uptake when injected into oocytes expressing GLUT1 and GLUT4, but not when added extracellularly. Prompted by the noted discrepancy, we found sequence similarity between GLUTs and porins, two of which are known from crystallography to form 16-stranded transmembrane antiparallel beta-barrels. Analysis of the hydrophobicity, amphiphilicity, and turn propensity of GLUT1 leads us to propose that GLUTs fold as porin-like transmembrane beta-barrels. This model is consistent with the results of the present antibody studies and also with previously published experimental evidence inconsistent with the 12-helix model.
Low Ba2+ and Ca2+ induce a sustained high probability of repolarization openings of L-type Ca2+ channels in hippocampal neurons: physiological implications.
Authors: Thibault O , Porter N M , Landfield P W
Published: May 2006

Openings of single L-type Ca2+ channels following repolarization to negative membrane potentials from a depolarizing step (repolarization openings, ROs) have been described previously in brain cell preparations. However, these ROs have been reported to occur only infrequently. Here we report that the frequency of ROs in cell-attached patches of cultured rat hippocampal neurons can be increased dramatically by lowering the pipette Ba2+ concentration to 20 mM from the usual 90-110 mM. This increased opening probability can last for hundreds to thousands of milliseconds following repolarization. Current-voltage analyses of open probability show that the depolarization pulse threshold for inducing ROs in 20 mM Ba2+ is -10 to 0 mV but that the probability of ROs reaches maximal levels following depolarizing pulses that approach the apparent null (equilibrium) potential for Ba2+. Comparable current-voltage curves in 110 mM Ba2+ from a more positive holding potential (-50 mV) indicate that membrane surface charge screening accounts for some, but not all, of the effect of lowering the Ba2+ concentration. Consequently, current-dependent inactivation or some other ion-dependent mechanism (e.g., ion binding inside the pore) also appears to regulate this potentially major pathway of Ca2+ entry. A high probability of ROs also can be induced under relatively physiological conditions (5-ms depolarizing steps, 2-5 mM Ca2+ in the pipette). Thus, the high open probability state at negative potentials may underlie the long Ca2+ tail currents in hippocampus that were described previously and appears to have major implications for physiological functions (e.g., the slow Ca(2+)-dependent afterhyperpolarization), particularly in brain neurons.
A neo-Darwinian algorithm: asymmetrical mutations due to semiconservative DNA-type replication promote evolution.
Authors: Wada K N , Doi H , Tanaka S , Wada Y , Furusawa M
Published: May 2006

Evolution is, in a sense, to resolve optimization problems. Our neo-Darwinian algorithm based on the mechanics of inheritance and natural selection uses double-stranded DNA-type genetic information to resolve the "knap-sack problem." The algorithm with asymmetrical mutations due to semiconservative DNA-type replication most effectively resolved the problem. Our results strongly suggest that disparity in mutations caused by the asymmetric machinery of DNA replication promotes evolution, in particular of diploid organisms with a high mutation rate, in a small population, and under strong selection pressure.
HLA-C is the inhibitory ligand that determines dominant resistance to lysis by NK1- and NK2-specific natural killer cells.
Authors: Colonna M , Borsellino G , Falco M , Ferrara G B , Strominger J L
Published: May 2006

Natural killer (NK) cells recognize alloantigens on normal cells. One of these alloantigens correlates with homozygosity for a dimorphism of HLA-C at positions 77-80, which is shared by a number of HLA-C alleles. A second allelic alloantigen correlates with homozygosity for the alternative HLA-C dimorphism, which is shared by the remaining HLA-C alleles. Moreover, NK1- and NK2-specific NK cell lines can be generated by mixed leukocyte cultures in which donor and stimulator are homozygous for the alternative dimorphisms at positions 77-80 of HLA-C. In the present work, the role of HLA-C in NK cell-mediated allorecognition was directly investigated by analyzing the effects produced by transfection of several HLA-C alleles on NK sensitivity of class I-deleted mutant cell lines. Transfection of cells with HLA-C alleles encoding Asn-77-Lys-80 (including HLA-Cw4, -Cw5, and -Cw6) inhibited the lysis of the targets by NK1-specific NK cells, whereas HLA-C alleles encoding Ser-77-Asn-80 (including HLA-Cw1, -Cw7, and -Cw13) protected the targets from NK2-specific NK cells. Thus, HLA-C alleles are the dominant inhibitory ligands that protect targets from lysis by these allospecific NK cells.
Deficient transcription of XIST from tiny ring X chromosomes in females with severe phenotypes.
Authors: Migeon B R , Luo S , Stasiowski B A , Jani M , Axelman J , Van Dyke D L , Weiss L , Jacobs P A , Yang-Feng T L , Wiley J E
Published: May 2006

The severe phenotype of human females whose karyotype includes tiny ring X chromosomes has been attributed to the inability of the small ring X chromosome to inactivate. The XIST locus is expressed only from the inactive X chromosome, resides at the putative X inactivation center, and is considered a prime player in the initiation of mammalian X dosage compensation. Using PCR, Southern blot analysis, and in situ hybridization, we have looked for the presence of the XIST locus in tiny ring X chromosomes from eight females who have multiple congenital malformations and severe mental retardation. Our studies reveal heterogeneity within this group; some rings lack the XIST locus, while others have sequences homologous to probes for XIST. However, in the latter, the locus is either not expressed or negligibly expressed, based on reverse transcription-PCR analysis. Therefore, what these tiny ring chromosomes have in common is a level of XIST transcription comparable to an active X. As XIST transcription is an indicator of X chromosome inactivity, the absence of XIST transcription strongly suggests that tiny ring X chromosomes in females with severe phenotypes are mutants in the X chromosome inactivation pathway and that the inability of these rings to inactivate is responsible for the severe phenotypes.
The unusual metal clusters of nitrogenase: structural features revealed by x-ray anomalous diffraction studies of the MoFe protein from Clostridium pasteurianum.
Authors: Bolin J T , Ronco A E , Morgan T V , Mortenson L E , Xuong N H
Published: May 2006

Nitrogenase (EC 1.18.6.1) catalyzes the conversion of dinitrogen to ammonia, the central reaction of biological nitrogen fixation. X-ray anomalous diffraction data were analyzed to probe the structures of the metal clusters bound by nitrogenase MoFe protein. In addition to one FeMo cofactor, each half-molecule of MoFe protein binds one large FeS cluster of a type not previously observed in a protein. The FeS cluster contains roughly eight Fe atoms, comprises two subclusters, and is separated from the FeMo cofactor by an edge-to-edge distance of 14 A. The inorganic framework of the FeMo cofactor is not resolved into subclusters, but the Mo atom is located at its periphery. FeMo cofactors in different half-molecules are 70 A apart and cannot promote binuclear activation of dinitrogen by two Mo atoms.
Single-stranded shuttle phagemid for mutagenesis studies in mammalian cells: 8-oxoguanine in DNA induces targeted G.C-->T.A transversions in simian kidney cells.
Authors: Moriya M
Published: May 2006

A single-stranded shuttle vector has been developed for the purpose of investigating translesional events in mammalian cells. The vector is designed to permit site-specific introduction of defined DNA lesions between a gene for neomycin resistance and its promoter. Efficiencies of translesional synthesis in simian kidney cells (COS) and Escherichia coli are established by determining the number of neomycin- and ampicillin-resistant colonies recovered, respectively, after introduction of a modified vector. Fidelity of translesional synthesis is evaluated by analyzing the nucleotide sequence of progeny phagemid DNA in the region corresponding to the lesion site. This experimental system, capable of detecting mutagenic and nonmutagenic events at and adjacent to the lesion site, was used to establish the mutagenic potential of a single 8-oxoguanine residue in DNA. This modified base, produced by attack of reactive oxygen species on cellular DNA, did not cause a decrease in the number of transformants when single-stranded DNA containing the lesion replicated in COS cells or E. coli. The predominant mutations observed (> 78%) were G-->T transversions targeted to the site of the lesion. The mutation frequencies for this event were 2.5-4.8% in COS cells and 1.8% in E. coli. It is concluded that a single-stranded shuttle vector, utilized in conjunction with a site-specific approach, can be used to investigate translesional events in mammalian cells and in bacteria.