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At least 811 records · Page 45Linked to original sources
PubMed2002

The mouse Muc5b mucin gene: cDNA and genomic structures, chromosomal localization and expression.

We report here the isolation and characterization of the mouse Muc5b mucin gene (mMuc5b). We determined its complete cDNA sequence, its genomic organization, and chromosomal localization. Moreover, we analyzed the expression of this gene by reverse-transcription PCR and in situ hybridization. The structure of the gene was determined from a genomic cosmid clone that encompasses the entire mMuc5b gene, including the 5'-flanking region. The mMuc5b gene spans approximately 36 kb and contains 49 exons. It is located on mouse distal chromosome 7. mMuc5b encodes at least two transcripts by alternative splicing of the second exon, the longest one being 14.9 kb in length. The deduced peptide contains 4782 amino acids. Its central region can be subdivided into 10 imperfect repeats, each composed of a cysteine-rich domain followed by a threonine, serine, and proline-rich mucin-type domain. It is flanked by cysteine-rich domains similar to cysteine-rich domains of pre-pro-von Willebrand factor. Comparison with its human homologue MUC5B revealed common features including high sequence similarities in the 5' and 3' regions, and the conservation of the genomic organization. In contrast, mMuc5b differs from its human homologue, since no highly tandemly repeated sequences could be identified within its central region. mMuc5b is expressed mainly in laryngeal mucous glands, and at a lesser extend in stomach and duodenum.

Alternative Splicing↗
PubMedDate not supplied

Human and mouse RAD17 genes: identification, localization, genomic structure and histological expression pattern in normal testis and seminoma.

Recently, the human orthologue to the cell cycle checkpoint genes rad17 (Schizosaccharomyces pombe) and RAD24 (Saccharomyces cerevisiae), called HRAD17, has been isolated and localized to chromosome 4. Independently, we have isolated the HRAD17 transcript and mapped it to chromosome 5q13 between the CCNB1 and BTF2p44cen genes. Furthermore, we have identified the complete exon-intron structure of HRAD17. The gene is organized into 14 exons, the translation initiation site lies within exon 2, and the stop codon within exon 14. Two further HRAD17 pseudogenes, HRAD17P1 and HRAD17P2, were identified on chromosomes 7p21 and 13q14.3, respectively, encompassing exons 3-14 and bearing 84% and 93% homology, respectively. Additionally, we have isolated the coding region of the mouse orthologue, Mrad17, and mapped it on chromosome 13 between Ccnb1 and Btf2p44, the same two genes between which it maps in human. The predicted Mrad17 polypeptide encompasses 687 amino acids and shows 89% similarity to HRAD17. Both genes are most highly expressed in testis compared to all other tissues, as shown by Northern blot hybridization. Histological studies, based on in situ hybridization with radioactively labeled antisense HRAD17 riboprobes, showed a strong expression within the germinal epithelium of the seminiferous tubuli in normal testis whereas in testicular tumors (seminomas) only weak, diffuse signals were seen. In light of the known function of the yeast orthologue at meiotic and mitotic checkpoints, as well as the strong expression in testis and weak expression in seminomas, we suggest a putative involvement of HRAD 17 in testicular tumorigenesis.

Animals↗
PubMed2001

Folding-unfolding of goat alpha-lactalbumin studied by stopped-flow circular dichroism and molecular dynamics simulations.

Folding reaction of goat alpha-lactalbumin has been studied by stopped-flow circular dichroism and molecular dynamics simulations. The effects of four single mutations and a double mutation on the stability of the protein under a native condition were studied. The mutations were introduced into residues located at a hydrophobic core in the alpha-domain of the molecule. Here we show that an amino acid substitution (T29I) increases the native-state stability of goat alpha-lactalbumin against the guanidine hydrochloride-induced unfolding by 3.5 kcal/mol. Kinetic refolding and unfolding of wild-type and mutant goat alpha-lactalbumin measured by stopped-flow circular dichroism showed that the local structure around the Thr29 side chain was not constructed in the transition state of the folding reaction. To characterize the local structural change around the Thr29 side chain to an atomic level of resolution, we performed high-temperature (at 400 K and 600 K) molecular dynamics simulations and studied the structural change at an initial stage of unfolding observed in the simulation trajectories. The Thr29 portion of the molecule experienced structural disruption accompanied with the loss of inter-residue contacts and with the water molecule penetration in the 400-K simulation as well as in four of the six 600-K simulations. Disruption of the N-terminal portion was also observed and was consistent with the results of kinetic refolding/unfolding experiments shown in our previous report.

Animals↗
PubMed2004

Structure changes in glassforming liquids upon cooling and compression.

Upon cooling and compression, both the thermodynamic and the kinetic properties of glassformers change. In fragile glassformers, these changes suggest changes in the local structure of the liquid. Thermodynamic data may then provide a measure of structure changes. Special criteria for structure changes in terms of energy and pressure fluctuations in the isochoric system are proposed; recent theoretical results allow one to rewrite the criteria in terms of long- and short-time thermodynamic characteristics of the liquid. Based on the proposed criteria, we discuss the changes in the local structure due to temperature and pressure variations.

Journal Article↗
PubMed2003

Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function.

The first step in metabolism of glucose (Glc) is usually phosphorylation, catalyzed by hexokinase. However, the Glc-6-P produced can then enter one or more of several alternative pathways. Selective expression of isozymic forms of hexokinase, differing in catalytic and regulatory properties as well as subcellular localization, is likely to be an important factor in determining the pattern of Glc metabolism in mammalian tissues/cells. Despite their overall structural similarity, the Type I, Type II and Type III isozymes differ in important respects. All three isozymes are inhibited by the product, Glc-6-P, but with the Type I isozyme, this inhibition is antagonized by P(I), whereas with the Type II and Type III isozymes, P(i) actually causes additional inhibition. Reciprocal changes in intracellular levels of Glc-6-P and P(i) are closely associated with cellular energy status, and it is proposed that the response of the Type I isozyme to these effectors adapts it for catabolic function, introducing Glc into glycolytic metabolism for energy production. In contrast, the Type II, and probably the Type III, isozymes are suggested to serve primarily anabolic functions, e.g. to provide Glc-6-P for glycogen synthesis or metabolism via the pentose phosphate pathway for lipid synthesis. Type I hexokinase binds to mitochondria through interaction with porin, the protein that forms channels through which metabolites traverse the outer mitochondrial membrane. Several experimental approaches have led to the conclusion that the Type I isozyme, bound to actively phosphorylating mitochondria, selectively uses intramitochondrial ATP as substrate. Such interactions are thought to facilitate coordination of the introduction of Glc into glycolysis, via the hexokinase reaction, with the terminal oxidative stages of Glc metabolism occurring in the mitochondria, thus ensuring an overall rate of Glc metabolism commensurate with cellular energy demands and avoiding excessive production of lactate. The Type II isozyme also binds to mitochondria. Whether such coupling occurs with mitochondrially bound Type II hexokinase in normal tissues, and how it might be related to the proposed anabolic role of this isozyme, remain to be determined. The Type III isozyme lacks the hydrophobic N-terminal sequence known to be critical for binding of the Type I and Type II isozymes to mitochondria. Immunolocalization studies have indicated that, in many cell types, the Type III has a perinuclear localization, the possible metabolic consequences of which remain unclear.

Animals↗
PubMed2000

Genomic structure, chromosomal localization, and expression pattern of the human LIM-homeobox3 (LHX 3) gene.

Lhx3 is a LIM-homeobox protein essential for pituitary development in mice. The human homologue gene spans 7.2 kb and contains 7 exons, including two alternatively spliced first exons. This structure encodes two distinct protein isoforms, LHX3a and LHX3b, that differ exclusively in their amino-terminus. The LHX3 gene was localized at 9q34.2-34.3. The predicted protein sequence is highly homologous to other known Lhx3 proteins, the highest degree of homology being in the conserved domains. The highest expression of LHX3 was found in pituitary gland, spinal cord, and lung. Among different pituitary cell types, corticotrophs appear to express preferentially LHX3b isoform, suggesting a distinct role of the b-form in the development of this cell lineage. Although the human LHX3 gene structure would provide a ground for clarification of the molecular basis of complete anterior pituitary deficiency, we were unable to identify any mutation in the LHX3 gene of 46 such patients.

Alternative Splicing↗
PubMed2004

XMCD under pressure at the Fe K edge on the energy-dispersive beamline of the ESRF.

The present paper demonstrates the feasibility of X-ray absorption spectroscopy (XAS) and X-ray magnetic circular dichroism (XMCD) at high pressure at the Fe K edge on the ID24 energy-dispersive beamline of the ESRF. In 3d transition metals, performing experiments at the hard X-ray K edge rather than at the magnetically interesting soft X-ray L edges represents the only way to access the high-pressure regime obtainable with diamond anvil cells. The simultaneous availability of a local structure (XAS) and of a magnetic (XMCD) probe on the sample under identical thermodynamical conditions is essential for studying correlations between local structural and magnetic properties. The state-of-the-art theoretical understanding of K-edge XMCD data is briefly summarized, and the set-up of beamline ID24 for high-pressure XMCD experiments is illustrated and the conditions required to perform measurements at the K edges of 3d transition metals are underlined. Finally, two examples of recent high-pressure results at the Fe K edge in pure Fe and Fe3O4 powder are presented.

Journal Article↗
PubMed2000

Fragile T-stem in disease-associated human mitochondrial tRNA sensitizes structure to local and distant mutations.

Mutations in human mitochondrial isoleucine tRNA (hs mt tRNA(Ile)) are associated with cardiomyopathy and opthalmoplegia. A recent study showed that opthalmoplegia-related mutations gave rise to severe decreases in aminoacylation efficiencies and that the defective mutant tRNAs were effective inhibitors of aminoacylation of the wild-type substrate. The results suggested that the effectiveness of the mutations was due in large part to an inherently fragile mitochondrial tRNA structure. Here, we investigate mutant tRNAs associated with cardiomyopathy, and a series of rationally designed second-site substitutions introduced into both opthalmoplegia- and cardiomyopathy-related mutant tRNAs. A source of structural fragility was uncovered. An inherently unstable T-stem appears susceptible to misalignments. This susceptibility sensitizes both domains of the L-shaped tRNA structure to base substitutions that are deleterious. Thus, the fragile T-stem makes the structure of this human mitochondrial tRNA particularly vulnerable to local and distant mutations.

Cardiomyopathy, Hypertrophic↗
PubMed1985

Localization of structural variations distinguishing I-Ak-related molecules to the alpha 1 and beta 1 domains.

The structural variations that distinguish the A molecules encoded by wild-derived H-2 complexes which express Ak-related molecules have been localized into the alpha 1 and beta 1 domains by radiochemical sequence analyses of tryptic peptides. The A alpha subunits of B10.STC90 (Akv1) and W12A (Akv2) differ from those of B10.BR (Ak) in two adjacent tryptic peptides spanning positions 43 to 71 in the alpha 1 domain. The A beta subunit of W12A differs from that of B10.BR in two peptides spanning positions 26 to 29 and 95 to 106. Isoleucine and leucine residues present at positions 28 and 95, respectively, in the B10.BR A beta subunit are not found in the corresponding positions in W12A A beta subunits. Both of these A beta sequence variations are in the beta 1 domain. B10.STC90 A beta subunits are identical to those of W12A except for a structural variation in the beta 1 domain affecting the HPLC retention time of a peptide spanning positions 49 to 63. These results suggest that these A molecules are encoded by closely related class II gene alleles which have diversified by the accumulation of discrete mutations within the exons encoding the alpha 1 and beta 1 domains of the A molecule. Our previous functional analyses of these minor variant A molecules have demonstrated that they are readily distinguished with A molecule-specific alloreactive T lymphocytes. Together, these findings suggest that minor structural variations in the alpha 1 and beta 1 domains of the A molecule can dramatically modify the allodeterminants recognized by alloreactive T lymphocytes.

Amino Acid Sequence↗
PubMed1994

Dynamical order and disorder in lipid bilayers.

Various order and disorder phenomena in lipid bilayers are considered as they arise due to the very many-particle character of the bilayer. Particular attention is paid to dynamically maintained order in terms of lateral density- and compositional fluctuations that lead to dynamic heterogeneity, local structure, and lipid-domain formation on length scales of 10-1000 A. The influence of cholesterol and various drugs on the local structure is described. A discussion is presented of the possible role played by lipid order and disorder phenomena for the functional dynamics of membranes.

Anesthetics↗
PubMed2001

Iron and copper K-edge XAS study of serotransferrin and ovotransferrin.

The active metal site structure of transferrin with iron and copper atoms is investigated using metal K-XANES. Theoretical analysis of experimental data has been performed on the basis of full multiple-scattering theory. This approach made it possible to study the origin of XANES fine details and to investigate the local structure around active metal sites. A deep insight into the local structure and electronic subsystem of Fe, Cu transferrins is obtained. For example, in the case of Cu substitution of Fe in the active centre, the best fit of theoretical spectra to experiment has been obtained for distances 3% smaller between the Cu atom and the nearest neighbours.

Animals↗
PubMed2004

The structure of the first coordination shell in liquid water.

X-ray absorption spectroscopy and x-ray Raman scattering were used to probe the molecular arrangement in the first coordination shell of liquid water. The local structure is characterized by comparison with bulk and surface of ordinary hexagonal ice Ih and with calculated spectra. Most molecules in liquid water are in two hydrogen-bonded configurations with one strong donor and one strong acceptor hydrogen bond in contrast to the four hydrogen-bonded tetrahedral structure in ice. Upon heating from 25 degrees C to 90 degrees C, 5 to 10% of the molecules change from tetrahedral environments to two hydrogen-bonded configurations. Our findings are consistent with neutron and x-ray diffraction data, and combining the results sets a strong limit for possible local structure distributions in liquid water. Serious discrepancies with structures based on current molecular dynamics simulations are observed.

Chemical Phenomena↗
PubMed2002

A human short-chain dehydrogenase/reductase gene: structure, chromosomal localization, tissue expression and subcellular localization of its product.

We have previously described the cloning of Hep27, a short-chain dehydrogenase/reductase, which is synthesized in human hepatoblastoma HepG2 cells following growth arrest induced by butyrate treatment. The present report describes the cloning, the structure and the physical and cytogenetic mapping of the gene coding for Hep27. We also show that Hep27 is synthesized in a limited number of human normal tissues and that it is localized in the nuclei and cytoplasm of HepG2 cells.

Alcohol Dehydrogenase↗
PubMed1990

Investigation of the structure and localization of the urease of Helicobacter pylori using monoclonal antibodies.

The urease of Helicobacter pylori (formerly Campylobacter pylori) has been partly purified by fast protein liquid chromatography. This material contained 10 nm doughnut-like structures when examined by electron microscopy and comprised three major polypeptides (61 kDa, 56 kDa and 28 kDa). Only two of these polypeptides (61 kDa and 28 kDa) were observed in urease-containing material isolated by preparative non-denatured PAGE. Monoclonal antibodies (mAbs) were produced which were directed against two of these polypeptides (56 kDa and 28 kDa). Only mAbs directed against the 28 kDa polypeptide inhibited or captured urease activity. These results suggest that the 56 kDa polypeptide is not essential for enzyme activity. Anti-urease mAbs were used in an indirect immunogold technique to localize the enzyme at the ultrastructural level. In both prefixed bacteria and ultrathin cryosectioned bacteria the enzyme was located on the cell surface and in material apparently shed from that surface.

Antibodies, Monoclonal↗
PubMed1992

Primary structure, chromosomal localization, and functional expression of a voltage-gated sodium channel from human brain.

A cDNA library derived from human cerebral cortex was screened for the presence of sodium channel alpha subunit-specific clones. Ligation of three overlapping clones generated a full-length cDNA clone, HBA, that provided the complete nucleotide sequence coding for a protein of 2005 amino acids. The predicted structure suggests four homologous repeats and exhibits greatest homology and structural similarity to the rat brain sodium channel II. A second cDNA clone, HBB, that encodes a different subtype of sodium channel was isolated. Hybridization of DNA fragments from the 3' untranslated region of HBA and PCR with primers derived from HBB with human-hamster somatic cell hybrids localized these clones to human chromosome 2. In situ hybridization to human metaphase chromosomes mapped the structural genes for both HBA and HBB sodium channels to chromosome 2q23-24.3. The sodium channel HBA gene product was expressed by transfection in CHO cells. Expressed HBA currents were voltage-dependent, sodium-selective, and tetrodotoxin-sensitive and, thus, exhibit the biophysical and pharmacological properties characteristic of sodium channels.

Amino Acid Sequence↗
PubMed1984

Spatial distribution of DNA loop attachment and replicational sites in the nuclear matrix.

Biochemical fractionation was combined with high resolution electron microscopic autoradiography to study the localization in rat liver nuclear matrix of attached DNA fragments, in vivo replicated DNA, and in vitro synthesized DNA. In particular, we determined the distribution of these DNA components with the peripheral nuclear lamina versus more internally localized structural elements of isolated nuclear matrix. Autoradiography demonstrated that the bulk of in vivo newly replicated DNA associated with the nuclear matrix (71%) was found within internal matrix regions. A similar interior localization was observed in isolated nuclei and in situ in whole liver tissue. Likewise, isolated nuclear lamina contained only a small amount (12%) of the total matrix-bound, newly replicated DNA. The structural localization of matrix-bound DNA fragments was examined following long-term in vivo labeling of the DNA. The radioactive DNA fragments were found predominantly within interior regions of the matrix structure (77%), and isolated nuclear lamina contained less than 15% of the total nuclear matrix-associated DNA. Most of the endogenous DNA template sites for the replicative enzyme DNA polymerase alpha (approximately 70%) were also sequestered within interior regions of the matrix. In contrast, a majority of the endogenous DNA template sites for DNA polymerase beta (a presumptive repair enzyme) were closely associated with the peripheral nuclear lamina. A similar spatial distribution for both polymerase activities was measured in isolated nuclei before matrix fractionation. Furthermore, isolated nuclear lamina contained only a small proportion of total matrix-bound DNA polymerase alpha endogenous and exogenous template activities (3-12%), but a considerable amount of the corresponding beta polymerase activities (47-52%). Our results support the hypothesis that DNA loops are both anchored and replicated at nuclear matrix-bound sites that are predominantly but not exclusively associated with interior components of the matrix structure. Our results also suggest that the sites of nuclear DNA polymerase beta-driven DNA synthesis are uniquely sequestered within the characteristic peripheral heterochromatin shell and associated nuclear envelope structure, where they may potentially participate in DNA repair and/or replicative functions.

Animals↗
PubMed1994

Cellular localization and structural characterization of natriuretic peptide-expressing ventricular myocytes from patients with dilated cardiomyopathy.

Although atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are expressed in the tissue of ventricles of failing hearts, the localization and histopathological features of natriuretic peptide-expressing ventricular myocytes have not been clearly described. This study was designed to characterize the ventricular cardiomyocytes that express both natriuretic peptides in 19 patients with dilated cardiomyopathy (DCM). Immunohistochemistry and in situ hybridization for ANP and BNP were performed with left ventricular biopsy specimens. Peptide-expressing myocytes were examined by hematoxylin-eosin staining and desmin immunohistochemistry and by in situ hybridization to characterize the corresponding cells histopathologically. The distribution of ANP- and BNP-expressing myocytes in the ventricle was identical and was located in the subendocardial layer, fibrous area, and perivascular region. Desmin was found in heavy deposits in the cytosol of peptide-expressing myocytes, and desmin mRNA was not always augmented in the peptide-expressing myocytes. These results indicate that the augmented expression of natriuretic peptides in the left ventricle of patients with DCM is not due solely to global stress on the ventricular wall but is also influenced by regional conditions and is associated with structural changes in the myocytes.

Atrial Natriuretic Factor↗