Test of the equivalence principle for ordinary matter falling toward dark matter.
Explore the source record for details and available documents.
Journal Article↗
Biomedical subjects
Y Su
Publications and source records attributed to Y Su.
Exclusion of linkage between schizophrenia and the D2 dopamine receptor gene region of chromosome 11q in 112 Irish multiplex families.
A leading theory hypothesizes that schizophrenia arises from dysregulation of the dopamine system in certain brain regions. As this dysregulation could arise from abnormal expression of D2 dopamine receptors, the D2 receptor gene (DRD2) on chromosome 11q is a candidate locus for schizophrenia. We tested whether allelic variation at DRD2 and five surrounding loci cosegregated with schizophrenia in 112 small- to moderate-size Irish families containing two or more members affected with schizophrenia or schizoaffective disorder, defined by DSM-III-R. Evidence of linkage was assessed using varying definitions of illness and modes of transmission. Assuming genetic homogeneity, linkage between schizophrenia and large regions of 11q around DRD2 could be strongly excluded. Assuming genetic heterogeneity, variation at the DRD2 locus could be rejected as a major risk factor for schizophrenia in more than 50% of these families for all models tested and in as few as 25% of the families for certain models. The DRD2 linkage in fewer than 25% of these families could not be excluded under any of the models tested. Our results suggest that the major component of genetic susceptibility to schizophrenia is not due to allelic variation at the DRD2 locus or other genes in the surrounding chromosomal region.
Alleles↗
Two crystal structures of rat liver dihydropteridine reductase.
Explore the source record for details and available documents.
Animals↗
Molecular characterization of human prorenin isoelectric forms.
OBJECTIVE: Mammalian renins and prorenins can be fractionated by isoelectric focusing into multiple species, the individual production and decay rates of which vary with certain physiological and pharmacological stimuli. The relative abundance of renin isoelectric species varies in certain pathological states, and the individual species have been reported to differ in their biological function. The present study was undertaken to clarify further the biochemical basis for isoelectric heterogeneity in human renin. DESIGN: Previous data have suggested that differences in glycosylation contribute to isoelectric heterogeneity in human renin. To determine whether glycosylation was solely responsible for the observed isoelectric heterogeneity, the isoelectric focusing patterns of either native or non-glycosylated human prorenin were compared. The specific activities of the three most abundant isoelectric species of human prorenin were also compared. METHODS: Tissue culture supernatants from transfected cells expressing either native recombinant human prorenin or human prorenin in which both glycosylation sites were eliminated were fractionated by isoelectric focusing. The expressed prorenins were detected in various fractions by trypsin activation and the angiotensin I generation assay. Specific activities of the various prorenins were estimated by direct comparison of immunoprecipitable radiolabeled prorenin and trypsin-activatable renin activity in the three most abundant fractions. RESULTS: Native human recombinant prorenin was fractionated into at least five isoelectric species, whereas non-glycosylated human recombinant prorenin migrated as a single isoelectric species, the migration of which was unaffected by forskolin treatment. There was a direct correlation between immunoprecipitable and enzymatically determined prorenin in the three major isoelectric species of native prorenin. CONCLUSIONS: The results suggest that isoelectric heterogeneity of human renin is due solely to differential glycosylation of the protein. The proposed different biological functions of the various isoelectric forms is not directly correlated with variations in their specific activities.
Animals↗
Analysis of a segment of the human glial fibrillary acidic protein gene that directs astrocyte-specific transcription.
To understand astrocyte-specific transcription, we have been studying the human gfa gene. This gene encodes glial fibrillary acidic protein (GFAP), an intermediate filament protein expressed primarily in astrocytes. A survey of the gfa 5' flanking region showed it to contain several segments that contribute to expression of a chloramphenicol acetyltransferase reporter gene in transfected cells. The most active of these was the 124-bp B region, which spans bp -1612 to -1489. We have now used site-directed mutagenesis to analyze this region in greater detail, and show that the B region itself contains several important elements. The most crucial of these is a consensus AP-1 sequence, the binding site for the Fos and Jun families of transcription factors. The presence of members of both these families in the glial fibrillary acidic protein-expressing U251 cell line used for our transfection studies was verified by gel mobility-shift experiments. This is the first demonstration of the functioning of a specific transcription factor site for astrocytes, and provides a focus for future studies of glial fibrillary acidic protein regulation during development and reactive gliosis.
Astrocytes↗
(Pro)collagenase (matrix metalloproteinase-1) is present in rodent osteoclasts and in the underlying bone-resorbing compartment.
Osteoclasts resorb the extracellular matrix of bone by secreting enzymes and acid into a sealed-off compartment that they form upon attachment to the bone surface. Although the lysosomal cysteine proteinases can degrade collagen after the demineralization of bone at low pH, several lines of evidence suggest that collagenase (matrix metalloproteinase-1, EC 3.4.24.7) may also be involved in this process. The question of whether collagenase is present in the osteoclast and/or in the bone-resorbing compartment has however not been resolved. We have prepared an anti-mouse collagenase antiserum and affinity-purified an IgG fraction that specifically immunoblots and immunoprecipitates (pro)collagenase. Using these antibodies, we demonstrate by immunolocalization the presence of (pro)collagenase both in the osteoclasts and in the extracellular subosteoclastic bone-resorbing compartment. These specific localizations were observed not only in mice but also in rat and rabbit osteoclasts and using not only the antibody we have prepared but also antibodies raised in other laboratories against rat (Jeffrey et al., J. Cell. Physiol. 143, 396-403, 1990) and rabbit (Brinckerhoff et al., J. Biol. Chem. 265, 22262-22269, 1990) collagenase. Intracellular collagenase was observed in the osteoclasts whether the cells were plated on bone or cultured on glass coverslips. It is proposed that osteoclastic collagenase is secreted in the resorbing compartment where it may cooperate with the lysosomal cysteine proteinases in the degradation of the collagen component of the matrix during the resorption of bone.
Animals↗
Possibility of signal transduction through microfilaments below the membrane following ligand-receptor interaction.
In this paper, we describe the changes of microfilament assembly and 3H-TdR incorporation in mouse ascites liver cancer cells under the action of concanavalin A (ConA) and laminin (LN). We have also studied the variation of 3H-TdR incorporation induced by destroying microfilaments with cytochalasin B (CB) following ConA and LN binding with their membrane receptors. It was found that ConA and LN interactions with their membrane receptors could induce the assembly of microfilaments below the membrane and promote DNA synthesis in these cells, but this effect was inhibited when microfilaments were destroyed by CB treatment. These results suggest that microfilaments might play a role in transferring signals from the membrane to the nucleus.
Actin Cytoskeleton↗
Biocompatibilities of organic solvents with Lactobacillus delbrueckii.
In the search for biocompatible extractants for extractive fermentation of lactic acid, the effect of twelve organic solvents on the activity of L. delbrueckii were studied at different concentration levels. On this basis, the compatibilities of twelve solvents with L. delbrueckii were summarized into four classes: completely compatible, compatible at molecular level, partially compatible and non-compatible. The characteristics were described qualitatively for each class in terms of relative values between the solubility of organic solvent in water (Cs) and the toxicity of organic solvent, which is indicated by a newly defined concentration parameter (Cm). The classification was helpful for the selection of extractant in extractive fermentation.
Fermentation↗
The effect of laminin on molecular motion in the cell membrane and on cell motility.
We have studied the variation of lateral diffusion of proteins in the cell membrane, of membrane lipid fluidity and of the electrophoretic motility (EPM) of macrophages after treatment with extrinsic laminin. The results showed that the lateral diffusion coefficient D value of membrane proteins, the fluidity of membrane lipids and the EPM of macrophages were decreased after laminin had bound to its membrane receptor on the macrophages. These results are important for developing an understanding of the early reaction of plasma membranes and cells in the presence of laminin.
Animals↗
Phosphofructokinase from white muscle of the rainbow trout, Oncorhynchus mykiss: purification and properties.
Phosphofructokinase was purified and characterized from the white skeletal muscle of rainbow trout Oncorhynchus mykiss. Purification involved three steps: ion-exchange chromatography on hydroxyapatite and affinity chromatography on phosphocellulose and ATP-agarose. A final specific activity of 75 units per mg of protein at 22 degrees C and pH 7.2 with 40% recovery was obtained. The purified enzyme gave a single band on SDS-PAGE with a subunit molecular mass of 76.5 +/- 0.6 kDa. Based on gel filtration analysis, the active form of the enzyme was found to be composed of six identical subunits. A high isoelectric point (7.1) was found for this enzyme. Arrhenius plots of the enzyme activity showed a sharp transition at 15-16 degrees C. The pH optimum of the enzyme was 8.0-8.5 at physiological level of ATP and positive modulators shifted the optimum to lower pH values. Amino-acid analysis revealed a lower content of the aromatic residues Phe, Tyr and Trp and higher level of Ser residue than in the rabbit muscle enzyme.
Amino Acids↗
Regulatory elements for the tissue-specific expression of the rat serine dehydratase-encoding gene.
L-Serine dehydratase (SDH; EC 4.2.1.13), the key enzyme for serine utilization in the rat, is synthesized primarily in the liver. Cis-acting DNA elements required for liver-specific expression of the SDH gene were identified by two approaches: (1) transient expression assays in primary cultured rat hepatocytes, and in rat fibrosarcoma and normal rat kidney epithelial (NRK-52E) cell lines; and (2) in vitro transcription assays with nuclear extracts prepared from rat liver and spleen. Deletion analyses of the 5' flanking sequences of the gene have defined two functionally different regions: (a) a cell-type-specific promoter located between positions -62 and +10, which is sufficient for liver-specific expression; and (b) a distal promoter region between bp -133 and -63 containing positive cis-acting elements that regulate the promoter activity in a non-tissue-specific fashion. No other cis-acting elements essential for liver-specific expression were found in the region of -134 to 2.1 kb upstream relative to the cap site of SDH.
Animals↗
Crystal structure of an actinidin-E-64 complex.
E-64, 1-(L-trans-epoxysuccinylleucylamino)-4-guanidinobutane, is a potent and highly selective irreversible inhibitor of cysteine proteases. The crystal structure of a complex of actinidin and E-64 has been determined at 1.86-A resolution by using the difference Fourier method and refined to an R-factor of 14.5%. The electron density map clearly shows that the C2 atom of the E-64 epoxide ring is covalently bonded to the S atom of the active-site cysteine 25. The charged carboxyl group of E-64 forms four H-bonds with the protein and thus may play an important role in favorably positioning the inhibitor molecule for nucleophilic attack by the active-site thiolate anion. The interaction features between E-64 and actinidin are very similar to those seen in the papain-E-64 complex; however, the amino-4-guanidinobutane group orients differently. The crystals of the actinidin-E-64 complex diffracted much better than the papain-E-64 complex, and consequently the present study provides more precise geometrical information on the binding of the inhibitor. Moreover, this study provides yet another confirmation that the binding of E-64 is at the S subsites and not at the S' subsites as has been previously proposed. The original actinidin structure has been revised using the new cDNA sequence information.
Binding Sites↗
Location and characterization of multiple glucocorticoid-responsive elements in the rat serine dehydratase gene.
Transcription of the gene coding for serine dehydratase (SDH, EC 4.2.1.13) in the rat in vivo is dramatically increased by glucocorticoid hormones. To identify DNA elements mediating the glucocorticoid-regulated expression of the SDH gene, we transiently transfected 7AD-7 rat hepatoma cells with fusion genes consisting of various regions of the SDH 5' flanking sequence linked to the coding sequence of the gene for chloramphenicol acetyltransferase (CAT). Analysis of the CAT activities from these 5' deletion mutants identified three closely associated glucocorticoid-responsive elements (GREs), located more than 5 kb upstream relative to the cap site. Two distal GREs act synergistically to confer strong glucocorticoid inducibility to the gene, whereas the proximal GRE functions independently of the distal GREs and confers only a weak hormone response to the gene. The purified DNA-binding domain of rat glucocorticoid receptor binds to the sequence of each GRE as shown by footprinting experiments. However, only one of these sequences contains the TGTTCT consensus sequence reportedly associated with many other GREs.
Animals↗
Identification of regions in the rat serine dehydratase gene responsible for regulation by cyclic AMP alone and in the presence of glucocorticoids.
Transcription of the rat serine dehydratase (SDH) gene is induced by glucagon, mediated by the action of cAMP. To identify the nucleotide sequences in the SDH gene responsible for this regulation, we constructed chimeric genes containing different portions of the 5' flanking region of the rat SDH gene fused to the structural sequence encoding the bacterial reporter enzyme, chloramphenicol acetyltransferase (CAT). The transcriptional activities of the fusion genes introduced into the rat hepatoma cell line 7AD-7 were assayed by measuring CAT activity in the cell lysates. Chlorophenylthio-cyclic AMP (CPT-cAMP), a potent protein kinase A activating agent, stimulated the expression of SDH-CAT fusion genes, and these inductions could be enhanced further by the addition of dexamethasone, although the glucocorticoid alone had no effect on CAT activity. Deletion analysis demonstrated that an 80 bp region located approximately 3.5 kb upstream from the transcription initiation site of the rat SDH gene was responsible for stimulation of transcription by CPT-cAMP, whereas the 120 bp region immediately upstream of the cAMP responsive element (CRE)-containing sequences is essential for the enhancement of CPT-cAMP induction by the glucocorticoid.
Animals↗
Application of automated DNA sizing technology for genotyping microsatellite loci.
Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.
DNA, Satellite↗
Differential effects of the 3',5'-cyclic adenosine monophosphate and protein kinase C pathways on the response of isolated rat osteoclasts to calcitonin.
Calcitonin (CT) activates both the cAMP and the protein kinase C (PKC) pathways in the kidney cell line LLC-PK1. Although CT also activates cAMP in osteoclasts, its effects on PKC in this cell type are unknown. In order to determine whether the response of osteoclasts to CT also involves the PKC pathway, the effects of activators and inhibitors of PKC on bone resorption and cell surface area were analyzed in isolated rat osteoclasts. As expected, CT inhibited in a dose-dependent manner bone resorption by rat osteoclasts cultured for 24 h on devitalized bovine bone slices and this effect could be mimicked by cAMP. The inhibitory effect of CT could however also be mimicked by phorbol-12,13-dibutyrate (PDBu) and blocked by the PKC inhibitor sphingosine, as well as by the less specific inhibitors H7 and H8, none of which had detectable effects in the absence of CT. No changes in the number of attached osteoclasts were observed under any of these conditions. These results indicate that CT activates PKC in osteoclasts and that this activation, like the activation of cAMP-dependent protein kinase, leads to an inhibition of bone resorption. Quantitative time-lapse videomicroscopy showed that the CT-induced retraction of osteoclasts also involved activation of the PKC pathway and could therefore be induced by phorbol esters. In contrast, (Bu)2 cAMP (1-200 microM) failed to induce rapid cell retraction. It is concluded that, in osteoclasts, CT receptors are coupled to both the cAMP-dependent protein kinase and the PKC pathways. Although these two second messengers can have additive inhibitory effects on bone resorption, only activation of the PKC pathway induces rapid cell retraction. These two effects of calcitonin on osteoclasts are therefore independent and may be functionally unrelated.
8-Bromo Cyclic Adenosine Monophosphate↗
Atrial natriuretic factor and renin synthesized in cultured aortic smooth muscle cells of rats.
This study was designed to determine whether or not atrial natriuretic factor (ANF) is present in the vascular walls and to observe the differences in ANF between control (WKY) and stroke-prone spontaneously hypertensive rats (SHRsp). It was found that ANF is indeed present in the vascular wall of the distal aorta. HPLC analysis of the extracts from cultured aortic smooth muscle cells (ASMC) and medium revealed that intracellular ANF was mainly in the form of ANF(1-126), at levels of 0.82 +/- 0.03 (SHRsp) and 1.04 +/- 0.10 ng/10(6) cells (WKY), while the major form in the medium was ANF(99-126), at levels of 0.40 +/- 0.06 and 0.60 +/- 0.06 ng/10(6) cells, respectively. Both forms were present in smaller amounts in SHRsp than in WKY rats. On the contrary, both renin activity and angiotensin I concentrations in SHRsp cells were significantly higher than those in the WKY controls. In addition, immunocytochemistry showed positive ANF staining in cultured ASMC of both strains. The results suggest that ANF can be synthesized and secreted by cultured ASMC from rats.
Animals↗
Nutritional regulation and tissue-specific expression of the serine dehydratase gene in rat.
The mechanism of dietary regulation and tissue-specific expression of the serine dehydratase gene in rat has been studied. The hepatic serine dehydratase activity and its mRNA showed a parallel increase with increasing protein content in the diet. However, when rats that had been maintained on a high protein diet were fed a protein-free diet, the mRNA level rapidly decreased to 0.5 in 3 h, whereas the enzyme activity gradually fell to a low level over a period of 5 days. With animals maintained on a high protein diet or on a protein-free diet, we examined the sites hypersensitive to DNase I in the 5'-flanking region of serine dehydratase gene in the liver chromatins. A series of DNase I-hypersensitive sites were located within 10.5 kilobase pairs upstream of the transcription start site. The DNA regions at -3050 and -3180 (region II) and -3600 to -3850 (region III) were more susceptible to the nuclease in the expressing than in the nonexpressing liver. A reverse situation obtained at -100 (region I). Kidney contained serine dehydratase mRNA at a level of 5% of liver as determined by Northern blotting. The kidney chromatin was found to be susceptible to DNase I only at region I. No conspicuous DNase I-hypersensitive sites were observed in the relevant regions of chromatins from brain and lung, in which serine dehydratase mRNA was scarcely transcribed. These results suggest that nutritional control and tissue-specific expression of the serine dehydratase gene is closely associated with the alteration of DNase I hypersensitivity at specific sites of the 5'-flanking region of the gene.
Animals↗