Biomedical subjects

W V Everson

Publications and source records attributed to W V Everson.

7 recordsLinked to original sources

PubMed1992

Changes in annexin (lipocortin) content in human amnion and chorion at parturition.

Arachidonic acid is mobilized from fetal membrane phospholipids at parturition leading to increased production of oxytocic prostaglandins which may initiate or maintain myometrial contractions. Phospholipid mobilization requires activation of phospholipase A2 or C, both of which require calcium for activity. The annexins (lipocortins) are a superfamily of proteins which bind to calcium and phospholipids and thereby may alter phospholipase activity through two mechanisms: modulation of intracellular free Ca2+ concentrations or regulation of the accessibility of phospholipids to hydrolyzing enzymes. Using Western immunoblotting with monospecific polyclonal antibodies, annexins I-VI were identified in human amnion and chorion/decidua at term in tissues obtained from patients in labor or not in labor. Each annexin was present in two distinct pools: a pool which only associated with the membrane in the presence of calcium (calcium-dependent pool) and a calcium-independent pool that remained membrane bound in the presence of calcium chelators. Annexin I was present as two species, resolving at 36 kDa and 68 kDa. The total concentration of annexin I in both amnion and chorion/decidua was significantly decreased with labor, while the total concentration of annexin V in chorion significantly increased with labor. The size of individual pools of annexins also changed with labor: the calcium-dependent pool of annexins I and II in both amnion and chorion significantly decreased; the calcium-dependent pool of annexin V increased in chorion; and calcium-independent pools of annexin I in amnion and annexins I, II, and V in chorion significantly decreased with labor. The decrease in total annexin I concentration with labor in amnion reflects a substantial decrease (80-90%) in the pool tightly bound to the membrane in a calcium-independent manner. This striking change distinguishes annexin I as a potential candidate inhibitor which is specifically downregulated at parturition, potentially leading to increased access of phospholipases to substrate phospholipids and increased prostaglandin production at labor.

Amnion↗

PubMed1992

Decrease in annexin I messenger ribonucleic acid expression in human amnion with labor.

OBJECTIVE: Annexins are a superfamily of proteins that are thought to inhibit phospholipase A2 activity and hence inhibit prostaglandin production. The purpose of this study was to test the hypothesis that annexin I concentration in human amnion is reduced with labor and that this reduction is mediated by a decrease in annexin I messenger ribonucleic acid expression. STUDY DESIGN: Amnion and choriodecidua were collected from term singleton pregnancies, eight after spontaneous vaginal delivery and eight from elective cesarean section without labor. Annexin I protein was quantitated by Western blotting. Ribonucleic acid was isolated from amnion, and then annexin I messenger ribonucleic acid was identified by Northern hybridization and quantitated by slot blotting. RESULTS: Annexin I (35 kd) was identified in amnion tissue. The concentration in the group undergoing labor (320 +/- 45 integrated optical density units, mean +/- SE) was significantly reduced (p < 0.05) compared with that in the group not undergoing labor (635 +/- 65 units). The size of the annexin I messenger ribonucleic acid was approximately 1.8 kb. The mean integrated optical density for the labor group (840 +/- 139 units, mean +/- SE) was significantly reduced (p < 0.05) compared with that of the nonlabor group (1912 +/- 464 units). CONCLUSION: There is a significant decrease in annexin I messenger ribonucleic acid expression in human amnion with labor, corresponding to a significant decrease in annexin I protein concentration. This may contribute to the increased phospholipase A2 activity, arachidonic acid mobilization, and prostaglandin production at labor in humans.

Amnion↗

PubMed1989

Effect of amino acid deprivation on initiation of protein synthesis in rat hepatocytes.

Conditions were defined for maintaining optimal protein synthetic activity in suspensions of freshly isolated rat hepatocytes. Under these conditions, isolated hepatocytes exhibited rates of protein synthesis and levels of polysomal aggregation equivalent to those observed in vivo and in perfused liver. Deprivation of total amino acids or single, essential amino acids resulted in a rapid decrease in the rate of protein synthesis, which was readily reversed by readdition of the deficient amino acid(s). The decrease was accompanied by a disaggregation of polysomes and an inhibition of 43S initiation complex formation, which was indicative of a limitation in the rate of initiation of protein synthesis. Extracts prepared from perfused liver deprived of amino acids were inhibitory to initiation of protein synthesis in reticulocyte lysate. The inhibition in reticulocyte lysate was accompanied by an increase in phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2), suggesting activation of an eIF-2 alpha kinase or inhibition of a phosphatase in amino acid-deprived hepatocytes. This suggestion was confirmed by prelabeling hepatocytes with 32Pi before amino acid deprivation. Incorporation of 32Pi into eIF-2 alpha was two- to threefold higher in lysine-deprived cells than in hepatocytes incubated in fully supplemented medium. Overall, the results indicated that an increase in eIF-2 alpha phosphorylation was responsible for the defect in initiation of protein synthesis caused by amino acid deprivation.

Adenosine Triphosphate↗

PubMed1989

Initiation of protein synthesis in a cell-free system prepared from rat hepatocytes.

A cell-free system, which maintained a linear rate of protein synthesis for up to 20 min of incubation, was prepared from isolated rat hepatocytes. The rate of protein synthesis in the cell-free system was approximately 20% of the rate obtained in isolated hepatocytes or perfused liver. More than 70% of total protein synthesis in the cell-free system was due to reinitiation, as indicated by addition of inhibitors of initiation, i.e., edeine or polyvinyl sulfate. The rate of protein synthesis and formation of 43S initiation complexes in the cell-free system were reduced to 60 and 30% of the control values, respectively, after incubation of hepatocytes in medium deprived of an essential amino acid. Therefore, the cell-free system maintained the defect in initiation induced in the intact cells by amino acid deprivation. The defect in initiation was corrected by addition of either rat liver eukaryotic initiation factor 2 or the guanine nucleotide exchange factor (GEF) to the cell-free system. A role for GEF in the defect in initiation was further implicated by experiments that showed that the activity of the factor was decreased in extracts from livers perfused with medium deficient in amino acids. The cell-free system should provide a valuable tool for investigation of mechanisms involved in the regulation of initiation of protein synthesis.

Adenosine Triphosphate↗

PubMed1988

Structure of the gene encoding the beta-subunit of chicken prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) converts peptidyl proline to peptidyl hydroxyproline in procollagen polypeptides during collagen biosynthesis. The active enzyme is a tetramer which is composed of two pairs of non-identical subunits in a molecular form of alpha 2 beta 2. In addition to the tetrameric prolyl 4-hydroxylase (alpha 2 beta 2), the free beta-subunit is also found inside cells. Recently it was shown that the beta-subunit of prolyl 4-hydroxylase is identical to the protein disulfide isomerase and cellular thyroid hormone-binding protein. We previously isolated and characterized cDNAs of the beta-subunit of chicken prolyl 4-hydroxylase. The cDNA of beta-subunit was used to screen a chicken genomic DNA library constructed with the lambda EMBL-3 vector. Two clones, lambda gCPH beta-22 and beta-50, were isolated and characterized by restriction enzyme analysis, heteroduplex analysis, and nucleotide sequencing. The results showed that the 2.5-kb mRNA of the beta-subunit is divided into eleven exons and that the gene is 9.0 kb long. The gene contains consensus sequence for TATA at -24 bp and four CAAT at -57, -157, -194 and -223 bp in the 5' end flanking sequence of the transcription start point. In addition, there are three GC boxes upstream from the TATA box and four GC boxes in the first intron. This is similar to the structure of the alpha 1(I) collagen coding gene (COL1A1). These elements may interact with nuclear factors and play important roles in expression regulation of the beta-subunit gene as has been described in COL1A1.

Amino Acid Sequence↗

PubMed1988

Isolation of cDNA clones and genomic DNA clones of beta-subunit of chicken prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis. The active enzyme is a tetramer composed of two pairs of non-identical subunits, alpha and beta. Sheep antiserum directed against chicken proly 4-hydroxylase was initially used to screen two cDNA expression libraries. The cDNA was prepared from chicken smooth muscle mRNA and cloned into the plasmids pUC8- and pUC9. Antibodies identified twenty-five clones among the approximately 2 x 10(5) clones in the libraries. Ten clones were isolated pure and used in the subsequent analysis. Monospecific antibodies directed against beta subunit of the enzyme were used in Western-blot analyses of extracts of bacteria carrying the cDNA clones. The results indicated that the clone CPH 9-10B encodes a portion of beta-subunit. The cDNA from CPH 9-10B was used to screen another cDNA library prepared from mRNA from chicken skeletal muscle. Several overlapping clones were isolated. Together the cDNAs correspond to 2.4 kb which is the same as the corresponding mRNA. Three regions of the amino acid sequence deduced from the cDNA sequence matched with that of the NH2-terminus of beta-subunit and two CNBr peptides derived from beta-subunit. The cDNA of CPH 9-10B was also used to screen a genomic DNA library constructed with lambda EMBL-3. Two overlapping genomic clones lambda gCPH beta-22 and beta-50 were isolated and characterized by restriction enzyme analysis. The results indicate that lambda gCPH beta-22 contains the portion of the beta-subunit gene that is transcribed into the 5' portion of beta-subunit mRNA, whereas lambda gCPH beta-50 contains the 3' portion.

Amino Acid Sequence↗

PubMed1987

Purification and characterization of eukaryotic initiation factor 2 and a guanine nucleotide exchange factor from rat liver.

Two polypeptide chain initiation factors, eukaryotic initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF), were isolated from rat liver. Two forms of eIF-2 were identified, one contained three subunits (alpha, beta, and gamma), and the other contained only the alpha- and gamma-subunits. The three-subunit form was similar to eIF-2 from rabbit reticulocytes with respect to the sedimentation coefficient, Stokes radius, molecular weight of the alpha- and gamma-subunits, ability to restore protein synthesis in hemin-deficient reticulocyte lysate, and immunological cross-reactivity of the alpha-subunits using antibodies against liver eIF-2. In contrast, the beta-subunits of the liver and reticulocyte factors were distinct; they had different molecular weights, and antibodies against rat liver eIF-2 beta did not recognize the beta-subunit of the reticulocyte factor. Furthermore, the GDP dissociation constant for reticulocyte eIF-2 was more than twice that of the liver factor. GEF from rat liver reversed GDP inhibition of the ternary complex assay and catalyzed the exchange of eIF-2-bound GDP for free GDP or GTP, characteristics ascribed to the corresponding protein from rabbit reticulocytes. However, its subunit composition and molecular weight were different from those reported for reticulocyte GEF. The T1/2 for GDP exchange mediated by GEF was about 5-fold slower with two-subunit than with three-subunit eIF-2. In addition, the KD for GDP was lower for two-subunit than for three-subunit eIF-2 when GEF was present. Taken together, these data demonstrate species-associated variability in the beta-subunit of eIF-2 and suggest a crucial role for the beta-subunit in the functional interaction of eIF-2 and GEF.

Animals↗