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Biomedical subjects

H Yu

Publications and source records attributed to H Yu.

At least 631 records · Page 35Linked to original sources
PubMed1994

Characterization of the interaction of N-acyl-L-tryptophan benzyl ester neurokinin antagonists with the human neurokinin-1 receptor.

We have recently shown that a series of N-acyl-L-tryptophan benzyl esters are potent substance P antagonists (Macleod, A. M., Merchant, K. J., Cascieri, M. A., Sadowski, S., Ber, E., Swain, C. J., and Baker, R. (1993) J. Med Chem. 14, 2044-2045). We now report the detailed characterization of the interaction of N-acetyl-L-tryptophan-3,5-bistrifluoromethyl benzyl ester (L-732,138) with the human neurokinin-1 (NK-1) receptor. L-732,138 inhibits the binding of 125I-substance P to the cloned human NK1 receptor expressed in Chinese hamster ovary cells with an IC50 of 2.3 +/- 0.7 nM. In contrast, it has 200-fold lower affinity for the cloned rat NK-1 receptor and has > 1000-fold lower affinity for the human NK-2 and NK-3 receptors. L-732,138 acts as a competitive antagonist of substance P, as shown by functional Schild analysis of the inhibition of substance P-induced inositol phosphate synthesis, by kinetic analysis of the dissociation rate, and by thermodynamic analysis of the equilibrium binding of 125I-substance P to the NK-1 receptor. L-732,138 also competitively inhibits the binding of the quinuclidine amine antagonist, [125I]L-703,606, to the receptor. The compound has 230- and 10-fold reduced affinity for mutant NK-1 receptors in which histidine 265 or histidine 197, respectively, are replaced with alanine. We have previously shown that these residues play key roles in the binding of quinuclidine antagonists to the NK-1 receptor. These results suggest that the tryptophan and quinuclidine series of NK-1 antagonists bind to similar binding sites on the human NK-1 receptor.

Amino Acid Sequence↗
PubMed1994

Primary structure of avian H+/K(+)-ATPase beta-subunit.

A cDNA encoding a beta-subunit of the avian H+/K(+)-ATPase was cloned from a chicken stomach cDNA library, and its nucleotide sequence determined. A comparison between all the available sequence data for the beta-subunits of P-type ATPases reveals several evolutionarily conserved regions. Overall identity was 66% when compared with mammalian H+/K(+)-ATPase beta-subunits, 34% identity when compared with the Na+/K(+)-ATPase beta 2-subunits, and 33% identity when compared with the Na+/K(+)-ATPase beta 1-subunits.

Amino Acid Sequence↗
PubMed1994

The role of histidine 265 in antagonist binding to the neurokinin-1 receptor.

Previous studies suggested that the antagonist binding site in the neurokinin-1 receptor is composed of phylogenetically conserved residues, while phylogenetically divergent residues affect the conformation of the binding site. To test this hypothesis, we investigated the role of conserved residues in antagonist binding. Histidine 197 in the human receptor was found to interact with CP-96,345 but not RP67580. In addition, a nearby residue, histidine 265 of the human receptor, is required for the binding of RP67580 but not CP-96,345 or substance P. The interaction between residue 265 and RP67580 is consistent with a hydrogen bonding interaction. Analysis of several analogs of CP-96,345 revealed that histidine 265 of the human receptor is in proximity to the substituted benzyl moiety of CP-96,345, and can interact with other analogs of CP-96,345. In contrast to the human neurokinin-1 receptor, both histidine 197 and histidine 265 in the rat neurokinin-1 receptor appear to interact with both CP-96,345 and RP67580. These results support a conformational difference between the antagonist binding sites of the rat and human neurokinin-1 receptors and provide a model for examining specific interactions between antagonists and the receptor.

Amino Acid Sequence↗
PubMed1994

Ectopic production of prostate specific antigen by a breast tumor metastatic to the ovary.

We have recently reported that about 30% of breast tumors produce prostate specific antigen (PSA). We examined here, 99 primary ovarian cancer tumors and found relatively low levels of PSA in only three tumors. One patient with metastatic ovarian cancer from a primary breast tumor, produced relatively high levels of PSA and responded well to antiestrogen treatment although she was steroid receptor-negative. Another patient with metastatic ovarian cancer from a primary breast tumor did not produce PSA and did not respond to treatment although she was steroid hormone receptor-positive. This data describe for the first time, ectopic PSA production by a breast tumor at the ovarian metastatic site and further support the view that PSA is a favourable prognostic indicator in breast cancer.

Breast Neoplasms↗
PubMed1994

Identification of a lipid transfer protein as the major protein in the surface wax of broccoli (Brassica oleracea) leaves.

Presence of proteins in the surface wax of plants has been noted but no protein has been identified heretofore. A 9-kDa protein was purified as the major protein from the surface wax of broccoli (Brassica oleracea var. italica) leaves and the N-terminal amino acid sequence was determined. A cDNA corresponding to the wax-associated protein, designated WAX9, was sequenced revealing an open reading frame that codes for an 11.9-kDa polypeptide of 118 amino acids including a 25-amino acid leader peptide. The amino acid sequence showed 40 to 50% identity with nonspecific lipid transfer proteins isolated from various plants. Northern blots showed that a wax9 transcript of 650 nucleotides was highly expressed in leaves, stems, and flower buds and only at a reduced level in open flowers and not in roots. The transcript level decreased as the leaf fully expanded. The results suggest that the wax9 gene is expressed at the highest level at the time of maximal synthesis of the surface wax. Immunogold labeling studies showed that the WAX9 protein was present mainly in the cell wall of the epidermis and mesophyll tissues as well as in the phloem. However, immunological measurements showed that 50 and 4% of the total WAX9 was in the surface wax in the young and old leaves, respectively. Since immunogold labeling method would involve the loss of the surface wax, it would not detect the WAX9 protein in the wax. Thus, the present results reveal for the first time that a major portion of lipid transfer protein is in the surface wax.

Amino Acid Sequence↗
PubMed1994

Genetic analyses of temperature-sensitive mutations in baculovirus late expression factors.

Two temperature-sensitive mutants of Autographa californica nuclear polyhedrosis virus were characterized with respect to late and very late gene expression. Cells infected with ts317 or ts538 at the nonpermissive temperature revealed slightly reduced intracellular levels of late structural gene products including gp64 envelope, vp39 capsid, and p6.9 DNA binding proteins. However, with the exception of gp64, there was a dramatic reduction in extracellular levels of the structural proteins, suggesting that assembly or transport of extracellular virions was impaired. Electron microscopy confirmed the dramatic effect the mutations exhibited on viral morphogenesis. The ts538 mutation was physically mapped to the 60-65 mu region and DNA sequence analysis identified a single nucleotide alteration converting Leu to Phe in amino acid 105 of the late expression factor called lef-4. The mutation was positively correlated with the temperature-sensitive phenotype by analysis of DNA from ts+ revertants generated by marker rescue. Transient expression assays confirmed that lef-4 was required for activation of the late vp39 gene in virus infected cells. The defect in activation of the ts317 and ts538 polyhedrin genes could be overcome by coinfection with helper virus indicating that the mutations affected functions involved in activating the very late class of baculovirus genes as well. These results demonstrated a clear functional differentiation between genes regulating viral DNA replication which was normal in the mutant infected cells and those regulating late gene expression and extracellular virus production which were defective in these same infected cells.

Animals↗
PubMed1994

Induction of prostate specific antigen production by steroids and tamoxifen in breast cancer cell lines.

We demonstrate that the steroid hormone receptor-positive breast carcinoma cell lines T-47D and MCF-7 can be induced by androgens, progestins, mineralocorticosteroids, glucocorticosteroids, and antiestrogens, to produce prostate specific antigen (PSA). Estrogens failed to induce such stimulation in both cell lines and, in addition, were able to block the induction by androgens in the cell line T-47D. These data support and extend our previous report on PSA production by breast tumors and describe an in vitro system which can be used to study the phenomenon for possible application in prognosis and design of new therapy.

Blotting, Western↗
PubMed1994

Detection of prostate-specific antigen immunoreactivity in breast tumors.

Prostate-specific antigen (PSA) is a glycoprotein produced by the epithelial cells of the prostate. PSA is currently used in clinical practice to facilitate diagnosis and monitoring of prostate carcinoma. The prostate is an organ that possesses androgen, estrogen, and progesterone receptors, and in this respect is similar to the breast. We postulated that breast tumors might also have the ability to produce PSA. We performed these studies on a collection of 525 tumor specimens collected for routine biochemical determination of estrogen and progesterone receptors. Using a highly sensitive immunofluorometric procedure, we measured the p53 tumor suppressor gene product and PSA. Twenty nine percent of the breast tumor extracts contained detectable levels of PSA immunoreactivity (> 0.05 microgram/L). The immunoreactive PSA content was associated with estrogen and/or progesterone receptor-positive tumors (P < 0.002). No association was found between PSA immunoreactivity and levels of the p53 tumor suppressor gene product (P = 0.37). High performance liquid chromatography and Western blot analysis revealed that the PSA immunoreactivity in the tumor had a molecular weight of 30 kDa, similar to that of seminal PSA. Immunoreactive PSA-positive tumors were associated with younger women (P = 0.012) and earlier disease stage (P = 0.064). We postulate that PSA immunoreactivity may be an additional marker of steroid hormone receptor-ligand action.

Age Factors↗
PubMed1994

Quantitative analysis of mutant p53 protein in breast tumor cytosols and study of its association with other biochemical prognostic indicators in breast cancer.

Breast tumors are thought to originate, grow, and metastasize in an environment which includes steroid hormone receptors, their cognate steroid ligands, and many gene products which are regulated by steroid hormone receptor-ligand complexes. In this paper we describe highly sensitive and quantitative immunofluorometric procedures for measuring three proteins that are candidate prognostic indicators in breast cancer, namely, the p53 tumor suppressor gene product, carcinoembryonic antigen (CEA), and prostate specific antigen (PSA). These proteins were quantified in over 950 cytosolic tumor extracts along with estrogen and progesterone receptors (ER, PR). Association analysis between all five biochemical parameters revealed strong negative associations between p53 and receptors and strong positive associations between CEA and receptors. Negative associations between p53 and CEA and between CEA and PSA were also found. These associations, not quantitatively studied in previous reports, are related to each other using a hypothetical model. The observed associations may further contribute to the understanding of the biology of breast tumors.

Breast Neoplasms↗
PubMed1994

Immunoreactive prostate-specific antigen levels in female and male breast tumors and its association with steroid hormone receptors and patient age.

Prostate-specific antigen (PSA) is believed to be a highly specific marker for normal or cancerous prostatic tissue. We recently found that immunoreactive PSA (IR-PSA) is present in 30% of breast tumor cytosols (from 525 breast cancer patients). In this paper we analyzed a new series of 750 breast tumor cytosols, obtained from 744 women and six men, for IR-PSA. The positivity rates in the old and new series were very similar (approximately 30%). Combining the two series of breast cancer patients, we examined the associations between IR-PSA and estrogen (ER) or progesterone (PR) receptors, or patient age. We found that IR-PSA positivity rate declines with age. PSA-positive tumors were highly associated with either ER-positive or PR-positive tumors alone. However, analysis in a subset of tumors that combine the two receptors, ER(-)/PR(-), ER(+)/PR(-), ER(-)/PR(+), and ER(+)/PR(+), revealed that IR-PSA was only associated with PR, and no relationship was found between IR-PSA and ER. We speculate that the presence of IR-PSA in breast cancer may be associated with the PR action and that the association between PSA and ER is indirect due to the known association between ER and PR. As five of the six male breast tumors were found negative for IR-PSA, it is suggested that androgen may not be involved in the presence of IR-PSA in breast tumor.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1994

Identification of protein A-binding components in Spisula oocytes.

Components involved in sustaining meiosis arrest of oocytes were determined. Proteins that bind to protein A from meiosis-arrested and 5-HT-matured Spisula oocytes were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Meiosis-arrested oocytes contained three doublets of proteins with estimated Mrs of 43 and 45, 38 and 40, and 21 and 23 kDa. In 5 HT-matured oocytes the 21 and 23 and 38 and 40 kDa proteins were retained; whereas the 43 and 45 kDa proteins were absent. The protein A-bound proteins did not interact with antibodies against the various subclasses of human, mouse, rat and rabbit IgG or human Fc fragment. The amino acid sequence of the N-terminus of the 43 kDa protein was determined to be NH2-VLRIGSGMXDT. Comparison of this sequence with existing database at Protein Identification Resource (R 32.0), GenBank (R 72.0), SWISS-PROT (R 22.0), and EMBL (R 31.0) showed no homology with any reported protein. The protein A-bound components from meiosis-arrested oocytes were incubated in vitro with [gamma-32P]ATP. Only the 68 kDa protein was radiophosphorylated. This protein was not detected in 5-HT-matured oocytes. The disappearance of the 43, 45, and 68 kDa proteins in 5-HT-matured oocytes suggests that these components may be involved in sustaining meiosis meiosis. A unique property of these proteins is that they interact with protein A and are distinctly different from immunoglobulin.

Adenosine Triphosphate↗
PubMed1994

Mechanisms of ATP-induced Ca2+ signaling in osteoclasts.

We investigate the mechanisms underlying the intracellular calcium pulse that occurs in response to extracellular adenosine triphosphate (ATP) in osteoclasts. We find that pre-loading of GDP-beta-S abolishes the response in Ca(2+)-free medium, demonstrating an internal release of Ca2+ via a pathway that involves a G protein. GDP-beta-S does not block in normal Ca(2+)-containing medium, suggesting that ATP also induces a Ca2+ influx across the cell membrane. We confirmed this using the Mn2+ quenching technique, which shows significant opening of Ca2+ channels. We find a smaller response to adenosine diphosphate (ADP) and 2-methylthio-ATP (2-MeSATP), but no response to beta, gamma-methylene-ATP (AMP-PCP), adenosine monophosphate (AMP) or uridine triphosphate (UTP). Prior application of AMP and UTP, but not AMP-PCP, blocks the response to ATP. Our results indicate that the receptor is a P2 subtype that is not characteristic of any previously reported P2 receptor or combination of P2 receptors.

Adenosine Diphosphate↗
PubMed1994

Antihypertensive effects of the neutral endopeptidase inhibitor SCH 42495 in essential hypertension.

The antihypertensive effects and safety of a novel neutral endopeptidase inhibitor, SCH 42495, were investigated in hypertensive patients. A multicenter, open clinical trial was conducted in 27 patients with essential hypertension, WHO Stage I or II. Mean age was 64 +/- 1 years. After 2 to 4 weeks of a placebo run-in, 50 mg twice daily, was started, with the dose increased to 100 mg twice daily, and 200 mg twice daily, every 2 weeks, if necessary, to achieve a predetermined response. Blood pressure and pulse rate were monitored every 2 weeks. Blood chemistry, plasma atrial natriuretic peptide (ANP), and plasma cGMP levels were determined before and after the 8-week treatment period. Blood pressure was significantly reduced, from 171 +/- 1/100 +/- 1 mm Hg to 146 +/- 3/84 +/- 2 mmHg (P < .001) at the end of the 8-week treatment period. No change in pulse rate was noted. Efficacy rate was evaluated in 25 patients treated for 4 weeks or more. Efficacy rate was 44% with 50 mg twice daily, 60% with 100 mg twice daily, and 80% with 200 mg twice daily. Adverse reactions such as headaches and palpitation were observed in six patients (22.2%), with treatment discontinued in five. Significant correlation was observed between increment in plasma ANP levels and blood pressure reductions (r = -0.53, P < .05). Increase in plasma cGMP was positively correlated with increments in plasma hANP (r = 0.80, P < .001). SCH 42495 has potent antihypertensive effect associated with an enhancement of endogenous hANP and may be clinically useful as a new class of antihypertensive drug.

Aged↗
PubMed1994

Germline RET mutations in MEN 2A and FMTC and their detection by simple DNA diagnostic tests.

Multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC) are two closely related cancer syndromes inherited in an autosomal dominant manner. Mutations in the RET proto-oncogene were found in MEN 2A and FMTC families. In this study we report seven different germline mutations in the RET proto-oncogene in five of five MEN 2A and five of six FMTC families. Each of the mutations involves a cysteine residue in the extracellular cysteine-rich domain of the RET receptor tyrosine kinase. We developed simple polymerase chain reaction based diagnostic tests for all seven mutations in these families.

Base Sequence↗
PubMed1994

Possible involvement of the lysine epsilon-aminotransferase gene (lat) in the expression of the genes encoding ACV synthetase (pcbAB) and isopenicillin N synthase (pcbC) in Streptomyces clavuligerus.

Streptomyces clavuligerus produces the beta-lactam antibiotics penicillin N, O-carbamoyldeacetylcephalosporin C and cephamycin C. We characterized a wild-type DNA region which restores antibiotic formation to a mutant strain named NP1, previously shown to exhibit depressed activities for two early enzymes of cephalosporin synthesis, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) and isopenicillin N synthase (IPNS). L-Lysine epsilon-aminotransferase (LAT) assays and alpha-AAA feeding experiments suggested that strain NP1 is a lat mutant. NP1 recovered LAT, ACVS and IPNS activities when transformed with the cloned region. DNA sequencing showed that this region encodes the entire LAT gene (lat), required for the conversion of L-lysine to the beta-lactam precursor L-alpha-aminoadipic acid (alpha-AAA), as well as the upstream half of the ACVS gene (pcbAB). The activities of ACVS and IPNS appear to depend upon LAT expression. Gene fusions constructed to investigate promoter activities in the cloned region support a model of interdependence in the expression of the genes for LAT, ACVS and IPNS (pcbC).

Amino Acid Sequence↗
PubMed1994

DNA polymerase III of Mycoplasma pulmonis: isolation and characterization of the enzyme and its structural gene, polC.

Mycoplasmas have originated from Gram-positive bacteria via rapid degenerative evolution. The results of previous investigations of mycoplasmal DNA polymerases suggest that the process of evolution has wrought a major simplification of the typical Gram-positive bacterial DNA polymerase profile, reducing it from three exonuclease (exo)-positive enzymes to a single exo-negative species. The objective of this work was to rigorously investigate this suggestion, focusing on the evolutionary fate of DNA polymerase III (Pol III), the enzyme which Gram-positive bacteria specifically require for replicative DNA synthesis. The approach used Mycoplasma pulmonis as the model organism and exploited structural gene cloning, enzymology, and Pol III-specific inhibitors of the HPUra class as investigative tools. Our results indicate that M. pulmonis has strongly conserved a single copy of a structural gene homologous to polC, the Gram-positive bacterial gene encoding Pol III. M. pulmonis was found to possess a DNA polymerase that displays the size, primary structure, exonuclease activity, and level of HPUra sensitivity expected of a prototypical Gram-positive Pol III. The high level of sensitivity of M. pulmonis growth to Gram-positive Pol III-selective inhibitors of the HPUra type strongly suggests that Mycoplasma has conserved not only the basic structure of Pol III, but also its essential replicative function. Evidence for a second, HPUra-resistant polymerase activity in M. pulmonis is also described, indicating that the DNA polymerase composition of Mycoplasma is complex and closer to that of Gram-positive bacteria than previously thought.

Amino Acid Sequence↗
PubMed1994

Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis.

An invertible DNA element of 6.8 kb, designated the hsd1 locus, was identified in the chromosome of Mycoplasma pulmonis. Infection of host cells with mycoplasma virus P1 revealed that the organism's restriction and modification (R-M) properties are controlled by inversion of hsd1. The nucleotide sequence of hsd1 revealed several genes, the predicted amino acids of which bear striking similarity to the subunits of the type I R-M enzymes previously found only in enteric bacteria.

Amino Acid Sequence↗