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R Wu

Publications and source records attributed to R Wu.

At least 289 records · Page 16Linked to original sources
PubMed1993

High-level production of the mouse epidermal growth factor in a Bacillus brevis expression system.

In order to determine the three-dimensional structure, the folding pathways, and the residues which are critical for biological functions of the mouse epidermal growth factor (mEGF), large amounts of wild-type and site-specific mutants are needed for biological and physiochemical studies. Genes coding for mEGF and its mutants were expressed in a Bacillus brevis system in which the expressed foreign proteins were secreted into the culture medium. However, proteases were also secreted that resulted in partial degradation of the desired foreign proteins. One mutant with much less protease secretion was isolated by applying nitrosoguanidine mutagenesis on a B. brevis strain. A new vector to facilitate DNA mutagenesis and sequencing was also constructed for this system. Methodologies for the transformation of B. brevis and the purification of expressed mEGF were developed. Under the optimized conditions, the production of the mEGF and its mutants was about 50 mg per liter, and yielded up to 10 mg of purified mEGF. Several mEGF mutants have been produced and their receptor binding abilities have been measured.

Amino Acid Sequence↗
PubMed1993

A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein.

A rice (Oryza sativa L.) cDNA clone coding for the cytoplasmic ribosomal protein L5, which associates with 5 S rRNA for ribosome assembly, was cloned and its nucleotide sequence was determined. The primary structure of rice L5, deduced from the nucleotide sequence, contains 294 amino acids and has intriguing features some of which are also conserved in other eucaryotic homologues. These include: four clusters of basic amino acids, one of which may serve as a nucleolar localization signal; three repeated amino acid sequences; the conservation of glycine residues. This protein was identified as the nuclear-encoded cytoplasmic ribosomal protein L5 of rice by sequence similarity to other eucaryotic ribosomal 5 S RNA-binding proteins of rat, chicken, Xenopus laevis, and Saccharomyces cerevisiae. Rice L5 shares 51 to 62% amino acid sequence identity with the homologues. A group of ribosomal proteins from archaebacteria including Methanococcus vanniellii L18 and Halobacterium cutirubrum L13, which are known to be associated with 5 S rRNA, also related to rice L5 and the other eucaryotic counterparts, suggesting an evolutionary relationship in these ribosomal 5 S RNA-binding proteins.

Amino Acid Sequence↗
PubMed1993

Systemic induction of a potato pin2 promoter by wounding, methyl jasmonate, and abscisic acid in transgenic rice plants.

To address the question whether common signal(s) and transduction pathways are used to mediate a systemic wound response in monocot and dicot plants, a fusion of the potato proteinase inhibitor II gene (pin2) promoter and the bacterial beta-glucuronidase gene (Gus)-coding region was introduced into rice. In transgenic rice plants, the expression of the pin2-Gus fusion gene displays a systemic wound response, although the expression level is relatively low. Incorporation of the first intron from the rice actin 1 gene (Act1) into the 5'-untranslated region of the pin2-Gus construct results in high-level, systemically wound-inducible expression of the modified construct in transgenic rice plants. Histochemical analysis shows that this high-level, wound-inducible expression is associated with the vascular tissue in both leaves and roots. Furthermore, the expression of the pin2-Act1 intron-Gus fusion gene in transgenic rice plants can be systemically induced by both methyl jasmonate (MJ) and the phytohormone abscisic acid (ABA). These results suggest that the signal(s) mediating the observed systemic wound response and certain steps of the transduction pathways are conserved between dicot and monocot plants. Transient expression assays show that the pin2-Act1 intron-Gus construct is also actively expressed in transformed cells and tissues of several other monocot plants. Thus, the wound-inducible pin2 promoter in combination with the rice Act1 intron 1 might be used as an efficient regulator for foreign gene expression in transgenic monocot plants.

Abscisic Acid↗
PubMed1993

Mononuclear leukocytes exposed to oxidized low density lipoprotein secrete a factor that stimulates endothelial cells to express adhesion molecules.

In animals fed a hypercholesterolemic diet, development of atherosclerosis is preceded by attachment of mononuclear leukocytes to the arterial endothelium. Early lesions begin to develop as monocytes migrate into the intima and ingest lipids. A major part of these lipids is believed to be derived from oxidatively modified low density lipoprotein (LDL). In the present study we demonstrate that human mononuclear leukocytes exposed to low concentrations of copper-oxidized LDL secrete one or several factors that stimulate the expression of intercellular adhesion molecule-1 (ICAM-1, CD54), vascular cell adhesion molecule (VCAM-1) and endothelial selectin (E-selectin-1, ELAM-1), whereas native LDL was found to be without effect. Exposure of endothelial cells to non-conditioned medium containing oxidized LDL did not influence the expression of adhesion molecules. Incubation of endothelial cells with conditioned medium from mononuclear cells grown in the presence of oxidized LDL also resulted in a three-fold increase in the binding of monocytoid U937 cells. The present findings suggest that mononuclear leukocytes exposed to oxidatively modified LDL in early atherosclerotic lesions may stimulate the recruitment of other leukocytes by secreting cytokines which induce the expression of adhesion molecules on the endothelium.

Cell Adhesion↗
PubMed1993

High-performance liquid chromatographic method for the determination of norfloxacin glutamate and glucuronate in solid and liquid dosage forms and its application to stability testing.

A simple, precise, stability-indicating reversed-phase high-performance liquid chromatographic method for norfloxacin glutamate and norfloxacin glucuronate in liquid and solid dosage forms is described. Chloronitrodiazepine was used as the internal standard. The eluent used with a C18 bonded phase column was methanol-water-diethylamine (50:50:0.4, v/v/v) (pH* 5.5). The effects of the eluent pH*, the ratio of methanol to water, and the quantity of diethylamine on the retention times of the sample and internal standard were investigated. The method showed good linearity in the range 1-45 micrograms ml-1 for norfloxacin. Solid samples were ground, dissolved in the eluent, filtered, and then determined by this method. Liquid samples were dissolved in the same solvent. The average recoveries of norfloxacin glutamate and norfloxacin glucuronate in their simulated preparations were 99.5% for solid products and 99.8% for liquid products. The method was applied to the study of the thermal stability of the drugs by following the degradation of norfloxacin glutamate and glucuronate in the four products in accelerated tests at 37-80 degrees C for up to 3 months. Shelf-lives at 25 degrees C of the four products were predicted from the results assuming zero- and first-order kinetics of decomposition, and were at least 1.5 years for liquid products and 2 years for solid products.

Chemistry, Pharmaceutical↗
PubMed1993

Light-regulated and cell-specific expression of tomato rbcS-gusA and rice rbcS-gusA fusion genes in transgenic rice.

A previously isolated rice (Oryza sativa) rbcS gene was further characterized. This analysis revealed specific sequences in the 5' regulatory region of the rice rbcS gene that are conserved in rbcS genes of other monocotyledonous species. In transgenic rice plants, we examined the expression of the beta-glucuronidase (gusA) reporter gene directed by the 2.8-kb promoter region of the rice rbcS gene. To examine differences in the regulation of monocotyledonous and dicotyledonous rbcS promoters, the activity of a tomato rbcS promoter was also investigated in transgenic rice plants. Our results indicated that both rice and tomato rbcS promoters confer mesophyll-specific expression of the gusA reporter gene in transgenic rice plants and that this expression is induced by light. However, the expression level of the rice rbcS-gusA gene was higher than that of the tomato rbcS-gusA gene, suggesting the presence of quantitative differences in the activity of these particular monocotyledonous and dicotyledonous rbcS promoters in transgenic rice. Histochemical analysis of rbcS-gusA gene expression showed that the observed light induction was only found in mesophyll cells. Furthermore, it was demonstrated that the light regulation of rice rbcS-gusA gene expression was primarily at the level of mRNA accumulation. We show that the rice rbcS gene promoter should be useful for expression of agronomically important genes for genetic engineering of monocotyledonous species.

Amino Acid Sequence↗
PubMed1993

Suppression of replication-deficient mutants of IncFII plasmid NR1 can occur by two different mechanisms that increase expression of the repA1 gene.

Replication-proficient (Rep+) revertants were isolated from mutants of IncFII plasmid NR1 that were replication defective (Rep-). The parental Rep- plasmids contained a mutation that inactivated promoter PE for transcription of RNA-E, a trans-acting repressor of translation of the essential RepA1 replication initiation protein of NR1. The PE mutation also introduced a nonsense codon into a leader peptide gene that precedes and slightly overlaps the repA1 translation initiation site in the mRNA. This reduced the rate of synthesis of RepA1 by uncoupling its translation from that of the leader peptide. The reduced rate of RepA1 synthesis was responsible for the Rep- phenotype. All Rep+ revertants retained the PE mutation and contained second-site mutations responsible for suppression of the Rep- phenotype. One Rep+ revertant contained a second mutation adjacent to the Shine-Dalgarno sequence of repA1. Another Rep+ revertant contained a mutation in the repA2 gene, which encodes the trans-acting repressor of transcription of repA1. By using translational lacZ gene fusions, it was found that both kinds of suppressor mutation increased the expression of repA1 to a level sufficient to support replication. In both cases, the synthesis of RepA1 remained uncoupled from that of the leader peptide. The Shine-Dalgarno mutation increased the rate of leader peptide-independent translation of repA1 mRNA and also reduced the sensitivity of repA1 mRNA to inhibition by RNA-E. The repA2 mutation inactivated the RepA2 repressor and increased the rate of transcription of repA1 mRNA. The translational lacZ gene fusions were used to assess the range of regulation of expression of repA1 provided by each of the RNA-E and RepA2 regulatory circuits. By constructing miniplasmids that contained various combinations of the mutations, the contributions of the RNA-E and RepA2 regulatory circuits were assessed with respect to control of plasmid copy number and stable inheritance. Plasmids that lacked either circuit were less stable than wild-type plasmids.

Amino Acid Sequence↗
PubMed1993

Elevated levels of serum mucin-associated antigen in adult patients with cystic fibrosis.

Previous studies using the CA 19-9 antibody have demonstrated that serum mucin levels in patients with cystic fibrosis (CF) are elevated and that the degree of elevation relates to the age of the patient and possibly to his or her clinical status. However, CA 19-9 only recognizes the mucin-associated blood group sialyl Le(a+) antigen, so mucin levels cannot be measured in patients without Lewis antigens. The present study used the 17B1 monoclonal antibody to measure serum mucin levels in normal subjects, and in patients with CF, patients with chronic obstructive pulmonary disease (COPD), and patients with lung transplants. Serum mucin levels were 25 ng/ml (+/- 1 SEM, n = 8) in normal subjects, 13,853 ng/ml (+/- 1,281, n = 25) in patients with CF, and 25.5 ng/ml (+/- 1.9, n = 17) in patients with COPD. Patients with CF who were sialyl Le(a-b-) also had elevated serum mucin levels (715 +/- 152, n = 2). Serum mucin levels of six lung transplant recipients with CF were elevated compared with those in normal subjects (4,621 +/- 765 ng/ml), but they were not different from serum mucin levels in six lung transplant recipients without CF (5,307 +/- 1.677 ng/ml). Preliminary characterization of the serum mucin antigen showed that: (1) in CF sera, the antigen is polydisperse and smaller than the antigen in normal sera; (2) the mucin antigen is distinct from ABO blood group antigens. Serum mucin levels may be a useful marker to follow a specific patient's response to therapy.

ABO Blood-Group System↗
PubMed1993

A small proline-rich protein regulated by vitamin A in tracheal epithelial cells is induced in lung tumors.

In cell-free translations of RNA from primary cultures of pig trachea surface epithelial cells, we observed that a 20 kD proline-rich protein (sPRP) is induced during culturing (Biochem. Biophys. Res. Commun. 1990; 172:1304-1309). Subsequently, a cDNA encoding sPRP has been cloned from pig tracheal cell mRNA and sequenced. This cDNA shows a high similarity to cDNAs cloned from monkey tracheal cells cultured in vitamin A-free medium and from UV-irradiated human epidermal keratinocytes. Amino acid sequences from these cDNAs are exceptionally rich in proline, glutamine, cysteine, and lysine but contain no aromatic amino acids. Two repeats of 12 amino acids on the N-terminus are followed by multiple 8 amino acid repeats. When compared with monkey trachea and human keratinocyte cDNAs, the sPRP cDNA from pig trachea has an additional 24 bp nucleotide repeat. Antiserum raised to a synthetic peptide (23 amino acids) on the C-terminus of sPRP (C23-antiserum) reacted with the 20 kD sPRP in immunoprecipitations from cell-free translations. On Northern blot analysis, sPRP cDNA hybridized to RNAs of similar sizes in tracheal cells from cat, rabbit, and lamb. sPRP was not detected in tracheal cells that were cultured with 10(-9) M arotinoid. Since sPRP is considered a putative squamous cell differentiation marker, experiments using lung tumors were performed. sPRP mRNA levels were dramatically increased in squamous lung tumors that were induced by injecting hamsters with 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone, a tobacco-specific nitrosamine. In situ hybridization with tissue sections prepared from these lung tumors revealed that cells around the keratin pearls contained high levels of sPRP mRNA.

Amino Acid Sequence↗
PubMed1993

Fractional analysis of the 120-ml bronchoalveolar lavage. Determination of the best specimen for diagnosis of sarcoidosis.

STUDY DESIGN: Bronchoalveolar lavage with seven 20-ml aliquots was performed on 9 patients with sarcoidosis and 11 patients with idiopathic pulmonary fibrosis (IPF). The returns from the first 20-ml aliquot (volume 1), the next 100 ml of bronchoalveolar lavage fluid (volume 2), and the last 20 ml of lavage fluid (volume 3) were analyzed separately for cell content and differential cell counts. The cell content was calculated four different ways, including absolute number of cells per milliliter of return and as a differential cell count using three different denominators. The denominators used were (1) neutrophils, lymphocytes, eosinophils, macrophages, and epithelial cells; (2) neutrophils, lymphocytes, eosinophils, and macrophages; and (3) neutrophils, lymphocytes and eosinophils. The data were analyzed to see what volume and arithmetic expression of cell content provided the best means of distinguishing sarcoidosis from IPF. RESULTS: The results confirmed a proximal distribution of the first 20-ml aliquot with more bronchial epithelial cells and neutrophils than volumes 2 and 3. There were more macrophages and lymphocytes in volumes 2 and 3 than in volume 1. Volumes 2 and 3 had similar cell content. Sequential fractional analysis showed that the cells in volume 2 and 3 more clearly distinguished sarcoidosis from IPF than the cell content of volume 1. CONCLUSION: The presence of lymphocytes, and especially a predominance of lymphocytes with the relative absence of neutrophils, correlated best with sarcoidosis. The percentages of differential cell counts proved superior to both the absolute number of neutrophils and of lymphocytes per milliliter of return in distinguishing sarcoidosis from IPF. A percentage differential cell count with a denominator of neutrophils+lymphocytes+eosinophils provided the best means of distinguishing between sarcoidosis and IPF.

Aged↗
PubMed1993

Bronchoalveolar lavage cell populations in the diagnosis of sarcoidosis.

STUDY DESIGN: Between February 1, 1984, and February 1, 1989, fiberoptic bronchoscopy was performed on 2,692 patients, 592 of whom had bronchoalveolar lavage (BAL). One hundred twenty-eight patients with 16 percent or more lymphocytes in BAL fluid (BALF) were selected for further study. The group included 27 patients with sarcoidosis, 28 with nonsarcoidosis interstitial lung disease (ILD), 22 with lung infection (organism isolated), 31 with inflammation (presumed infection, no organism isolated), 14 with neoplasm, and 6 with bronchial hyperreactivity. METHODS: The percentages of lymphocytes, B lymphocytes, and T lymphocytes, the CD4/CD8 ratio and the percentages of neutrophils and eosinophils were analyzed individually and in combination for discrimination between the sarcoidosis and nonsarcoidosis patients and compared with the diagnostic accuracy of multiple noncaseating granuloma (MNG) on a simultaneous transbronchial biopsy (Tbbx). RESULTS: Neither the percentages of lymphocytes, T lymphocytes, or B lymphocytes discriminated sarcoidosis from nonsarcoidosis patients. Sarcoidosis patients had higher CD4/CD8 ratios, fewer neutrophils, and 1 percent or less eosinophils in the BAL cell populations. An analysis of CD4/CD8 ratios, and percentages of neutrophils and eosinophils individually revealed that a CD4/CD8 ratio of 4:1 or greater had a positive predictive value of 94 percent in distinguishing sarcoidosis from other ILD but a sensitivity of only 59 percent. The positive predictive value of CD4/CD8 ratio of 4:1 or greater fell to 50 percent in separating sarcoidosis from all other diseases. A CD4/CD8 ratio of less than 1:1 has a 100 percent negative predictive value to exclude the diagnosis of sarcoidosis. Finding 1 percent or less neutrophils in BAL had an 80 percent positive predictive value in distinguishing sarcoidosis from nonsarcoidosis ILD and 51 percent for distinguishing sarcoidosis from all other disease groups. The CD4/CD8 ratio and the percentages of neutrophils and eosinophils also were combined and analyzed for the diagnosis of sarcoidosis. CONCLUSIONS: Results showed a BALF with a CD4/CD8 ratio of 2:1 or greater, 1 percent or less neutrophils, and 1 percent or less eosinophils has essentially the same specificity and positive predictive value as MNG on Tbbx in distinguishing sarcoidosis from nonsarcoidosis disease. The combination of finding MNG in a Tbbx specimen plus a BALF CD4/CD8 ratio of 4:1 or greater had a 100 percent positive predictive value in separating sarcoidosis from other ILD and an 81 percent value in separating sarcoidosis from all other disease. Finding MNG in a Tbbx specimen plus a BALF with a CD4/CD8 ratio of 2:1 or greater, 1 percent or less neutrophils, and 1 percent or less eosinophils had a 93 percent positive predictive value in distinguishing sarcoidosis from both nonsarcoidosis ILD and all other diseases.

Bronchial Hyperreactivity↗
PubMed1993

Mucin synthesis and secretion by cultured tracheal cells: effects of collagen gel substratum thickness.

We previously demonstrated that human tracheobronchial epithelial (TBE) cells synthesize mucin and form mucous granules in culture when they are maintained on a collagen gel (CG) substratum, but not on a plastic tissue culture surface or a thin collagen-coated surface (Wu et al., Am. J. Respir. Cell Mol. Biol., 3:467-478; 1990). This observation led us to examine the effects of CG thickness on cell growth and differentiation in primary human/monkey TBE cell cultures. Using the same CG preparation, culture dishes with different thicknesses of CG substratum were prepared. In general, equivalent degrees of cell attachment and proliferation were observed in all cultures maintained on a collagen gel, independent of the thicknesses of CG substratum. However, a greater degree of mucin synthesis and secretion by the cells was observed as the thickness of the CG substratum was increased. Cultures maintained on a thick collagen gel (1 mm) exhibited greater apical membrane complexity, more pseudostratification, and more mucous granules than did cultures maintained on a thin CG substratum. The optimal culture surface for airway mucous cell differentiation contains more than 1-mm thickness of collagen gel substratum.

Animals↗
PubMed1992

Regulation of alpha-amylase-encoding gene expression in germinating seeds and cultured cells of rice.

Four alpha-amylase-encoding cDNA (alpha Amy-C) clones were isolated from a cDNA library derived from poly(A)+RNA of gibberellic acid (GA3)-treated rice aleurone layers. Nucleotide sequence analysis indicates that the four cDNAs were derived from different alpha Amy genes. Expression of the individual alpha Amy gene in germinating seeds and cultured suspension cells of rice was studied using gene-specific probes. In germinating seeds, expression of the alpha Amy genes is positively regulated by GA3 in a temporally coordinated but quantitatively distinct manner. In cultured suspension cells, in contrast, expression of the alpha Amy genes is negatively and differentially regulated by sugars present in the medium. In addition, one strong and one weak carbohydrate-starvation-responsive alpha Amy genes have been identified. Interactions between the promoter region (HS501) of a rice alpha Amy gene and GA3-inducible DNA binding proteins in rice aleurone cells were also studied. A DNA mobility-shift assay showed that the aleurone proteins interact with two specific DNA fragments within HS501. One fragment is located between nt -131 to -170 and contains two imperfect directly repeated pyrimidine elements and a putative GA3-response element. The other fragment is located between nt -92 to -130 that contains a putative enhancer sequence. The interactions between aleurone proteins and these two fragments are sequence-specific and GA-responsive.

Base Sequence↗