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Biomedical subjects

R Wu

Publications and source records attributed to R Wu.

At least 361 records · Page 20Linked to original sources

Keratin protein expression during the development of conducting airway epithelium in nonhuman primates.

Keratin protein expression during the development of Rhesus monkey conducting airway epithelium was investigated by both biochemical and immunohistochemical methods. Keratin proteins were extracted from tracheal and intrapulmonary airway tissues of fetal (at 80- and 140-day gestational ages), neonatal, and adult animals. Using immunoblot analyses and immunohistochemistry with various monoclonal (AE1, AE3, AE8, 6.01 and 6.11) and monospecific antibodies (anti-50/55 and anti-40 kDa), the presence of keratins 5, 6, 8, 13, 14, and 19 in adult airway epithelium were demonstrated. Except for keratin 13 (51 kDa), the remaining keratins could be immunologically detected in fetal and neonatal tissues. To further understand the nature of the synthesis of keratin 13 during development, airway epithelial cells from different ages were isolated and cultured in vitro. Cultured cells were labeled with 35S-methionine, and the patterns of keratin protein were analyzed by one- and two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Results indicated that the cultured airway cells synthesized additional keratins including 7, 15, 16, 17, and 18. However, consistent with the in vivo finding, fetal cells synthesized less or no keratin 13. These in vivo and in vitro studies strongly suggest that the synthesis of the keratin 13 in monkey conducting airway epithelium is developmentally regulated.

Animals↗

The use of sputum cultures in the evaluation of immigrants classified as tuberculosis suspects.

Because of possible deficiencies in the evaluation, based on symptoms and chest roentgenogram review, of new immigrants classified during the visa application process as tuberculosis suspects, a prospective (cohort) and a retrospective (case control) study were done to test the usefulness of routinely obtaining sputum specimens for culture in that setting. In the prospective study, 249 consecutive classified immigrants who were considered on the basis of clinical and roentgenographic findings to have nonprogressive tuberculosis submitted at least two sputums for culture: 13 (5.2%) had at least one culture positive for M. tuberculosis. Immigrants younger than 50 yr of age and refugees from Kampuchea and Laos had a fivefold to tenfold elevated risk of having a positive sputum culture. The cost per case detected of obtaining and processing sputum cultures was estimated to be +1,996 to +2,994. In the case-control study, 37 classified immigrants evaluated from 1981 through 1986 who had sputum cultures positive for M. tuberculosis even though they fulfilled clinical and roentgenographic criteria for nonprogressive tuberculosis served as control subjects. Several demographic, clinical, and roentgenographic factors were associated with an increased risk of being culture-positive: age younger than 50 yr, a positive tuberculin test, report of a cough, and a cavitary lesion on chest roentgenogram. The history of prior receipt of antituberculosis drugs was associated with having a negative culture, including a marked dose-response effect.

Asia, Southeastern↗

An ELISA method for the quantitation of tracheal mucins from human and nonhuman primates.

Monoclonal antibodies, 17B1 and 17Q2, which are specific for large molecular weight mucous glycoproteins of airway epithelium, have been used to develop an ELISA method to quantitate the tracheal mucins of humans and rhesus monkeys. The assay is a double-sandwich system that does not depend on either the binding of mucous antigens to the microtiter plate or the use of a second antibody. The assay protocol includes (1) coating the microtiter well with purified IgG of 17B1 or 17Q2, (2) incubating the wells with mucous samples, (3) binding of alkaline phosphatase-conjugated IgG to the wells, and (4) developing the color with phosphate substrate. This ELISA method is very sensitive for human and rhesus monkey tracheal mucins. Quantitation is not affected by the presence of various proteoglycans (keratan sulfate, hyaluronate, heparin, heparan sulfate, and chondroitin sulfate). However, the quantitation is affected by the treatment of antigen with periodic acid and endo-beta-galactosidase. Other enzymes (e.g., neuraminidase, hyaluronidase, chondroitinase, heparitinase, heparinase, fucosidase, keratanase) have no effect on the antigenicity of substrate. The quantitation is linear, with a concentration from 0.2 to 4 ng protein/sample. The ELISA method developed in this study should be useful for quantitating the mucin content of various biologic fluids, such as sputum, bronchoalveolar lavage, and media from cultures following various pharmacologic and physiologic manipulations.

Amino Acids↗

[Cold dysmenorrhea with blood stagnation treated with zhuyu wengong decoction].

Sixty-six women with stagnant blood and cold dysmenorrhea were treated with Zhuyu Wengong Decoction (ZWD). The results showed that 47 of them were cured, 13 improved, the total effective rate was 90.9%. Before treatment, the value of amplitude and the volume of blood flow in the left pelvic cavity with blood flow graph was very lower in the dysmenorrhea patients than those in the normal women (P less than 0.01), while the number of amplitude difference between the left and the right pelvic cavity being over 30% and of abnormal wave was higher in the patients than that in the normal women (P less than 0.01). After treatment, the value of amplitude and the volume of blood flow in the left pelvic cavity had a marked increase (P less than 0.01). These data suggested that blood circulation in the pelvic cavity took a turn for the better after the treatment. The results in animal experiment also showed that ZWD had strongly suppressive effect on the automatic action of uterine muscle of rats in vitro and of rabbits in vivo. The suppressive rates were 30.75% and 20% respectively, and the contractive range was at a lower level. The decoction also had strong suppressive effect on the contract induced by MPGF2 alpha, suppressive rate decreased to 24.4% and 34.75% respectively in contrast to the group which patients had not taken this decoction. The results in this study also proved that ZWD could improve the reduction of blood flow volume induced by MPGF2 alpha and had analgesic effect on dysmenorrhea model of rats induced by oxytocin.

Adolescent↗

Dietary cravings and aversions in the postpartum period.

Although food cravings and aversions are commonly experienced by pregnant women, the degree to which they persist in the postpartum period is unknown. This study examines this issue in both lactating and nonlactating women. A random sample of 463 subjects was selected from all prenatal patients receiving care at a large health maintenance organization (HMO). Even though almost all of the subjects reported craving some food or beverage during the last trimester of pregnancy and half reported some aversion, those phenomena dropped markedly in the postpartum period. Evaluation of dietary quality as related to food cravings revealed no clear association; that is, the data provided no evidence to support the hypothesis that cravings are caused by dietary deficiencies or that they result in significantly augmented intake of key nutrients found in those foods. Overall, food cravings and aversions do not continue into the postpartum period at a higher rate than one would expect in the adult female population; manifestation of food cravings/aversions is not related to dietary quality.

Female↗

[Effects of human chorionic gonadotrophin and progesterone on small intestinal migrating myoelectric complex in rats].

The effects of human chorionic gonadotrophin (HCG) and progesterone (P) on migrating myoelectric complex (MMC) of the small intestine in ovariectomized rats were investigated by implanted monopolar-electrode in the intestinal wall of the duodenum, jejunum and ileum. The results showed (1) after i.v. administration of HCG the periodicity of the duodenal and upper jejunal MMC was interrupted by irregularly prolonged phases II occurring intermittently; (2) after i.m. administration of P the duration of phases I and II of the duodenal MMC was significantly prolonged, but the periodicity was not affected; (3) the effect of P plus HCG on MMC was similar to that of HCG alone, but the combined effect was spread over the whole small intestine. These results suggest that HCG can induce remarkable changes of the small intestinal MMC, which are similar to those observed in pregnant rats. In the presence of P, the effect of HCG is enhanced markedly.

Animals↗

Organization and nucleotide sequence of genes at both junctions between the two inverted repeats and the large single-copy region in the rice chloroplast genome.

We describe the isolation and organization, at the nucleotide sequence level, of genes located at the two junctions of the large single-copy region (LSCR) and the two inverted repeats (IRA and IRB) in the rice chloroplast genome. This is the first example where the two junctions are precisely located in a monocot. In rice, a ribosomal protein gene cluster, rpl23-rpl2-rps19, which codes for the ribosomal proteins L23 (rpl23), L2 (rpl2) and S19 (rps19), lies at the ends of the two IRs near the LSCR. The inverted repeats end 45 bp from the translation stop codon of rps19. The gene for the 32-kDa photosystem II protein, psbA, is located at the extremity of the LSCR near IRA, and transcribed towards IRA. The translation stop codon of psbA is 68 bp from the right-hand junction (JLA). Thus, JLA is located within the intergenic sequence of the two genes, rps19 and psbA. Around the left-hand junction (JLB), there is a typical ribosomal protein gene cluster, rpl23-rpl2-rps19-rpl22 (rpl22 for the ribosomal protein L22). The translation start codon of rpl22 is located in the LSCR 25 bp from JLB. Therefore, JLB is located within the intergenic sequence between rps19 and rpl22.

Chloroplasts↗

Structure-function studies of murine epidermal growth factor: expression and site-directed mutagenesis of epidermal growth factor gene.

Wild-type murine epidermal growth factor (mEGF) and mutants with Leu47 replaced by serine and valine, respectively, have been produced by recombinant DNA methodology. A synthetic gene for mEGF was fused to the coding sequence for the signal peptide of the outer membrane protein A (ompA) of Escherichia coli in the secretion vector pIN-III-ompA3, and the recombinant plasmid was used to transform E. coli. Upon induction of gene expression, mEGF and the mutants was expressed and secreted into the periplasmic space. Purification of the wild-type Leu47-mEGF and the mutants was carried out by reversed-phase and anion-exchange high-performance liquid chromatography (HPLC). Amino acid analysis and Western blot analysis further confirmed the identities of the proteins. Specific activities for wild-type and mutant proteins were measured in both mEGF receptor binding and autophosphorylation assays. The recombinant mEGF has specific activities identical with that of mEGF purified from mouse submaxillary glands, while both mutants have reduced specific activities in both bioassays. The data demonstrate the importance of the highly conserved Leu47 residue in mEGF for full biological activity.

Amino Acid Sequence↗

Retinol-regulated gene expression in human tracheobronchial epithelial cells. Enhanced expression of elongation factor EF-1 alpha.

Conducting airway epithelial cells requires vitamin A or its synthetic chemicals (retinoids) for their survival and for the expression of normal mucociliary functions. By using molecular cloning, we have shown that one of the effects of retinol on cultured human tracheobronchial epithelial (HTBE) cells is the enhancement (from 2- to 4-fold) of the mRNA encoding the elongation factor EF-1 alpha. Sequence analysis has shown that clone HT7, which was identified by differential hybridization procedures, contained a cDNA insert which encoded a protein closely resembling (81%) elongation factor EF-1 alpha from brine shrimp and completely identical to the published sequence of human elongation factor EF-1 alpha (Brands, H.H.G.M., Maassen, J.A., Van Hemert, F.J., Amons, R., and Moller, W. (1986) Eur. J. Biochem. 155, 167-171). Regions of homology of HT7 to EF-Tu from yeast mitochondria, plant chloroplasts, and Escherichia coli are also evident. A single RNA band at 1700 bases was observed for both untreated and retinol-treated HTBE cells, and for mouse liver and parotid glands when Northern transfer from denaturing agarose gel was probed with a 32P-labeled HT7 insert. An enhanced amino acid incorporation and increased protein content per cell for HTBE cells grown in the presence of retinol were observed. Results presented by these studies indicate that retinol may regulate the transcription of a factor required for translation.

Amino Acid Sequence↗

Rice mitochondrial genome contains a rearranged chloroplast gene cluster.

We have previously reported the isolation and partial sequence analysis of a rice mitochondrial DNA fragment (6.9 kb) which contains a transferred copy of a chloroplast gene cluster coding for the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL), beta and epsilon subunits of ATPase (atpB and atpE), methionine tRNA (trnM) and valine tRNA (trnV). We have now completely sequenced this 6.9 kb fragment and found it to also contain a sequence homologous to the chloroplast gene coding for the ribosomal protein L2 (rpl2), beginning at a site 430 bp downstream from the termination codon of rbcL. In the chloroplast genome, two copies of rpl2 are located at distances of 20 kb and 40 kb, respectively, from rbcL. We have sequenced these two copies of rice chloroplast rpl2 and found their sequences to be identical. In addition, a 151 bp sequence located upstream of the chloroplast rpl2 coding region is also found in the 3' noncoding region of chloroplast rbcL and other as yet undefined locations in the rice chloroplast genome. Hybridization analysis revealed that this 151 bp repeat sequence identified in rice is also present in several copies in 11 other plant species we have examined. Findings from these studies suggest that the translocation of rpl2 to the rbcL gene cluster found in the rice mitochondrial genome might have occurred through homologous recombination between the 151 bp repeat sequence present in both rpl2 and rbcL.

Base Sequence↗

A biphasic chamber system for maintaining polarity of differentiation of cultured respiratory tract epithelial cells.

A simple, disposable, biphasic cultivation chamber has been developed for respiratory tract epithelial cells. This chamber, the Whitcutt chamber, contains a movable, transparent, permeable gelatin membrane that can be employed either submerged in the culture medium, thereby feeding the cells by the traditional immersion method, or raised to the surface of the culture medium, to bring the apical surfaces of the cells into contact with air and provide nutrients only from below (basal feeding). The effects of biphasic cultivation on the growth and differentiation of respiratory tract epithelial cells from different sources have been studied in Whitcutt chambers. Primary hamster tracheal epithelial (HTE) cells grown to confluence with basal feeding developed a ciliated columnar morphology, with differentiated features (cilia and mucous granules) located in the apical region of the epithelial layer. These cells secreted mucinlike molecules from the apical surface (i.e. the surface in contact with air). Although the apical localization of differentiation features was greater, mucous cell differentiation achieved by basal feeding was quantitatively not greater than that achieved by continuous immersion feeding. Similarly, basal feeding did not alter the degree of epithelial cell differentiation in cultures derived from rat, rabbit, and monkey tracheas or from human bronchial and nasal tissues. In contrast, the differentiation of guinea pig tracheal epithelial cells in culture was significantly influenced by the feeding method employed. When fed basally, guinea pig tracheal epithelial cell cultures expressed various mucociliary functions with resemblance to mucociliary layers in vivo, whereas constantly immersed cultures seemed stratified and squamous. These results suggest that, at least for guinea pigs, the combination of feeding methods provided by the Whitcutt chamber can be used to achieve differentiated cultures of tracheal epithelial cells with a polarity of differentiation that is similar to that observed in intact airways in vivo.

Animals↗

Base-change analysis of revertants of the hisD3052 allele in Salmonella typhimurium.

This report is an investigation of the specific sequence changes in the DNA of Salmonella hisD3052 revertants induced by a set of specific frameshift mutagens found in our diet. They include B[a]P, aflatoxin B1, and the cooked-food mutagens, IQ, MeIQ, and PhIP. The Salmonella DNA was cleaved with restriction enzymes Sau3A, EcoR1, and Alu1 to give a 620-bp fragment containing the hisD3052 site. The size-fractionated fragments were ligated to the bacteriophage vector M13mp8. After transformation into E. coli, the recombinants were screened with a nick-translated hisD+ gene probe, and the isolated single-stranded DNA was sequenced. All IQ (13), MeIQ (3), PhIP (5), and aflatoxin B1 (3) induced revertants isolated had a 2-base (-CG- dinucleotide) deletion situated 10 bases upstream from the original hisD3052 -C- deletion. In contrast, 9 of 24 revertants induced by B[a]P had extensive deletions varying from 8 to 26 nucleotides in length and located at various sites along a 45-base-pair sequence beginning at nucleotide 2085 of the his operon. The other 15 B[a]P-induced revertants had a -CG- deletion at the same location as the revertants induced by the other food mutagens. 7 spontaneous revertants were also analyzed; they showed 3 -CG- deletions, 1 insertion and 3 distinct deletions (varying from 2 to 11 bases in size). In total, 13 distinct base changes are described which lead to reversion of the hisD3052 mutation.

Aflatoxin B1↗

2-deoxy-D-glucose modulation of T-lymphocyte reactivity: differential effects on lymphoid compartments.

This study was designed to evaluate the effect of glucoprivation, as induced by 2-deoxy-D-glucose (2-DG) administration, on lymphocyte mitogen reactivity in Sprague-Dawley rats. The results showed that a single injection of 2-DG decreased reactivity in both whole-blood and spleen lymphocytes, as determined by mitogenic stimulation to concanavalin A (Con A) and phytohemagglutinin (PHA). However, the suppressed reactivity for the spleen lymphocytes attenuated with repeated injections, but the whole-blood lymphocytes did not show attenuation. Mitogen assessments of lymphocytes obtained from the thymus indicated that a single injection did not induce suppressed reactivity, but repeated injections induced a pronounced suppression of responsiveness. Furthermore, mitogen assessments of mesentery lymph nodes did not show any effect of 2-DG injections. These results corroborate other findings using electric shock as the stressor, namely that different compartments of the immune system are differentially affected by a stressor.

Animals↗

Interaction of a gibberellin-induced factor with the upstream region of an alpha-amylase gene in rice aleurone tissue.

The interaction between the DNA sequences of an alpha-amylase (EC 3.2.1.1) gene and a tissue-specific factor induced in rice (Oryza sativa L.) aleurone tissue by gibberellin was studied. DNA mobility-shift during electrophoresis indicated that a 500-base-pair sequence (HS500) of a rice alpha-amylase genomic clone (OSamy-a) specifically interacted with a factor from gibberellin-induced rice aleurone tissue. The amount of complex formed between the HS500 DNA fragment and the gibberellin-induced factor increased in proportion to increasing amounts of aleurone extract and decreased with the addition of unlabeled HS500 DNA fragment. A specific segment of the HS500 fragment was protected from exonuclease III digestion by protein(s) in the aleurone extract, revealing the approximate position of the protein-DNA interaction. In the protected region, there is a direct repeat that overlaps with a potential stem-loop structure. Protein in extracts from leaves, roots, or deembryonated seeds incubated without gibberellin A3 did not bind to the HS500 DNA fragment. However, the binding factor was induced in deembryonated seeds when exogenous gibberellin A3 was added during seed imbibition. This suggests that activation of alpha-amylase synthesis by gibberellin may be mediated by the formation of a factor that interacts with a specific sequence of the alpha-amylase gene.

Base Sequence↗

The positive and negative transcriptional regulation of the Drosophila Gapdh-2 gene.

One of the genes encoding glyceraldehyde 3-phosphate dehydrogenase of Drosophila melanogaster, Gapdh-2, is expressed in all cell types examined, but its level of expression is regulated developmentally. Here we report the analysis of the regulatory sequences for the transcription of Gapdh-2. We have generated Gapdh-2-LacZ fusion genes in which the 5'-flanking sequence of Gapdh-2 has been mutated. Examination of the expression of these fusion genes, which have been introduced by transfection into the Schneider II cell line and by germ-line transformation into flies, led to the identification of two distinct regulatory regions, URS-1 and URS-2, within the first 145 bp of the 5'-flanking sequence of Gapdh-2. URS-1 activates transcription throughout the development of Drosophila. However, URS-2 exhibits a dual function during the development. It clearly represses transcription in Schneider II cells, and perhaps also in mid-stage embryos. In contrast, it activates transcription in larvae and adult flies. Thus, URS-2 plays a key role in the developmental regulation of Gapdh-2. Additionally, the efficient transcription of Gapdh-2 in larval and adult stages appears to depend on a synergistic function of URS-1 and URS-2.

Animals↗

Differential regulation of the two glyceraldehyde-3-phosphate dehydrogenase genes during Drosophila development.

Drosophila melanogaster contains two genes encoding glyceraldehyde-3-phosphate dehydrogenase, Gapdh-1 and Gapdh-2. The two genes are highly conserved in their coding sequences but not in their noncoding and flanking sequences. We report that both genes are expressed at higher levels in larval, late pupal, and adult stages than in embryonic, early, and midpupal stages. However, a major difference in the expression of the two genes is observed in the adult stage, during which the level of the Gapdh-1 transcript decreases over fourfold, while that of the Gapdh-2 transcript remains at a constant high level. In addition, the Gapdh-1 transcript appears highly enriched in the thorax section compared with the head and abdomen sections, while the Gapdh-2 transcript is evenly distributed. Analyses of the expression patterns of the two Gapdh hybrid genes, GAP1/2 and GAP2/1, revealed that the two genes have a distinct organization of their regulatory sequences. The principle regulatory sequences of Gapdh-2 reside upstream of the translation start, while the principle sequences specifying the level and developmental pattern of Gapdh-1 expression reside downstream of the translation start.

Animals↗