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Biomedical subjects

W Liu

Publications and source records attributed to W Liu.

At least 19 recordsLinked to original sources
PubMed1992

Enhancement of the chemical and antimicrobial properties of subtilin by site-directed mutagenesis.

Subtilin and nisin are gene-encoded antibiotic peptides that are ribosomally synthesized by Bacillus subtilis and Lactococcus lactis, respectively. Gene-encoded antibiotics are unique in that their structures can be manipulated by mutagenesis of their structural genes. Although subtilin and nisin share considerable structural homology, subtilin has a greater tendency than nisin to undergo spontaneous inactivation. This inactivation is a accompanied by chemical modification of the dehydroalanine at position 5 (DHA5) with a kinetic first-order t1/2 of 0.8 days. It was hypothesized that the R group carboxyl of Glu4 in subtilin participates in the chemical modification of the adjacent DHA5. Noting that nisin has Ile at position 4, site-directed mutagenesis was used to change Glu4 of subtilin to Ile, in order to eliminate this carboxyl-group participation. The DHA5 of this mutant subtilin (E4I-subtilin) underwent modification with a t1/2 of 48 days, which is 57-fold slower than natural subtilin, and the rate of loss of biological activity dropped by a like amount. These results suggest that an intact DHA5 is critical for subtilin activity against bacterial spore outgrowth. A double mutant of subtilin, in which the DHA5 residue of E4I-subtilin was mutated to Ala was devoid of detectable inhibition against spore outgrowth. The specific activity of E4I-subtilin was 3-4-fold higher than natural subtilin, suggesting that an increase in the hydrophobicity of the N-terminal end of the molecule enhances activity. These are the first mutants of subtilin that have been reported, and E4I-subtilin is the first example of any lantibiotic whose properties have been improved by mutagenesis. In order to carry out the mutagenesis, a host-vector pair was constructed that permits a deletion replacement in which the natural subtilin gene is replaced by the mutant gene at the normal location in the chromosome. This maintains normal gene dosage and regulatory responses, as well as eliminates ambiguities caused by expression of the normal and mutant genes in the same cell.

Amino Acid Sequence↗
PubMed1992

Enzymatic semisynthesis of a superpotent analog of human growth hormone-releasing factor.

A superpotent analog of human growth hormone-releasing factor, [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 (4), was prepared from the precursor, [Ala15,29]-GRF(4-29)-OH (1), by a two-step enzymatic semisynthesis. The amidated C-terminus, essential for high biological potency, was obtained via a carboxypeptidase Y-catalyzed exchange of Ala29-OH for Arg29-NH2 to produce [Ala15]-GRF(4-29)-NH2 (2). The N-terminal desNH2Tyr-D-Ala moiety, which greatly increases in vivo duration of action, was then incorporated by V8 protease-catalyzed condensation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR [R = CH3CH2- (3a) or 4-NO2C6H4CH2-(3b)]. The main focus of this report was to develop conditions to use the V8 protease-catalyzed coupling while avoiding a competing cleavage of the proteolytically-sensitive Asp25-Ile26 bond in GRF. Conversion of 2 to 4 in couplings employing the alpha-ethyl ester of the acyl component 3a was limited to about 60% by competing proteolysis at Asp25-Ile26. This system was adequate for preparing, isolating, and fully characterizing the target analog 4 and identifying the side products. The 4-nitrobenzyl ester 3b proved to be a superior substrate, resulting in 90% conversion of 2 to 4 with no detectable loss to proteolysis and requiring significantly lesser amounts of catalyst. These results demonstrate that enzymatic semisynthesis of a biologically-active peptide amide which contains unnatural amino acids at the N-terminus can be achieved from a biosynthetic precursor in good yield and purity.

Amino Acid Sequence↗
PubMed1992

High-performance liquid chromatographic determination of the herbicide imazapyr residues in water and soil.

A rapid method for determining residues of the herbicide imazapyr in water and soil is described. The soil samples were extracted with aqueous methanol and, after concentrating, the residue was estimated by HPLC with UV detection at 250 nm. Imazapyr dissolved in water was quantified by injecting the samples without any previous treatments. Analyses were performed without clean-up of soil and water samples. The pre-concentration of samples was required only at very low herbicide amounts. The maximum recovery was achieved by methanol/water (3:2 v/v) with both soils after three extraction procedures. The detection limits were 0.01 mg/l and 0.05 mg/kg in water and soil, respectively.

Chromatography, High Pressure Liquid↗
PubMed1992

Localization of the C termini of the Rh (rhesus) polypeptides to the cytoplasmic face of the human erythrocyte membrane.

We have raised a rabbit antiserum to a synthetic peptide corresponding to the C terminus (residues 400-416) of the Rh30A polypeptide. The rabbit antiserum reacted with the Rh30B (D30) polypeptide in addition to the Rh30A (C/c and/or E/e) polypeptide(s), indicating that these proteins share homology at their C termini. The antiserum did not react with erythrocyte membranes from an individual with Rh(null) syndrome. The rabbit antiserum immunoprecipitated Rh polypeptides from erythrocyte membranes and alkali-stripped membranes, but not from intact erythrocytes. Treatment of intact red cells with carboxypeptidase Y did not affect the reactivity of the antiserum, whereas treatment of alkali-stripped and unsealed erythrocyte ghost membranes resulted in the loss of antibody binding. Carboxypeptidase A treatment of intact erythrocytes and alkali-stripped membranes had no effect on antibody binding, indicating that the C-terminal domains of the Rh polypeptides contain lysine, arginine, proline, or histidine residues. These results show that the C termini of the Rh polypeptides are located toward the cytoplasmic face of the erythrocyte membrane. Treatment of intact radioiodinated erythrocytes with bromelain followed by immunoprecipitation with monoclonal anti-D gave a band of M(r) 24,000-25,000, indicating that the Rh30B (D30) polypeptide is cleaved at an extracellular domain close to the N or C terminus, with loss of the major radioiodinated domain. Immunoblotting of bromelain treated D-positive erythrocyte membranes with the rabbit antiserum to the C-terminal peptide revealed a new band of M(r) 6000-6500, indicating that the extracellular bromelain cleavage site is located near the C terminus of the molecule. The band of M(r) 6000-6500 was not obtained in erythrocyte membranes derived from bromelain treated D-negative erythrocytes. Erythrocytes of the rare -D- phenotype appear to either totally lack, or have gross alterations in, the Cc/Ee polypeptide(s), since the bromelain treatment of these cells resulted in the total loss of staining in the M(r) 35,000-37,000 region and the concomitant appearance of the new band of M(r) 6000-6500.

Amino Acid Sequence↗
PubMed1992

Dissection of the enhancer activity of beta-globin 5' DNase I-hypersensitive site 2 in transgenic mice.

The beta-globin locus control region (LCR) consists of four erythroid-specific DNase I-hypersensitive sites, which are necessary for high-level expression of the beta-like globin genes in erythroid tissues. One of these sites, designated 5'HS-2, functions as an erythroid-specific enhancer element in transfection and transgenic mouse experiments. Recent transfection experiments and studies of DNA-protein interactions have localized the 5'HS-2 enhancer to 18 nucleotides that contain a binding site for both the erythroid-specific factor nuclear factor erythroid 2 (NFE-2) and for activator protein 1 (AP-1). To define the sequences necessary for in vivo enhancer activity, several deletion mutants of 5'HS-2 were linked to the human beta-globin gene and their activity was tested in transgenic mice. Three upstream fragments of 5'HS-2 [341, 374, and 412 base pairs (bp)], each of which contained the NFE-2/AP-1 sequences, resulted in beta-globin expression at levels equivalent to or higher than those observed with the entire 732-bp 5'HS-2 fragment. In contrast, a 358-bp downstream portion of 5'HS-2, which lacked the NFE-2/AP-1 sequences, resulted in beta-globin expression at the low levels seen with the beta-globin gene alone. Removal of the NFE-2/AP-1 sequences by a 67-bp internal deletion resulted in similar low levels of beta-globin expression. A 100-bp 5' fragment that contained the NFE-2/AP-1 sequences resulted in beta-globin expression that was higher than the beta-globin gene alone but lower than the entire 5'HS-2 fragment or the three larger upstream fragments. These studies demonstrate that the NFE-2/AP-1 sequences are essential for enhancer activity of 5'HS-2 but that other sequences are required for full activity in vivo.

Animals↗
PubMed1992

Inhibition of benzo[a]pyrene-induced mouse forestomach neoplasia by a principal flavor component of Japanese-style fermented soy sauce.

A refined diet supplemented with Japanese-style fermented soy sauce (shoyu) inhibits benzo[a]pyrene-induced forestomach neoplasia in mice (Cancer Res., 51:2940-2942, 1991). In the present study, soy sauce was extracted with ethyl acetate. The soluble fraction contained flavor/aroma compounds and antioxidants, whereas amino-carbonyl compounds that impart color were concentrated in the ethyl acetate insoluble fraction. Both fractions inhibited benzo[a]pyrene-induced forestomach neoplasia in a protocol in which the test material was fed following exposure to the carcinogen. A principal flavor/aroma component of soy sauce, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone, was fed to mice following benzo[a]pyrene administration and found to inhibit the subsequent development of forestomach neoplasia. 4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone was effective when fed at 4 mg/kg body weight/day, indicating that it is a potent anticarcinogen.

Animals↗
PubMed1992

Mutation of the high affinity calcium binding sites in cardiac troponin C.

Fast skeletal and cardiac troponin C (TnC) contain two high affinity Ca2+/Mg2+ binding sites within the C-terminal domain that are thought to be important for association of TnC with the troponin complex of the thin filament. To test directly the function of these high affinity sites in cardiac TnC they were systematically altered by mutagenesis to generate proteins with a single inactive site III or IV (CBM-III and CBM-IV, respectively), or with both sites III and IV inactive (CBM-III-IV). Equilibrium dialysis indicated that the mutated sites did not bind Ca2+ at pCa 4. Both CBM-III and CBM-IV were similar to the wild type protein in their ability to regulate Ca(2+)-dependent contraction in slow skeletal muscle fibers, and Ca(2+)-dependent ATPase activity in fast skeletal and cardiac muscle myofibrils. The mutant CBM-III-IV is capable of regulating contraction in permeabilized slow muscle fibers but only if the fibers are maintained in a contraction solution containing a high concentration of the mutant protein. CBM-III-IV also regulates myofibril ATPase activity in fast skeletal and cardiac myofibrils but only at concentrations 10-100-fold greater than the normal protein. The pCa50 and Hill coefficient values for Ca(2+)-dependent activation of fast skeletal muscle myofibril ATPase activity by the normal protein and all three mutants are essentially the same. Competition between active and inactive forms of cardiac and slow TnC in a functional assay demonstrates that mutation of both sites III and IV greatly reduces the affinity of cardiac and slow TnC for its functionally relevant binding site in the myofibrils. The data indicate that although neither high affinity site is absolutely essential for regulation of muscle contraction in vitro, at least one active C-terminal site is required for tight association of cardiac troponin C with myofibrils. This requirement can be satisfied by either site III or IV.

Adenosine Triphosphatases↗
PubMed1992

Time trend and age-period-cohort effects on incidence of esophageal cancer in Connecticut, 1935-89.

The purpose of this study was to examine the incidence pattern of esophageal cancer in Connecticut (USA) during the past decades, and to identify components of birth cohort, period, and and age as determinants of the observed time trends by regression modelling. This study is based on all of the esophageal cancer cases reported to the Connecticut Tumor Registry between 1935 and 1989. A total of 6,310 incident cases were included. Results indicate that among males, the overall age-adjusted incidence rate of esophageal cancer increased after 1935 and peaked between 1955 and 1959. Since then, incidence rates have been relatively stable. Among females, the overall esophageal cancer rate has not changed markedly since 1935. Analysis by histologic type indicates that the incidence rate of squamous cell carcinoma has been declining in this population; adenocarcinoma, however, showed a continuous increase. A fivefold increase among males and a threefold increase among females were observed for adenocarcinoma of the esophagus between 1970 and 1989. If cancers of the esophagus and gastric cardia are considered together, the incidence rate of adenocarcinoma exceeds that of squamous cell carcinoma among males during 1985-89. The observed increasing trend for adenocarcinoma of the esophagus is mainly from cancers arising in the lower third of the esophagus and primarily among Whites, especially White males. The results from regression modeling indicate that both period and birth cohort may have contributed to the observed increasing trend, and adenocarcinoma of the esophagus is likely to increase continuously in this population in the coming years.

Adenocarcinoma↗
PubMed1992

Transforming growth factor beta 1 is an epithelial-derived signal peptide that influences otic capsule formation.

Interactions between epithelial and mesenchymal tissues in the developing inner ear direct the formation of its cartilaginous capsule. Recent work indicates that many growth factors are distributed in the early embryo in vivo in a temporal-spatial pattern that correlates with sites of ongoing morphogenetic events. We report here that the localization of transforming growth factor beta 1 (TGF-beta 1) in both epithelial and mesenchymal tissues of the mouse inner ear between 10 and 16 days of embryonic development (E10-E16). In addition, utilizing a high-density culture system as an in vitro model of otic capsule chondrogenesis, we show that modulation of chondrogenesis by TGF-beta 1 in cultured mouse periotic mesenchyme mimics the in vitro effects of otic epithelium on the expression of chondrogenic potential. We provide evidence of a causal relationship of this growth factor to otic capsule formation in situ by demonstrating that the actual sequence of chondrogenic events that occur in the developing embryo is reproduced in culture by the addition of exogenous TGF-beta 1 peptide. Furthermore, in cultures of mesenchyme containing otic epithelium, we demonstrate the localization of endogenous TGF-beta 1, first within the epithelial tissue and later within both the epithelium and its surrounding periotic mesenchyme, contrasted to an absence of endogenous TGF-beta 1 in cultures of mesenchyme alone. Our results suggest that TGF-beta 1 is one of the signal molecules that mediate the effects of otic epithelium in influencing the formation of the cartilaginous otic capsule.

Animals↗
PubMed1992

First phase insulin release in the non-obese diabetic mouse: correlation with insulitis, beta cell number and autoantibodies.

The ontogenic variation of beta cell function and its relationship with the degree of islet damage and levels of autoantibodies have been studied in the non-obese diabetic (NOD) mouse model. We conducted in vivo first phase insulin release (FPIR) in response to intravenous glucose and studied its correlation with the degree of insulitis, islet cell antibody (ICA) and insulin autoantibody (IAA) levels in female NOD mice cross-sectionally at days 40 (n = 19), 90 (n = 21), 150-160 (n = 21) and day 250 (n = 20). The mean +/- SEM FPIR values showed an age-related decline from day 40 (46.2 +/- 5.3 microU/ml) to day 150-160 (17.8 +/- 2.5 microU/ml) and then doubled at day 250 (34.5 +/- 5 microU/ml), while the mean +/- SEM insulitis scores increased progressively until day 150-160 (61.7 +/- 6.1%) and then declined slightly at day 250 to 50.2 +/- 6.2%. In female NOD mice with spontaneous diabetes (n = 4) and streptozotocin-induced diabetic Swiss mice (n = 5) FPIR was either absent or greatly attenuated. A statistically significant inverse correlation between FPIR and insulitis was found among NOD mice at days 90 (P = 0.02; r = -0.52) and 150-160 (P = 0.03; r = -0.48). However, no statistically significant correlation was observed at days 40 and 250. Morphometric techniques applied to day 150-160 pancreatic sections showed a statistically significant negative correlation between insulitis and beta cell number per unit area of islet tissue (P = 0.0001; r = - 0.75). At this age some islet beta cells showed different intensities of staining by immunofluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1992

Comparison of the virulence of two isolates of porcine parvovirus in 72-day-old porcine fetuses.

Two strains of porcine parvovirus (PPV), designated Kresse and NADL-8, were compared for relative virulence in porcine fetuses. Strain Kresse was injected into the amniotic fluid of all fetuses of 1 uterine horn of each of 2 pregnant gilts at 72 days of gestation. Strain NADL-8 was administered similarly to fetuses of 4 other gilts at the same stage of gestation. All gilts were killed and necropsied 35 days later. Selected tissues of all fetuses were tested for infectious virus and viral antigen. Sera from live fetuses were tested for antibody to PPV. These tests confirmed that most fetuses exposed to PPV by intra-amniotic injection became infected. All of 11 fetuses exposed to strain Kresse by intra-amniotic injection were alive at the time of necropsy, and all appeared clinically normal. In contrast, 8 of 24 fetuses exposed similarly to strain NADL-8 were dead. Many of the fetuses from the uterine horns contralateral to the uterine horns inoculated with virus were infected after 72 days of gestational age by intrauterine spread of the virus. Four such fetuses, 3 infected with the NADL-8 strain and 1 infected with the Kresse strain, were dead at the time of necropsy. These findings were inconsistent with those of a previous report, which indicated that the Kresse strain of PPV was markedly more virulent than the NADL-8 strain of PPV for porcine fetuses. A possible reason for this apparent discrepancy is discussed.

Animals↗
PubMed1992

Effects of the expression of mammalian annexins in yeast secretory mutants.

The hypothesis that calcium-dependent membrane-binding proteins of the annexin family can influence intracellular membrane trafficking was tested by expressing five mammalian annexins in wild-type yeast cells (Saccharomyces cerevisiae) and in 13 yeast secretory (sec) mutants. Expression of human synexin (annexin VII) inhibited the growth of sec2, sec4 and sec15 mutants at a semi-permissive temperature. These three sec mutants are defective in the final step in the secretory pathway, the process of exocytosis. The inhibition of growth correlated with reduced viability and increased accumulation of internal invertase in these mutants when expressing synexin. Bovine endonexin (annexin IV) partially suppressed the growth defect of a sec2 mutant incubated at a semi-permissive temperature. Human synexin, human lipocortin (annexin I), and murine p68 (annexin VI) reduced the lag time associated with adaptation of sec2 mutants to galactose-containing medium. These interactions suggest that the annexins may influence specific steps in membrane trafficking associated with cell growth, secretion and plasma membrane remodelling.

Annexins↗
PubMed1992

Primary care arrangements and access to care among African-American women in three Chicago communities.

African-American women of child-bearing age residing in three high-risk communities in Chicago were surveyed regarding their primary care arrangements and access to care (n = 552). This study examined factors which differentiated women who used office-based practices from those who used institutional settings (community clinics, health department clinics, hospital-based clinics) for primary care. Results of multivariate analysis indicate that women who used office-based practices were more likely than those who used institutional settings to see the same provider, to walk to their provider, to have less travel time and to walk in without an appointment. They were less likely to be hospitalized in the past year and less likely to report the availability of family planning at their usual source of care. Satisfaction with care, insurance status and sociodemographic characteristics were not associated with use of a particular facility type. Implications for organizing comprehensive health services for this population are discussed.

Adolescent↗
PubMed1992

The effect of IH764-3 on fibroblast proliferation and function.

The effect of IH764-3, a potent component isolated from Salvia miltiorrhiza, on the proliferation and function of cultured fibroblasts was studied. It was found that the fibroblast growth curve had a dose-dependent relationship with IH764-3 concentration. The incorporation of 3H-TdR and 3H-proline into fibroblasts was significantly inhibited by IH764-3, and calmodulin, fibronectin and thrombospondin contents in the test group were obviously lower than those in the control group. Flow cytometry showed that in the IH764-3-treated group, the percentage of cells in G0/G1 phase was higher than that in the control. Electron microscopic observation (TEM and SEM) showed that in the treated group, collagen secretion was decreased. All of these results indicate that IH764-3 exerts a direct inhibitory effect on fibroblast proliferation and affects their ability to synthesize collagen.

Animals↗
PubMed1992

[Effect of oxytocin and cholecystokinin octapeptide (CCK-8) on electroacupuncture (EA) analgesia].

The effect of oxytocin (OT) and cholecystokinin octapeptide (CCK-8) on EA analgesia was studied in rats. The increase of 20.8-39.8% and 9.0-45.0% in pain threshold was observed respectively when ICV of CCK-8 or naloxone was combined with EA, these increases were lower than that in saline-EA group significantly, while the simultaneous ICV of OT and CCK-8 or OT and naloxone in combination with EA produced the increase of 76.2-116.6% and 41.8-104.5% in pain threshold separately. These results showed that only a small part in the role of OT enhancing EA analgesia was blocked by CCK-8 and naloxone. The data suggest that the role of OT in EA was not entirely dependent upon the endogenous opiate peptides.

Acupuncture Analgesia↗
PubMed1992

Cloning, characterization and partial sequence analysis of the 5'-flanking region of bovine alpha-sl casein gene.

We have isolated a clone from a EMBL3 constructed bovine genomic library using a PCR-generated DNA fragment as a probe covering-660bp to + 158 bp of boving alpha-sl casein gene. The results obtained from hybridization assay, restriction endonucleases map and partial DNA sequence analysis indicated that the cloned fragment spanned 5.7kb of the first part of the gene and 8.0 kb of its upstream flanking region. Some divergent restriction endonuclease recognition sites and a few mutations and deletions were found in exons, introns and 5'-flanking region of the gene we cloned when compared with the PCR-generated fragment and those which had been published [6,10]. All the deletions occurred at repeat sequences.

Animals↗
PubMed1992

[The preparation and clinical use of a radioimmunoassay CA125 kit for the diagnosis of epithelial ovarian cancer].

A self-made radioimmunoassay CA125 kit (using OC125 monoclonal antibody ascites offered by Dr. Bast Laboratory which was purified, solidified and labelled with 125I) was used for serum determination in 80 patients with epithelial ovarian cancer and in 40 standard antigen samples. The results demonstrated a statistically significant correlation between our self-made CA125 kit and an imported CENTOCOR CA125 kit (P < 0.001). Clinical experience before operation in 40 patients with epithelial ovarian cancer, 34 with endometriosis and 39 with other malignant ovarian tumors suggested that the self-made CA125 kit was more sensitive in diagnosing epithelial ovarian cancer than the other two diseases. Post-operative follow-up in 86 non-recurrent cases and 101 recurrent cases showed a 96% positive predictive rate and 91% negative predictive rate. It is concluded that self-made CA125 kit is very useful in monitoring epithelial ovarian cancer.

Antibodies, Monoclonal↗