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Biomedical subjects

L Wu

Publications and source records attributed to L Wu.

At least 667 records · Page 37Linked to original sources
PubMed1993

[A preliminary report on the preparation of antisera against Haemophilus influenzae].

The antigenicity of 5 strains of serotype b Haemophilus influenzae was compared. Strain 58534 showed a higher titer than the rest four. Three lots of antisera were made by immunizing rabbits with 6 strains of Haemophilus influenzae. The results indicated that the antibody titer against strain 58534 also showed a higher level, and the titer only dropped by one dilution after stored in cool place for half a year. We considered that Haemophilus influenzae serotype b strain 58534 is a good strain for the preparation of antiserum against Haemophilus influenzae.

Animals↗
PubMed1993

[Construct of the stable-produced strain for the thermostable alpha-amylase].

The plasmid pE194 as a vector is used to subclone the thermostable alpha-amylase gene of B. licheniformis and construct recombinant plasmid pNW102. The plasmid pNW102 was transduced into B. subilis BF 7658 by bacteriophage PBS1. The B. subtilis BF7658 (pNW102) strain was treated at non-permissive temperature for a long time, and obtained many recombinant strains. This is the homologous recombination results between alpha-amylase genes of pNW102 and BF7658. The homologous recombination occurs at any site in alpha-amylase gene but there are some heat-spots. We have already screened 2 strains from the recombination strains which can stably produce the thermostable alpha-amylase in B. subtilis BF7658. The analysis of enzymology shows that the characters of the thermpstable alpha-amylase produced from the recombinants strains are the same with that of B. licheniformis.

Bacillus subtilis↗
PubMed1993

Two potentially oncogenic cyclins, cyclin A and cyclin D1, share common properties of subunit configuration, tyrosine phosphorylation and physical association with the Rb protein.

Originally identified as a 'mitotic cyclin', cyclin A exhibits properties of growth factor sensitivity, susceptibility to viral subversion and association with a tumor-suppressor protein, properties which are indicative of an S-phase-promoting factor (SPF) as well as a candidate proto-oncogene. Other recent studies have identified human cyclin D1 (PRAD1) as a putative G1 cyclin and candidate proto-oncogene. However, the specific enzymatic activities and, hence, the precise biochemical mechanisms through which cyclins function to govern cell cycle progression remain unresolved. In the present study we have investigated the coordinate interactions between these two potentially oncogenic cyclins, cyclin-dependent protein kinase subunits (cdks) and the Rb tumor-suppressor protein. The distribution of cyclin D isoforms was modulated by serum factors in primary fetal rat lung epithelial cells. Moreover, cyclin D1 was found to be phosphorylated on tyrosine residues in vivo and, like cyclin A, was readily phosphorylated by pp60c-src in vitro. In synchronized human osteosarcoma cells, cyclin D1 is induced in early G1 and becomes associated with p9Ckshs1, a Cdk-binding subunit. Immunoprecipitation experiments with human osteosarcoma cells and Ewing's sarcoma cells demonstrated that cyclin D1 is associated with both p34cdc2 and p33cdk2, and that cyclin D1 immune complexes exhibit appreciable histone H1 kinase activity. Immobilized, recombinant cyclins A and D1 were found to associate with cellular proteins in complexes that contain the p105Rb protein. This study identifies several common aspects of cyclin biochemistry, including tyrosine phosphorylation and the potential to interact directly or indirectly with the Rb protein, that may ultimately relate membrane-mediated signaling events to the regulation of gene expression.

Amino Acid Sequence↗
PubMed1993

G1 expression and multistage dynamics of cyclin A in human osteosarcoma cells.

Cyclin A was initially characterized as a 'mitotic cyclin', believed to function exclusively at the G2-to-M phase transition; however, recent studies have provided compelling evidence that cyclin A additionally functions earlier in the mammalian somatic cell cycle as a putative 'S-phase-promoting factor'. Moreover, numerous inconsistencies have arisen concerning the temporal induction, subcellular localization, subunit configuration, covalent modification and proteolytic destruction of cyclin A, as well as the physiological function of the cyclin A-associated protein kinase complexes. Utilizing precisely synchronized human MG-63 osteosarcoma cells, the present study demonstrates that cyclin A mRNA and protein are clearly expressed in late G1 prior to S-phase entry, as is cyclin A-associated kinase activity and concomitant phosphorylation of the Rb protein. A series of monospecific cyclin A antibodies were generated and utilized to confirm that multiple covalent modifications of cyclin A occur during the course of the cell cycle, and to characterize the subcellular dynamics in additional detail. Pharmacological blockade with mimosine was utilized to further delineate cyclin A expression and to distinguish the temporal induction from the mechanisms of enzyme activation. Subcellular fractionation and immunocytochemical staining localized nascent cyclin A to the cytoplasm, and revealed a distinct translocation to the nucleus during the G1-to-S phase transition. The results of these studies support a multistage model of cyclin A metabolism and enzyme activation.

Amino Acid Sequence↗
PubMed1992

Alternative exon usage and processing of the major histocompatibility complex-encoded proteasome subunits.

The finding that two subunits of the proteasome, LMP2 and LMP7, are encoded in the major histocompatibility complex (MHC) has linked the proteasome which represents a major extralysosomal proteolytic system to the processing of intracellular antigens. Here we describe a second form of the human LMP7 cDNA, LMP7-E2, which has been identified during the characterization of novel genes in the MHC. The analysis of the genome organization of LMP7 revealed that LMP7-E1 and LMP7-E2 arise by alternative exon usage. Using specific antibodies against LMP2 and LMP7, we show that they are co-expressed with class I MHC molecules as well as a putative peptide transporter. The polypeptides encoded by LMP7 and LMP2 undergo proteolytic processing when incorporated into proteasomes, and the LMP7 precursor is derived mainly from LMP7-E2. Furthermore, our data suggest that LMP7 and LMP2 are mutually dependent for their incorporation into the proteasomal complex.

Amino Acid Sequence↗
PubMed1992

Superinduction of CYP1A1 transcription by cycloheximide. Role of the DNA binding site for the liganded Ah receptor.

Nuclear run-off experiments reveal that four distinct DNA domains, each of which contains a binding site for the liganded Ah receptor, can mediate the superinduction of transcription by 2,3,7,8-tetrachlorodibenzo-p-dioxin plus cycloheximide. Superinduction requires substantial inhibition of protein synthesis by cycloheximide. Gel retardation analyses of nuclear extracts and methylation protection studies in intact cells reveal no evidence for cycloheximide-sensitive protein-DNA interactions that might mediate the superinduction response. These observations suggest the existence of a labile protein(s) that acts to inhibit the function of the dioxin-responsive receptor/enhancer regulatory system via protein-protein interactions.

Base Sequence↗
PubMed1992

Use of nuclear morphometry, gleason histologic scoring, clinical stage, and age to predict disease-free survival among patients with prostate cancer.

BACKGROUND: Currently, there are no accurate methods for predicting metastases or time to disease progression for patients with clinically localized prostate cancer after surgery. METHODS: In this report, histologic sections were studied from prostate cancer specimens from 100 men with clinically localized prostate cancer (clinical Stages A1 [9 cases], A2 [24 cases], B1 [27 cases], and B2 [40 cases]; pathologic Stages A1 [9 cases], A2 [22 cases], B [23 cases], C1 [8 cases], and D1 [38 cases]) to determine whether nuclear morphometry--when analyzed with clinical stage, pathologic parameters, and age in a multivariate fashion--would predict time to disease progression. RESULTS: These patients were treated with surgery alone for their clinically localized disease and were observed after surgery until disease progression or death. For each of the 100 specimens, 16 different mathematical descriptors described the shape of 150 nuclei. A series of 17 different statistical measurements were calculated to accurately describe the distribution, extremes, and variability within each descriptor. As univariate predictors, the variance of nuclear roundness, the mean of ellipticity, the Gleason score, age, and clinical stage were statistically significant predictors of disease progression when analyzed with Kaplan-Meier survival curves. A prognostic factor score calculated with multivariate analysis of clinical stage, Gleason score, age, and variance of nuclear roundness separated the patients into three statistically distinct groups and predicted time to progression by the Kaplan-Meier life table and Cox proportional hazards analysis. CONCLUSIONS: This prognostic factor score may aid in stratifying patients into high-risk and low-risk groups for testing adjuvant therapies for prostate cancer.

Adenocarcinoma↗
PubMed1992

The surface phenotype of dendritic cells purified from mouse thymus and spleen: investigation of the CD8 expression by a subpopulation of dendritic cells.

A new procedure for rapid isolation of dendritic cells (DC) was devised, involving collagenase digestion of tissues, dissociation of lymphoid-DC complexes, selection of light-density cells, then depletion of lymphocytes and other non-DC by treatment with a mixture of lineage-specific monoclonal antibodies (mAbs) and removal with anti-immunoglobulin-coupled magnetic beads. This enriched population (approximately 80% DC) was further purified when required by fluorescence-activated cell sorting for cells expressing high levels of class II major histocompatibility complex (MHC). The isolated DC were characterized by immunofluorescent staining using a panel of 30 mAbs. Thymic DC were surface positive for a number of markers characteristic of T cells, but they were distinct from T-lineage cells in expressing high levels of class II MHC, in lacking expression of the T cell receptor (TCR)-CD3 complex, and having TCR beta and gamma genes in germline state. Splenic DC shared many markers with thymic DC, but were negative for most T cell markers, with the exception of CD8. A substantial proportion of DC from both thymus and spleen expressed CD8 at high levels, comparable with that on T cells. This appeared to be authentic CD8, and was produced by the DC themselves, since they contained CD8 alpha mRNA. Thymic DC presented both the CD8 alpha and beta chains on the cell surface (Ly-2+3+), although the alpha chain was in excess; the splenic DC expressed only the CD8 alpha chain (Ly-2+3-). It is suggested that the expression of CD8 could endow certain antigen-presenting DC with a veto function.

Animals↗
PubMed1992

Major histocompatibility complex (MHC)-encoded HAM2 is necessary for antigenic peptide loading onto class I MHC molecules.

The mutant murine lymphoma cell line RMA-S is unable to present endogenous antigens due to its inability to efficiently assemble class I major histocompatibility complex molecules and antigenic peptides. Therefore, it has been suggested that RMA-S cells are defective either in peptide generation or in peptide transport into the endoplasmic reticulum, where class I major histocompatibility complex molecule assembly is believed to occur. As proteasomes and the putative peptide transporters HAM1 and HAM2 have been implicated in class I antigen processing, we have investigated their expression in RMA-S and its wild-type counterpart RMA. Both proteasomes and HAM1 proteins are expressed at similar levels and show identical subcellular distributions in the two cell lines. However, only one copy of the HAM2 gene is present in RMA-S cells, and it contains a point mutation that leads to a premature stop codon. Thus, the HAM2 protein is absent from RMA-S cells. These data demonstrate that HAM2 is essential for peptide loading onto class I molecules.

Amino Acid Sequence↗
PubMed1992

Mechanism of dioxin action: Ah receptor-mediated increase in promoter accessibility in vivo.

We have analyzed dioxin-inducible, Ah receptor-dependent changes in protein-DNA interactions at the CYP1A1 transcriptional promoter in intact mouse hepatoma cells. Our findings indicate that in uninduced cells, the promoter is inaccessible to its cognate binding proteins, which are known to be expressed constitutively. Dioxin induces, in Ah receptor-dependent fashion, an increase in promoter accessibility, which occurs rapidly and does not require ongoing transcription of the CYP1A1 gene. The change in promoter accessibility is not due to an altered pattern of cytosine methylation at the promoter; it probably reflects a 2,3,7,8-tetrachlorodibenzo-p-dioxin- induced change in the chromatin structure. These findings provide new insight into the mechanism of dioxin action and contribute to a better understanding of the regulation of inducible gene transcription in mammalian cells.

Animals↗
PubMed1992

Multidrug-resistant phenotype of disease-oriented panels of human tumor cell lines used for anticancer drug screening.

Disease-oriented panels of human tumor cell lines used by the National Cancer Institute for large-scale in vitro anticancer drug screening were evaluated for multidrug-resistant phenotype at the functional (in vitro drug sensitivity) and molecular levels. The cell line panels manifested a broad range of sensitivities to drugs typically associated with multidrug resistance (MDR) as well as to drugs not associated with MDR. Individual cell lines displayed unique and characteristic profiles of response. Patterns of correlated response were observed among, but not between, MDR and non-MDR drugs. Strong evidence of correlated response was limited to drugs sharing an intracellular mechanism of action. Several tumor cell lines exhibited a high degree of resistance to MDR drugs and relative sensitivity to non-MDR drugs, contained high levels of MDR-1 mRNA, and expressed cell surface P-glycoprotein detectable with one or more monoclonal antibodies. Parallel expression of all of these features representing the classic MDR phenotype was observed among members of the colon and renal tumor panels. Certain individual cell lines among other panels (lung, ovarian, melanoma, and central nervous system) also manifested some aspects of the MDR phenotype to various extents. Identification of MDR cell lines used for large-scale in vitro anticancer drug screening will facilitate interpretation of data in a way which may allow identification of new drug leads of potential value in treatment of MDR tumor cell populations.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Evaluation of metastatic human tumor burden and response to therapy in a nude mouse xenograft model using a molecular probe for repetitive human DNA sequences.

A sensitive DNA dot-blot assay for repetitive human DNA sequences was developed and applied to the quantitative determination of spontaneous metastases of a human melanoma in various tissues of nude mice. The assay was useful for defining the time course and pattern of tissue distribution of metastatic cells as well as for assessing response to therapy. The methodology is relatively simple, can be performed using nonradioactive DNA probes, and should be broadly applicable to studies of metastasis of human tumors in nude mice.

Animals↗
PubMed1992

Non-contacted bases affect the affinity of synthetic P22 operators for P22 repressor.

The affinity of synthetic P22 operators for P22 repressor varies with the base sequence at the operator's center. At 100 mM KCl, the affinity of these operators for P22 repressor varies over a 10-fold range. Dimethylsulfate protection experiments indicate that the central bases of the P22 operator are not contacted by the repressor. The KD for the complex of P22 repressor with an operator bearing central T-A bases (9T) increases less than 2-fold between 50 and 200 mM KCl, whereas the KD for the complex of repressor with an operator bearing central C-G bases (9C) increases 10-fold in the same salt range. The DNase I cleavage patterns of both bound and unbound P22 operators also vary with central base sequence. The DNase I pattern of the repressor-9C operator complex changes markedly with salt concentration, whereas that of the 9T operator-repressor complex does not. These changes in nuclease digestion pattern thereby mirror the salt-dependent changes in the P22 operator's affinity for repressor. P22 repressor protects the central base pair of the 9T operator from cleavage by the intercalative cleavage reagent Cu(I)-phenanthroline, while repressor does not protect the central bases of the 9C operator. Together these data indicate that central base pairs affect P22 operator strength by altering the structure of the unbound operator and the repressor-operator complex.

Amino Acid Sequence↗
PubMed1992

Assay of the Ca pump ATPase activity of intact red blood cells.

An assay for the Ca pump ATPase of intact human red blood cells (RBCs) was developed. The assay utilized a small volume (typically 10 microliters) of packed RBCs in 1 ml of a buffer of known composition. The assay was based on the exposure of intact RBCs to the ionophore, A23187, in the presence of Ca. Such exposure caused a rapid degradation of ATP in RBCs. This degradation process is modeled in a numerical simulation in a companion paper (Vincenzi, F. F. and Hinds, T. R. (1992) Biochim. Biophys. Acta 1105, 63-70). The loss of ATP followed pseudo-first-order kinetics, and the rate constants for ATP degradation was taken as a measure of the capacity of the Ca pump ATPase. A number of variables were examined to optimize the activity of the ATPase. These variables included the concentrations of Ca and A23187. Because A23187 can promote loss of cellular Mg, it was necessary to include MgCl2 in the incubation medium to optimize ATPase activity. Likewise, it was determined that inclusion of iodoacetic acid optimized the rate of ATP loss, presumably by preventing the resynthesis of ATP from ADP and inorganic phosphate. Cobalt inhibited the ionophore-dependent loss of ATP by apparent competition with Ca for binding to A23187. Results of many assays demonstrated substantial differences in the rate constant for ATP loss in RBCs from different individuals. RBCs were selected according to density. Density associated loss of Ca pump ATPase activity was observed both by the intact RBC assay, and by assay of Ca pump ATPase activity in saponin lysates of RBCs. The correlation coefficient between the two assays was 0.93. It is suggested that the rate constant for ATP loss in intact RBCs exposed to A23187 and Ca can be taken as a measure of the Ca pump ATPase activity. This may be useful when isolated membrane ATPase assays fail (e.g., dog RBCs). The intact cell assay can also be carried out on very small volumes of cells and may be of particular value when RBC volumes are limited.

Adenosine Triphosphatases↗