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Biomedical subjects

H Yu

Publications and source records attributed to H Yu.

At least 19 recordsLinked to original sources
PubMed2025

HLA-E and NKG2A Mediate Resistance to BCG Immunotherapy in Non-Muscle-Invasive Bladder Cancer.

Bacillus Calmette-Guérin (BCG) is the first-line therapy for high-grade non-muscle-invasive bladder cancer (NMIBC), yet many patients experience recurrence due to immune evasion. We identify HLA-E and NKG2A as mediators of adaptive resistance involving chronic activation of NK and T cells in BCG-unresponsive tumors. Prolonged IFN-γ exposure enhances HLA-E and PD-L1 expression on recurrent tumors, accompanied by the accumulation of NKG2A+ NK and CD8 T cells. HLA-Ehigh tumor cells preferentially cluster near CXCL12-rich stromal regions with dense effector cell presence, underscoring a spatially segregated tumor architecture. Although cytotoxic lymphocytes retain effector potential, their activity is restrained by HLA-E/NKG2A and PD-L1/PD-1 pathways located in their immediate neighborhood within the bladder tumor microenvironment. These data reveal a spatially organized immune escape program that limits anti-tumor immunity. Our findings support dually targeting NKG2A and PD-L1 checkpoint blockade as a rational, bladder-sparing strategy for patients with BCG-unresponsive NMIBC.

BCG-unresponsive↗
PubMed1995

Biochemical characterization and posttranslational modification of AlgU, a regulator of stress response in Pseudomonas aeruginosa.

AlgU is homologous to the extreme heat shock sigma factor sigma E from enteric bacteria. In this work, AlgU was overproduced and purified and its function investigated at the biochemical level. AlgU was shown to associate with RNA polymerase and direct transcription of a target promoter. AlgU also exhibited multiple isoforms detected by 2D gel analysis. Treatment with a Ser/Thr phosphatase shifted the distribution of isoforms towards the basic side on 2D gels, suggesting that posttranslational modifications of AlgU may involve phosphorylation. The underphosphorylated forms of AlgU copurified with RNA polymerase. It is possible that phosphorylation affects AlgU activity or its stability.

Amino Acid Sequence↗
PubMed1995

A general method for mapping tertiary contacts between amino acid residues in membrane-embedded proteins.

A general method for mapping tertiary interactions in membrane proteins using the visual pigment rhodopsin as a model is presented. In this approach, the protein is first assembled from two separately expressed gene fragments encoding nonoverlapping segments of the full-length polypeptide. Cys residues are then introduced into each of the two fragments such that juxtaposed residues are able to form disulfide cross-links in the protein either spontaneously or with the assistance of a Cu(2+)-(phenanthroline)3 oxidant. The cross-linked polypeptides are identified from a characteristic mobility shift on sodium dodecyl sulfate (SDS) gels as detected by Western blot analysis where the covalently bound heterodimer migrates with a mobility essentially identical to that of the native, full-length protein. Three different split rhodopsin mutants were prepared: one with a split in the loop connecting helices 3 and 4 (the 3/4 loop), one with a split in the 4/5 loop, and one with a split in the 5/6 loop. Each of these proteins when purified from transfected COS cells bound 11-cis-retinal, had a native absorption maximum at 500 nm, and activated transducin in a light-dependent manner. The cross-linking assay was tested with the rhodopsin mutant split in the 5/6 loop using the rho-1D4 antibody (which recognizes the carboxy terminal eight amino acids of rhodopsin) to detect the proteins on Western blots of SDS gels. Cys residues were substituted for Val-204 in the amino terminal fragment and Phe-276 in the carboxy terminal fragment of the rhodopsin mutant because Schwartz and co-workers [Elling et al.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1995

Recognition of type 1 chain oligosaccharides and lacto-series glycolipids by an antibody to human secretory component.

Binding of the mouse IgM antibody 6C4 is lost after treatment of human free secretory component with peptide N-glycosidase F (Bakos et al. (1991) J. Immunol. 146, 162-168) or periodate, suggesting that asparagine-linked oligosaccharides contain the epitope recognized by this antibody. Inhibition of antibody binding to free secretory component by milk oligosaccharides established that lacto-N-tetraose is the minimum structure recognized by the antibody, but larger oligosaccharides with terminal Gal beta 1-3GlcNAc sequences bind with much higher affinity. Antibody binding is enhanced by substitution with the Lewis Fuc alpha 1-4 and is inhibited by Fuc alpha 1-2Gal substitution. Free secretory component, however, does not bind other antibodies that recognize Le(a) or Leb oligosaccharides, and binding is lost after digestion with a beta-galactosidase that cleaves Gal beta 1-3 linkages but not after digestion with alpha-L-fucosidase. Therefore, the major epitope recognized by 6C4 on free secretory component is probably not an asparagine-linked Le(a) oligosaccharide. The antibody also binds to human milk lactoferrin, some human mucins, and lacto-series glycolipids including III4 alpha Fuc-lactotetraosyl ceramide and lactotetraosyl ceramide. Based on affinity chromatography of oligosaccharides released from free secretory component, the epitope recognized by antibody 6C4 is present on approximately 3.5% of the asparagine-linked oligosaccharides.

Animals↗
PubMed1995

Isolation and sequencing of two cDNA clones encoding rat spleen cathepsin E and analysis of the activation of purified procathepsin E.

Cathepsin E (CE) is an intracellular, nonlysosomal aspartic proteinase consisting of two identical subunits with a molecular mass of approximately 42 kDa and has a unique subcellular distribution in various rat tissues. In this study, we determined the complete amino acid sequence of rat spleen CE and examined the activation mechanism of the proenzyme purified from this tissue. Two cDNA clones encoding rat CE, termed pTN05 and pTN1, were isolated. Comparison of the amino acid sequences predicted from the respective cDNA sequences revealed that they were essentially identical, except that pTN1 lacked a sequence corresponding to residues Tyr293-Pro325 of the longer cDNA clone (pTN05). Based on the structural analysis of purified enzyme forms, the CE precursor was found to comprise a signal peptide, a prosequence, and a mature protein region of 19, 39, and 337 (pTN05) or 304 (pTN1) residues, respectively. Despite a high degree of similarity in the overall structure of CE between rat and other mammalian species, the first 11 residues in the NH2-terminal sequence of rat mature enzyme were significantly different from those of other species. The purified pro-CE was analyzed for its conversion to the mature form. The results indicated that the maximal conversion occurred at pH 3.0-4.0 in a temperature- and time-dependent manner by autocatalytic cleavage at the site between Phe39-Ser40. This conversion was highly dependent on the protein concentrations of pro-CE and delayed by the presence of exogenous substrates, suggesting the predominance of intermolecular reaction for its conversion to the mature form.

Amino Acid Sequence↗
PubMed1995

Structure of guanine-nucleotide-exchange factor human Mss4 and identification of its Rab-interacting surface.

Guanine-nucleotide-exchange factors (GEFs) promote the exchange of GDP for GTP in Ras GTPases, and thereby positively regulate their functions. Members of the Sec4/Ypt1/Rab branch of the Ras superfamily are essential for vesicular transport. A GEF for a subset of Rab proteins, termed mammalian suppressor of Sec4 (Mss4), has been identified. Here we use multidimensional NMR to determine the structure of human Mss4 (hMss4), which is the first tertiary structure established for a protein with GEF activity. Mss4 contains a central beta-sheet sandwiched between two small sheets. It also binds a Zn2+ ion through Cys 23, Cys 26, Cys 94 and Cys 97. The Rab-binding surface of hMss4 has subsequently been delineated using chemical-shift perturbation experiments and site-directed mutagenesis. The active site of hMss4 involves the Zn(2+)-binding region and a neighbouring loop.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of the human ryudocan promoter.

The promoter region of human ryudocan was isolated from a human lambda dash genomic library and cloned into pBluescript. The 5'flanking region contained a classical TATA box and GC rich regions that are commonly found in constitutively expressed genes. Two fusion gene constructs, one of 898 bp and the other of 480 bp of the 5' flanking DNA coupled to the luciferase gene, were transiently expressed in a mouse endothelioma cell line and in human umbilical endothelial cell cultures. Both constructs were capable of driving luciferase expression; the 898 bp construct produced greater levels of luciferase activity than the 480 bp construct in both cell types examined. Analysis of the sequence revealed the presence of several potential sites for nuclear transcription factor binding; the relevance of these sites is presently unknown.

Base Sequence↗
PubMed1995

Cloning, Zn2+ binding, and structural characterization of the guanine nucleotide exchange factor human Mss4.

The Sec4/Ypt1/Rab family of small GTP-binding proteins are involved in the regulation of intracellular vesicular transport. A rat gene, mss4, that encodes a guanine nucleotide exchange factor (GEF) for Sec4 was recently cloned by its ability to rescue defects in protein transport of a yeast temperature-sensitive (ts) mutant, sec4-8. We describe herein the cloning, bacterial expression, and biochemical characterization of human Mss4. As expected, both the cDNA and its encoded amino acid sequences are highly conserved between the human and rat mss4. Soluble and functional Mss4 protein was obtained by expressing the gene as a glutathione-S-transferase fusion protein in Escherichia coli. Subsequent biochemical analysis revealed that Mss4 binds 1 equiv of Zn2+, and zinc is essential for the stability of the protein. Utilizing multidimensional heteronuclear NMR techniques, we assigned most of the 1H, 15N, and 13C resonances of this 14-kDa protein. Its secondary structure was also deduced from slowly exchanging amide protons, characteristic NOEs, and 3JNH-C alpha H coupling constants. The protein contains a central seven-stranded antiparallel beta sheet, flanked by two small beta sheets. Many resonances pertaining to a loop region of the molecule cannot be identified, suggesting that it might be involved in local movements. These studies provide the first structural insights into a protein possessing GEF activity.

Amino Acid Sequence↗
PubMed1995

Dissociation of mitogen-activated protein kinase activation from the oxidative burst in differentiated HL-60 cells and human neutrophils.

In human polymorphonuclear leukocytes (PMNs), mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (Erks), are activated within minutes upon stimulation with either chemoattractant formyl-Met-Leu-Phe (fMLP) or phorbol 12-myristate 13-acetate (PMA). This activation of MAPKs coincides with the formation of superoxide anion, which occurs through the activation of a multiple-component NADPH oxidase pathway. MAPKs have thus been suggested to be involved in signal transduction leading to the oxidative burst. To investigate whether MAPK activation plays a central role in the oxidative burst, we evaluated the effect of cAMP on MAPK activation induced by fMLP and PMA. cAMP inhibits many PMN functional responses, including the oxidative burst, and has recently been shown to reduce growth factor- and PMA-induced MAPK activities in a variety of cells. We found that in differentiated, neutrophil-like HL-60 cells, while cAMP reduced PMA-induced MAPK activation, it had no effect on fMLP-induced MAPK activation. Despite the presence of unchanged levels of activated MAPKs, the fMLP-induced oxidative burst was substantially diminished by cAMP. By contrast, O2-production induced by PMA remained the same even though MAPK activation was inhibited. In PMNs, although the levels of O2-induced by either 10 ng/ml or 100 ng/ml PMA were similar, only 100 ng/ml could stimulate MAPK activation, suggesting that the oxidative burst could occur in the absence of detectable activation of MAPKs. As in HL-60 cells, cAMP inhibited the O2-production in fMLP-stimulated PMNs but had no effect on MAPK activity. These results demonstrate that, while MAPK activation coincides with PMN activation, it can be dissociated from the oxidative burst.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1995

Purification and properties of wild-type and exonuclease-deficient DNA polymerase II from Escherichia coli.

Wild-type DNA polymerase II (pol II) and an exonuclease-deficient pol II mutant (D155A/E157A) have been overexpressed and purified in high yield from Escherichia coli. Wild-type pol II exhibits a high proofreading 3'-exonuclease to polymerase ratio, similar in magnitude to that observed for bacteriophage T4 DNA polymerase. While copying a 250-nucleotide region of the lacZ alpha gene, the fidelity of wild-type pol II is high, with error rates for single-base substitution and frameshift errors being < or = 10(-6). In contrast, the pol II exonuclease-deficient mutant generated a variety of base substitution and single base frameshift errors, as well as deletions between both perfect and imperfect directly repeated sequences separated by a few to hundreds of nucleotides. Error rates for the pol II exonuclease-deficient mutant were from > or = 13- to > or = 240-fold higher than for wild-type pol II, depending on the type of error considered. These data suggest that from 90 to > 99% of base substitutions, frameshifts, and large deletions are efficiently proofread by the enzyme. The results of these experiments together with recent in vivo studies suggest an important role for pol II in the fidelity of DNA synthesis in cells.

Base Sequence↗
PubMed1995

Pacemaker current i(f) in adult canine cardiac ventricular myocytes.

1. Single cells enzymatically isolated from canine ventricle and canine Purkinje fibres were studied with the whole-cell patch clamp technique, and the properties of the pacemaker current i(f) compared. 2. Steady-state i(f) activation occurred in canine ventricular myocytes at more negative potentials (-120 to -170 mV) than in canine Purkinje cells (-80 to -130 mV). 3. Reversal potentials were obtained in various extracellular Na+ (140, 79 or 37 mM) and K+ concentrations (25, 9 or 5.4 mM) to determine the ionic selectivity of i(f) in the ventricle. The results suggest that this current was carried by both sodium and potassium ions. 4. The plots of the time constants of i(f) activation against voltage were 'bell shaped' in both canine ventricular and Purkinje myocytes. The curve for the ventricular myocytes was shifted about 30 mV in the negative direction. In both ventricular and Purkinje myocytes, the fully activated I-V relationship exhibited outward rectification in 5.4 mM extracellular K+. 5. Calyculin A (0.5 microM) increased i(f) by shifting its activation to more positive potentials in ventricular myocytes. Protein kinase inhibition by H-7 (200 microM) or H-8 (100 microM) reversed the positive voltage shift of i(f) activation. This effect of calyculin A also occurred when the permeabilized patch was used for whole-cell recording. 6. These results indicate i(f) is present in ventricular myocytes. If shifted to more positive potentials i(f) could play a role in ischaemia-induced ventricular arrhythmias. The negative shift of i(f) in the ventricle might play a role in differentiating non-pacing regions of the heart from those regions that pace.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Prostate-specific antigen is a new favorable prognostic indicator for women with breast cancer.

Prostate-specific antigen (PSA) is thought to be produced exclusively by prostatic epithelial cells and is currently used as a tumor marker of prostatic adenocarcinoma. We recently found that 30% of breast cancers contain PSA immunoreactivity (IR-PSA). To examine the prognostic value of PSA in female breast cancer, we measured IR-PSA in tumor cytosols of 174 breast cancer patients and classified the breast cancers as either PSA positive or PSA negative based on an IR-PSA cutoff level of 0.03 ng/mg. IR-PSA was present in 27% of the patients. IR-PSA presence was associated with early disease stage, small tumors, and estrogen receptor-positive tumors. We used the Cox proportional hazards regression model to analyze survival of patients in association with PSA status and found that patients with IR-PSA-positive tumors had a reduced risk for relapse and death in univariate analysis (P = 0.02 and 0.06, respectively) and a reduced risk for relapse in multivariate analysis (P = 0.03). Further analysis indicated that the effect of IR-PSA on relapse-free survival was evident in node-positive or estrogen receptor-negative patients. Our study suggests that IR-PSA is an independent favorable prognostic marker for breast cancer and may be used to identify a subgroup of estrogen receptor-negative and/or node-positive patients who have good prognoses.

Adult↗
PubMed1995

Expression of the prostate-specific antigen gene by a primary ovarian carcinoma.

We describe a patient with primary ovarian carcinoma that developed after liver transplantation whose tumor was highly positive for prostate-specific antigen (PSA). PSA in tumor tissue was characterized by two immunoassays, HPLC, immunohistochemistry, reverse transcription-PCR, Southern blotting, and DNA sequencing. PSA in the ovarian tumor was present as free, M(r) 33,000 protein (> 90%) and as PSA bound to alpha 1-antichymotrypsin (M(r) 100,000; < 10%). Immunohistochemistry localized PSA in the cytoplasm of epithelial cells of the tumor. Two separate reverse transcription-PCRs for PSA amplified the expected products which hybridized specifically to a PSA cDNA probe on Southern blots. Sequencing of the PCR products, representing the whole coding sequence of the PSA gene, revealed identity with the sequence of PSA cDNA from prostate tissue. These data suggest that the PSA produced by the ovarian tumor was identical in molecular weight and sequence to prostatic PSA. Based on data of tissue culture experiments with breast carcinoma cell lines, we speculate that the PSA gene in the tumor of this patient was up-regulated by the therapeutically administered glucocorticoids after liver transplantation.

Base Sequence↗
PubMed1995

Oral contraceptive-induced expression of prostate-specific antigen in the female breast.

Prostate-specific antigen (PSA) is widely used as a tumor marker of prostatic adenocarcinoma. We recently found that 30% of breast tumors produce PSA and that PSA is a favorable prognostic marker in female breast cancer. We measured immunoreactive PSA in cytosolic extracts of normal breast tissue from eight women receiving no medication and one woman who was receiving no medication and one woman who was receiving the progestin-containing oral contraceptive Brevicon. None of the eight cytosolic extracts of normal breast tissue contained appreciable amounts of immunoreactive PSA. However, left and right breast tissues from the woman receiving Brevicon contained high levels of PSA. This immunoreactive species was shown to have a molecular weight identical to that of seminal PSA. Furthermore, reverse transcription of RNA and polymerase chain reaction amplification produced a 571-base pair cDNA that hybridized to a labeled cDNA PSA probe. Upon sequencing, the cDNA polymerase chain reaction product was found to have 100% homology with cDNA from prostatic tissue. PSA production by breast carcinoma cell lines was achieved after in vitro stimulation with norethindrone and ethinylestradiol. Our data suggest that PSA can no longer be regarded as a specific prostatic protein because it is produced by breast tumors with good prognosis and by normal breast tissue after steroid hormone stimulation.

Base Sequence↗
PubMed1995

Kinectin, an essential anchor for kinesin-driven vesicle motility.

The membrane anchor for the molecular motor kinesin is a critical site involved in intracellular membrane trafficking. Monoclonal antibodies specific for the cytoplasmic surface of chick brain microsomes were used to define proteins involved in microtubule-dependent transport. One of four antibodies tested inhibited plus-end-directed vesicle motility by approximately 90 percent even as a monovalent Fab fragment and reduced kinesin binding to vesicles. This antibody bound to the cytoplasmic domain of kinectin, an integral membrane protein of the endoplasmic reticulum that binds to kinesin. Thus, kinectin acted as a membrane anchor protein for kinesin-driven vesicle motility.

Animals↗
PubMed1995

Mutant p53 protein overexpression is associated with poor outcome in patients with well or moderately differentiated ovarian carcinoma.

BACKGROUND: It has been shown that the p53 gene is mutated in 30-80% of ovarian carcinomas and that the genetic alterations most often manifest as an accumulation of mutant p53 protein in tumor tissue. The prognostic significance of these findings for patients with ovarian cancer, however, must be established clearly. METHODS: Mutant p53 protein in 90 consecutive epithelial ovarian carcinomas was quantitatively analyzed using a time-resolved immunofluorometric procedure. In contrast to immunohistochemical techniques, this method uses two anti-p53 antibodies. The Cox model was used to evaluate the strength of the associations between the prognostic markers and disease relapse or death at univariate and multivariate levels. Kaplan-Meier survival curves were calculated for patients who were p53-positive or negative and for subgroups with a different clinical stage, histologic grade, or residual postsurgical tumor. RESULTS: The positivity rates for p53 included 1/21 (5%) with Stage I disease, 1/6 (17%) with Stage II, 29/51 (57%) with Stage III, and 8/12 (67%) with Stage IV (total = 39/90, 43%). Patients with p53-negative tumors had a significantly longer disease free survival than did patients with p53-positive tumors (P = 0.03); these results were similar for overall survival (P = 0.06). Multivariate analysis revealed that the presence of postsurgical residual tumor was the only predictor significantly associated with poor patient outcome. However, when patients were divided into groups based on histologic grade, patients with well (G1) and moderately (G2) differentiated tumors had a significantly higher risk of cancer relapse and death if mutant p53 protein was present in their tumors compared with patients who were negative for mutant p53 protein (< 0.01). CONCLUSIONS: The immunofluorometric measurement of mutant p53 protein accumulation in epithelial ovarian carcinomas of a low histologic grade was associated significantly with an increased risk for cancer relapse and death. A similar trend also was suggested for early stage disease and in the absence of residual tumor after surgery. These increased risks, however, were not found for patients with high grade or advanced stage cancer or for those with residual tumor. To the authors' knowledge, this is the first report suggesting that p53 tumor protein accumulation is a marker of poor prognosis in a subset of patients with ovarian cancer.

Adult↗