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Biomedical subjects

L Chu

Publications and source records attributed to L Chu.

At least 37 records · Page 2Linked to original sources
PubMed1998

Genetic alterations in primary bladder cancers and their metastases.

Bladder cancer progression is thought to be associated with sequential genetic events. To search for the specific genetic changes associated with the metastatic process, comparative genomic hybridization was performed on 22 primary tumors and 24 metastases (10 distant and 14 nodal metastases) from 17 patients with stage pT2-4 bladder cancer. There was a striking similarity between the genetic alterations present in the primary and metastatic tumor samples from the same patient. The mean number of genetic changes/tumor was 12.2 for primary tumors and 11.7 for metastases. There was a strong concordance in the specific aberrations present in each patient's primary and metastatic lesions (mean, 75%). Concordance was also high among multiple sites from an individual primary tumor (mean, 96%) and multiple metastases from the same patient (mean, 75%). There were no specific genetic changes overrepresented in the metastases compared with their primary tumors. Genetic alterations present in more than 40% of tumors included gains on 6p, 8q, 10q, and 17q and losses involving 8p, 10q, and Y. Two regions of high-level amplification were common: (a) 10q22.1-q23.1 (32.6%); and (b) 17q11-21.3 (23.9%; the locus of erbB-2). A summary statistic was developed to quantitate the degree of clonal relationships between biopsies from the same patient. These data support a model in which minimal clonal evolution occurs in the metastatic tumor cell population after the metastatic event. When comparing primary cancers from patients with and without metastases, however, several unique genetic changes were identified in those cancers with metastases, suggesting that these loci may harbor genes important to the metastatic process.

Aged↗
PubMed1998

A critical role for complement in maintenance of self-tolerance.

The role of complement in the maintenance of self-tolerance has been examined in two models: an immunoglobulin transgenic model of peripheral tolerance and a lupus-like murine model of CD95 (Fas) deficiency. We find that self-reactive B lymphocytes deficient in complement receptors CD21/CD35 or transferred into mice deficient in the complement protein C4 are not anergized by soluble self-antigen. In the second model, deficiency in CD21/CD35 or C4 combined with CD95 deficiency results in high titers of anti-nuclear antibodies leading to severe lupus-like disease. These findings suggest a novel role for the complement system in B cell tolerance and provide insight into the genetic association of complement deficiency with susceptibility to systemic lupus erythematosus.

Animals↗
PubMed1997

Genetic alterations in lobular breast cancer by comparative genomic hybridization.

Infiltrating lobular carcinoma (ILC) and infiltrating ductal carcinoma (IDC) are distinguished by their histopathological appearance. However, little is known about the differences in genetic changes between lobular cancers and ductal cancers. We used comparative genomic hybridization (CGH) and compared aberrations in 19 ILCs and 46 IDCs. The total number of aberrations was lower in ILC than in IDC. While the average number of DNA copy number losses did not reach significance between them, copy number gains were significantly lower in ILCs. Fifteen of 19 ILCs (79%) showed increased copy number of 1q, and 12 cases (63%) revealed loss of 16q. The presence of these aberrations was independent of nodal status, histologic subtypes (pleomorphic or classic ILC), or BrdUrd-labeling index. ILCs had a higher frequency of 16q loss than did ductal cancers, and a lower frequency of 8q and 20q gains. Our data suggest that the altered growth pattern and clinical presentation which characterize infiltrating lobular cancers are correlated with distinct genetic alterations.

Adult↗
PubMed1997

Long-term erythropoietin expression in rodents and non-human primates following intramuscular injection of a replication-defective adenoviral vector.

Erythropoietin (Epo)-responsive anemia is a debilitating complication of chronic renal failure and human immunodeficiency virus (HIV) infection that effects more than 150,000 Americans. Patients with Epo-responsive anemias are currently treated with repeated injections of recombinant human Epo. In the studies described in this report, we have examined the safety and efficacy of using a single intramuscular (i.m.) injection of replication-defective adenoviral vectors (RDAd) encoding Epo for the treatment of Epo-responsive anemias in both mice and non-human primates. Our results demonstrate that there is a threshold dose of virus (2.5-8 x 10(7) pfu/gram of body weight) which is required to obtain long-term Epo expression and polycythemia in both species. A single i.m. injection of mice with 10(9) pfu of an RDAd encoding murine Epo (AdmEpo) resulted in elevations in hematocrits from control values of 49 +/- 0.9% to treated values of 81 +/- 3%, which were stable for more than 1 year. Similarly, a single i.m. injection of a monkey with 4 x 10(11) pfu of an RDAd-encoding simian Epo (AdsEpo) resulted in elevations of hematocrits from control levels of 40% to treated levels of > or =70%, which were stable for 84 days. Intramuscular injection of monkeys with AdsEpo appeared to be safe in that we did not detect abnormalities in chest X-rays, serum chemistries, hematologic, or clotting profiles (apart from elevated hematocrits) or organ histologies during the 84-day time course of the experiment. Taken together, these results suggest the feasibility of using i.m. injection of RDAd for the treatment of Epo-responsive anemias in humans.

Adenoviridae↗
PubMed1997

Transcriptional targeting of replication-defective adenovirus transgene expression to smooth muscle cells in vivo.

Gene transfer using replication-defective adenoviruses (RDAd) holds promise for the treatment of vascular proliferative disorders, but is potentially limited by the capacity of these viruses to infect multiple cell lineages. We have generated an RDAd vector, designated AdSM22-lacZ, which encodes the bacterial lacZ reporter gene under the transcriptional control of the smooth muscle cell (SMC)-specific SM22alpha promoter. Here, we show that in vitro AdSM22-lacZ programs expression of the lacZ reporter gene in primary rat aortic SMCs and immortalized A7r5 SMCs, but not in primary human umbilical vein endothelial cells (HUVECs) or NIH 3T3 cells. Consistent with these results, after intraarterial administration of AdSM22-lacZ to control and balloon-injured rat carotid arteries, beta-galactosidase activity was detected within SMCs of the tunica media and neointima, but not within endothelial or adventitial cells. Moreover, intravenous administration of AdSM22-lacZ did not result in lacZ gene expression in the liver or lungs. Finally, we have shown that direct injection of AdSM22-lacZ into SMC-containing tissues such as the ureter and bladder results in high-level transgene expression in visceral SMCs. Taken together, these results demonstrate that transgene expression after infection with an RDAd vector can be regulated in an SMC lineage-restricted fashion by using a transcriptional cassette containing the SMC-specific SM22alpha promoter. The demonstration of an efficient gene delivery system targeted specifically to SMCs provides a novel means to restrict expression of recombinant gene products to vascular or visceral SMCs in vivo.

Adenoviridae↗
PubMed1997

Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities.

A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa proteins were purified to homogeneity. Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(cat) = 12 s(-1). Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by beta-mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme, with activity of an alphaC-N and betaC-S lyase (cystathionase) type. Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule.

Cystathionine gamma-Lyase↗
PubMed1997

The human cytomegalovirus UL97 protein is a protein kinase that autophosphorylates on serines and threonines.

The product of the human cytomegalovirus (CMV) UL97 gene, which controls ganciclovir phosphorylation in virus-infected cells, is homologous to known protein kinases but diverges from them at a number of positions that are functionally important. To investigate UL97, we raised an antibody against it and overexpressed it in baculovirus-infected insect cells. Recombinant baculovirus expressing full-length UL97 directed the phosphorylation of ganciclovir in insect cells, which was abolished by a four-codon deletion that confers ganciclovir resistance to CMV. When incubated with [gamma-32P]ATP, full-length UL97 was phosphorylated on serine and threonine residues. Phosphorylation was severely impaired by a point mutation that alters lysine-355 in a motif that aligns with subdomain II of protein kinases. However, phosphorylation was impaired much less severely by the four-codon deletion. A UL97 fusion protein expressed from recombinant baculovirus was purified to near homogeneity. It too was phosphorylated upon incubation with [gamma-32P]ATP in vitro. This phosphorylation, which was abolished by the lysine 355 mutation, was optimal at high NaCl and high pH. The activity required either Mn2+ or Mg2+, with a preference for Mn2+, and utilized either ATP or GTP as a phosphate donor, with Kms of 2 and 4 microM, respectively. The phosphorylation rate was first order with protein concentration, consistent with autophosphorylation. These data strongly argue that UL97 is a serine/threonine protein kinase that autophosphorylates and suggest that the four-codon deletion affects its substrate specificity.

Animals↗
PubMed1997

Adherence of mononuclear cells to endothelium in vitro is increased in patients with NIDDM.

OBJECTIVE: To compare the binding to cultured endothelial cells of mononuclear cells isolated from healthy volunteers and patients with NIDDM. RESEARCH DESIGN AND METHODS: Mononuclear cells were isolated from healthy volunteers (n = 11) and patients with NIDDM (n = 14) and incubated with ECV 304 cells, a human umbilical endothelial cell-derived transformed cell line. Following a period of incubation, the adherence of mononuclear cells to endothelial cells was determined. RESULTS: Adherence of mononuclear cells from patients with NIDDM was significantly greater (P < 0.05) than that of cells isolated from the healthy volunteers, and this difference persisted when adjusted for age, sex, and degree of obesity. Mononuclear cell binding to ECV 304 cells correlated significantly with fasting plasma glucose (r = 0.52, P < 0.01), insulin (r = 0.51, P < 0.01), triglyceride (r = 0.54, P < 0.01), and VLDL (r = 0.54, P < 0.01) and HDL cholesterol (r = -0.45, P < 0.05) levels, but not with either total or LDL cholesterol levels or blood pressure. CONCLUSIONS: Since the adherence of mononuclear cells to the endothelium represents the earliest step in atherogenesis, the observation that mononuclear cells from patients with NIDDM bind more avidly to cultured endothelial cells may help explain why accelerated atherosclerosis occurs in patients with NIDDM. The metabolic abnormality, or abnormalities, present in patients with NIDDM that is responsible for the enhanced adhesiveness of mononuclear cells requires further examination.

Cell Adhesion↗
PubMed1997

Inhibition of platelet-derived growth factor-mediated signal transduction and tumor growth by N-[4-(trifluoromethyl)-phenyl]5-methylisoxazole-4-carboxamide.

Many reports have cited coexpression of platelet-derived growth factor (PDGF) and its receptors by tumor cells or cells supporting tumor growth, suggesting both autocrine and paracrine mechanisms for PDGF-mediated tumor growth. We found that a small organic molecule, N-[4-(trifluoromethyl)phenyl] 5-methylisoxazole-4-carboxamide (SU101, leflunomide), inhibited PDGF-mediated signaling events, including receptor tyrosine phosphorylation, DNA synthesis, cell cycle progression, and cell proliferation. SU101 inhibited PDGF-stimulated tyrosine phosphorylation of PDGF receptor (PDGFR) beta in C6 (rat glioma) and NIH3T3 cells engineered to overexpress human PDGFRbeta (3T3-PDGFRbeta). SU101 blocked both PDGF- and epidermal growth factor (EGF)-stimulated DNA synthesis. Previously, this compound was shown to inhibit pyrimidine biosynthesis by interfering with the enzymatic activity of dihydroorotate dehydrogenase. In the current study, EGF-stimulated DNA synthesis was restored by the addition of saturating quantities of uridine, whereas PDGF-induced DNA synthesis was not, suggesting that the compound demonstrated some selectivity for the PDGFR pathway that was independent of pyrimidine biosynthesis. Selectivity was further demonstrated by the ability of the compound to block the entry of PDGF-stimulated cells into the S phase of the cell cycle, without affecting cell cycle progression of EGF-stimulated cells. In cell growth assays, SU101 selectively inhibited the growth of PDGFRbeta-expressing cell lines more efficiently than it inhibited the growth of PDGFRbeta-negative cell lines. SU101 inhibited the s.c., i.p., and intracerebral growth of a panel of cell lines including cells from glioma, ovarian, and prostate origin. In contrast, SU101 failed to inhibit the in vitro or s.c. growth of A431 and KB tumor cells, both of which express EGF receptor but not PDGFRbeta. SU101 also inhibited the growth of D1B and L1210 (murine leukemia) cells in syngeneic immunocompetent mice, without causing adverse effects on the immune response of the animals. In an i.p. model of tumor growth in syngeneic immunocompetent mice, SU101 prevented tumor growth and induced long-term survivors in animals implanted with 7TD1 (murine B-cell hybridoma) tumor cells. Because PDGFRbeta was detected on most of the tumor cell lines in which in vivo growth was inhibited by SU101, these data suggest that SU101 is an effective inhibitor of PDGF-driven tumor growth in vivo.

3T3 Cells↗
PubMed1996

p53 gene alterations and p53 protein in oral epithelial dysplasia and squamous cell carcinoma.

To examine the expression of p53 protein and gene alterations in oral epithelial lesions including epithelial dysplasias and primary squamous cell carcinomas, immunohistochemical and temperature gradient gel electrophoresis (TGGE) methods were applied to formalin-fixed and paraffin-embedded tissues. Morphologically normal mucosal epithelium stained negatively for p53 protein. Three out of 11 (27.3 per cent) epithelial dysplasias and 19 out of 57 (33.3 per cent) primary squamous cell carcinomas stained positively for p53 protein. Although more than half of the cases were positive for p53 protein in stage I, the positive cancer cases were found at other stages with variable frequency. Immunoreactive products were localized in the nucleus, especially in the basal and suprabasal layers. The analysis by TGGE revealed gene alterations in exons 5-8 in 3 out of 3 epithelial dysplasias and 17 out of 19 (89.5 per cent) primary squamous cell carcinomas which were immunohistochemically positive for p53 protein. These results suggest that p53 gene mutation may be involved in carcinogenesis in the oral squamous epithelium even in the early stage of the dysplasia-carcinoma sequence.

Adult↗
PubMed1996

Isolation and characterization of a hemin-binding cell envelope protein from Porphyromonas gingivalis.

A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is both hemin and iron regulated was identified and purified in Porphyromonas gingivalis 381. A strong hemin-binding function was found by LDS-PAGE and TMBZ staining when cells were grown under hemin (iron)-limited conditions. N-terminal amino acid sequence analysis of CNBr-digested 24 kDa hemin binding protein revealed that this protein belongs to a new, so far undescribed hemin-binding class of proteins.

Amino Acid Sequence↗
PubMed1996

Feasibility of non-invasive measurement of tissue pH using near-infrared reflectance spectroscopy.

OBJECTIVE: Tissue pH measurement has a number of clinical applications, including the monitoring of both muscle pH and organ pH as an indicator of compromised blood flow and anaerobic metabolism. The objective of this work was to demonstrate the feasibility of a noninvasive measurement of deep tissue pH using near-infrared reflectance spectroscopy and multivariate calibration techniques. METHODS: Six studies were done on five New Zealand white rabbits. Two pH electrodes were implanted in the teres major muscle and a vascular clamp placed across the single artery feeding the muscle. Reflected light was collected through the skin from a site between the two electrodes as the pH was lowered by closing the clamp and raised by opening the clamp. Partial least squares analysis with cross-validation techniques was used to relate pH to light absorption at 201 evenly spaced wavelengths between 700 and 1100 nm. RESULTS: On average, the tissue pH started at 7.13 +/- 0.09 and decreased to 6.74 +/- 0.09, returning to 7.13 +/- 0.09 after reperfusion. Calibration models fit for each rabbit had an average of nine factors with an R2 of 0.98 and a prediction error of 0.016 +/- 0.002 pH units. CONCLUSIONS: We believe this to be the first in vivo demonstration of a non-invasive method for measuring tissue pH in skin-covered muscle using near-infrared reflectance spectroscopy and multivariate calibration techniques.

Animals↗
PubMed1996

Intracellular inducer Hg2+ concentration is rate determining for the expression of the mercury-resistance operon in cells.

Experiments involving mercury resistance mer operon-lacZ fusions, point mutations in the mercuric ion reductase merA gene, and transcomplementation have revealed that in Hg2+-resistant cells, the inducer Hg2+ concentration is rate determining for activation of transcription. mer operon expression is activated by the presence of nanomolar concentrations of Hg2+ in liquid media only when the mercuric ion reductase function is artificially inactivated in cells, whereas cells with active mercuric ion reductase require micromolar concentrations of Hg2+ for effective induction of the operon.

Bacterial Proteins↗
PubMed1996

Continuous infusion 5-fluorouracil as first-line therapy for metastatic breast cancer.

Previous phase II studies of continuous infusion Fluorouracil (5-FU) (CI 5-FU) in refractory metastatic breast cancer have shown modest activity with low toxicity. Its activity in a first-line setting has not been formally tested. Patients were eligible if they fulfilled the following criteria: metastatic breast cancer; measurable or evaluable disease, no prior chemotherapy in the metastatic setting; ECOG performance status of 0, 1, or 2: adequate bone marrow and liver function. Patients were treated with 5-FU 250 mg/m2 per day by continuous intravenous infusion for 5 weeks in a 6-week cycle. Treatment was continued until disease progression or unacceptable toxicity. In addition to the traditional endpoints of response, survival, and toxicity, quality of life was assessed with the Functional Living Index-Cancer (FLIC) and the Symptom Distress Scale (SDS). Twenty-one patients were enrolled. Among the 16 patients with measurable disease, the objective response rate was 44% (95% CI 20%, 68%) with CR rate 13% and PR rate 31%. The median duration of response was 37 weeks. Responses were not observed in patients with visceral (lung or liver) disease. Among all 21 patients in the study, the median time to disease progression was 12 weeks, and median overall survival was 64 weeks. Grade 1 or 2 mucosal and cutaneous toxicity were common. Only 4 patients (19%) had toxicity greater than grade 2; three patients had grade 3 mucositis, and 1 patient developed an indwelling catheter infection requiring its removal. Among responding patients, mean FLIC scores improved from 114.3 at baseline to 128.7 at week 8 (p = 0.11). Symptoms reported on the SDS generally improved in responding patients. Continuous infusion 5-FU as a first-line therapy for metastatic breast cancer has moderate activity and low toxicity. Its use should be considered in the first-line setting when toxicity needs to be minimized.

Ambulatory Care↗
PubMed1995

Do the saccadic system and the vestibulo-ocular reflex of monkeys share a common neural integrator?

A common 'neural integrator' has been hypothesized to perform mathematical integration of velocity-coded inputs for all oculomotor subsystems. Studies in cats suggested that the integrators for some subsystems may be differentially affected by drugs. Our tests in macaques show that the fixation is unaffected and saccadic and vestibulo-ocular systems are affected in parallel. Thus, the common integrator hypothesis cannot be ruled out on the basis of these results.

Animals↗
PubMed1995

The 46-kilodalton-hemolysin gene from Treponema denticola encodes a novel hemolysin homologous to aminotransferases.

The 46-kDa hemolysin produced by Treponema denticola may be involved in the etiology of periodontitis. In order to initiate a genetic analysis of the role of this protein in disease, its gene has been cloned. Synthetic oligonucleotides, designed on the basis of the previously reported amino-terminal amino acid sequence of the 45-kDa hemolysin, were used as primers in a PCR to amplify part of the hemolysin (hly) gene. This PCR product was then used to clone the entire hly gene from libraries of T. denticola genomic DNA. Constructs containing the entire cloned region on plasmids in Escherichia coli produced both hemolysis and hemoxidation activities either on sheep blood agar plates or in liquid assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis revealed that the constructs synthesized a protein with molecular size of about 46 kDa which was reactive with anti-T. denticola hemolysin. Nucleotide sequence analysis indicated that the largest open reading frame could encode a protein with a calculated molecular size of 46.2 kDa. The first 31 amino acids encoded by this open reading frame were identical to the experimentally determined amino-terminal sequence of the 45-kDa hemolysin. These results indicate that the entire hly gene has been cloned. The deduced amino acid sequence of the T. denticola hly gene is homologous (23 to 37% identity) to those of proteins that are members of a family of pyridoxal-phosphate-dependent aminotransferases. This suggests that the 46-kDa hemolysin may be related to an aminotransferase and have a novel mechanism of hemolysis. However, the functional aspects of this relationship remain to be investigated.

Amino Acid Sequence↗
PubMed1995

Glomus tumor of the upper lip.

A case of glomus tumor of the upper lip in a 57-year-old Japanese male is described. The patient had a tender swelling of the upper lip, and this was completely excised. The lesion was found to be encapsulated by fibrous connective tissue and composed of blood capillaries surrounded by sheets of epitheloid cells. Immunohistochemical examination revealed that the neoplastic cells were positive for S-100 protein, vimentin, desmin and actin. Although the endothelial cells of blood capillaries in the tumor tissue were positive for factor VIII, no reaction product was found in the tumor cells. These results suggested that the tumor cells had characteristics of smooth muscle cells.

Actins↗