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Biomedical subjects

L Chen

Publications and source records attributed to L Chen.

At least 937 records · Page 52Linked to original sources
PubMed1996

Paraquat toxicity and oxidative damage. Reduction by melatonin.

The ability of melatonin to protect against paraquat-induced oxidative damage in rat lung, liver, and serum was examined. Changes in the levels of malondialdehyde (MDA) plus 4-hydroxyalkenals (4-HDA) and reduced and oxidized glutathione concentrations were measured. Paraquat (50 mg/kg) was injected i.p. into either Sprague-Dawley or Wistar rats with or without the co-administration of 5 mg/kg melatonin. Paraquat alone increased MDA + 4-HDA levels in serum and lungs of both rat strains, with these increases being abolished by melatonin co-treatment. Paraquat also decreased reduced glutathione levels and increased oxidized glutathione concentrations in lung and liver; these changes were negated by melatonin. The effect of melatonin on paraquat-induced mortality was also studied. Paraquat at a dose of 79 mg/kg was lethal for 50% of animals within 24 hr; when administered together with melatonin, the LD50 for paraquat increased to 251 mg/kg.

Animals↗
PubMed1996

Impaired classical eyeblink conditioning in cerebellar-lesioned and Purkinje cell degeneration (pcd) mutant mice.

Converging lines of evidence from rabbits, rats, and humans argue for the crucial involvement of the cerebellum in classical conditioning of the eyeblink/nictitating membrane response in mammals. For example, selective lesions (permanent or reversible) of the cerebellum block both acquisition and retention of eyeblink conditioning. Correspondingly, electrophysiological and brain-imaging studies indicate learning-related plasticity in the cerebellum. The involvement of the cerebellum in eyeblink conditioning is also supported by stimulation studies showing that direct stimulation of the two major afferents to the cerebellum (the mossy fibers emanating from the pontine nucleus and climbing fibers originating from the inferior olive) can substitute for the peripheral conditioned stimulus (CS) and unconditioned stimulus (US), respectively, to yield normal behavioral learning. In the present study, we examined the relative contribution of the cerebellar cortex versus deep nuclei (specifically the interpositus nucleus) in eyeblink learning by using mutant mice deficient of Purkinje cells, the exclusive output neurons of the cerebellar cortex. We report that Purkinje cell degeneration (pcd) mice exhibit a profound impairment in the acquisition of delay eyeblink conditioning in comparison with their wild-type littermates. Nevertheless, the pcd animals did acquire a subnormal level of conditioned eyeblink responses. In contrast, wild-type mice with lesions of the interpositus nucleus were completely unable to learn the conditioned eyeblink response. These results suggest that both cerebellar cortex and deep nuclei are important for normal eyeblink conditioning.

Animals↗
PubMed1996

In vivo replication-deficient adenovirus vector-mediated transduction of the cytosine deaminase gene sensitizes glioma cells to 5-fluorocytosine.

Viral vector-mediated transfer of chemosensitization genes represents a promising new approach to the treatment of cancer. Previous reports have demonstrated that transfection of the bacterial cytosine deaminase (cd) gene into mammalian cells can sensitize them to the otherwise nontoxic nucleoside, 5-fluorocytosine (5-FC). We now report that a replication-deficient adenovirus vector that transduces the cd gene (Ad.CMV-cd) highly sensitizes 9L gliosarcoma cells to 5-FC, and that gene transduction is associated with a potent bystander effect that is not dependent on direct cell-to-cell contact. Stereotactic injection of Ad.CMV-cd into established rat gliomas, followed by systemic administration of 5-FC in vivo, results in prolongation of survival.

Adenoviruses, Human↗
PubMed1996

Potent low molecular weight substrates for protein-tyrosine phosphatase.

The ability of protein-tyrosine phosphatases (PTPases) to catalyze the hydrolysis of simple aromatic phosphates has been recognized for some time. However, these compounds are significantly poorer substrates than their peptide-based counterparts containing phosphotyrosine. Consequently, the effort to create potent PTPase substrates has predominantly focused on the use of peptidic carriers to deliver the phosphotyrosine moiety to the enzyme active site. We now report the synthesis and evaluation of several low molecular weight aromatic phosphates that serve as robust substrates for the rat PTPase, PTP1. We initially surveyed the ability of PTP1 to catalyze the hydrolysis of a variety of phenyl phosphate structural variants. Sterically demanding substituents positioned ortho and (to a lesser extent) meta to the phosphate group severely compromise the ability of these species to serve as phosphatase substrates. However, both benzylic and negatively charged substituents para to the hydrolyzable phosphate dramatically promote hydrolytic efficiency, which appears to be augmented through a dramatic enhancement in the affinity of the substrate for PTP1. The best substrate examined in this study exhibits a Km of 16 +/- 3 microM. In addition, it serves as an inhibitor of the PTP1-catalyzed hydrolysis of p-nitrophenyl phosphate with a Ki of 4.9 +/- 0.7 microM. The extraordinary structural simplicity of this compound, as well as those of several others described herein, provides a promising starting point for the design of potent PTPase inhibitors.

Animals↗
PubMed1996

Sensitization of human breast cancer cells to cyclophosphamide and ifosfamide by transfer of a liver cytochrome P450 gene.

The cancer chemotherapeutic agent cyclophosphamide (CPA) and its isomer ifosfamide (IFA) are alkylating agent prodrugs that require metabolism by liver cytochrome P450 (P450) enzymes for antitumor activity. The therapeutic effectiveness of these oxazaphosphorines is limited by the hematopoietic, renal, and cardiac toxicity that accompanies the systemic distribution of liver-derived activated drug metabolites. Transfer of a liver cytochrome P450 gene, CYP2B1, into human breast MCF-7 cancer cells is presently shown to greatly sensitize these cells to oxazaphosphorine toxicity as a consequence of the acquired capacity for intratumoral CPA and IFA activation. Thus, CPA and IFA were highly cytotoxic to MCF-7 cells following stable transfection of CYP2B1 but exhibited no toxicity to parental tumor cells or to a beta-galactosidase-expressing MCF-7 transfectant. This cytotoxicity could be appreciably blocked by the CYP2B1 inhibitor metyrapone. Cell cycle analysis revealed that CPA arrested the CYP2B1-expressing cells, but not CYP2B1-negative cells, at G(2)-M phase. A strong bystander cytotoxicity effect that does not require direct cell-cell contact was mediated by CYP2B1-expressing MCF-7 cells on non-CYP2B1 cells. Intratumoral CYP2B1 expression conferred a distinct therapeutic advantage when treating MCF-7 tumors grown in nude mice with CPA, as revealed by a 15-20-fold greater in vivo cytotoxicity, determined by tumor excision/colony formation assay, and by the substantially enhanced antitumor activity, monitored by tumor growth delay, for CYP2B1-e xpressing MCF-7 tumors as compared to CYP2B1-negative control tumors. These enhanced therapeutic effects were obtained without any apparent increase in host toxicity. To evaluate the extent to which a CPA/P450 gene therapy strategy may be generally applicable to other tumor cell types, a replication-defective recombinant adenovirus carrying the CYP2B1 gene driven by the cytomegalovirus (CMV) promotor ad.CMV-2B1 was constructed and used to infect a panel of human tumor cell lines. Ad.CMV-2B1 infection rendered each of the cell lines highly sensitive to CPA and IFA cytotoxicity, with substantial chemosensitization seen at multiplicities of infection as low as 10. The CPA/P450 prodrug activation system may thus serve as a useful paradigm for further development of novel cancer gene therapy strategies that utilize drug susceptibility genes to significantly potentiate the antitumor activity of conventional cancer chemotherapeutic agents.

Adenoviridae↗
PubMed1996

B7-CD28 costimulation unveils the hierarchy of tumor epitopes recognized by major histocompatibility complex class I-restricted CD8+ cytolytic T lymphocytes.

Immunization of mice with tumors genetically engineered to express the B7 costimulatory molecules amplifies the antitumor immune response mediated by CD8+ cytolytic T lymphocytes (CTL). In this report, we examined the effect of B7-CD28 costimulation on the hierarchy of tumor epitopes. Using a combination of affinity chromatography/reversed-phase high performance liquid chromatography and CTL cloning, we show that major histocompatibility complex (MHC) class I molecules from EL4 lymphoma cells can present at least six distinct CTL epitopes presented by MHC class I molecules. Nevertheless, mice immunized with wild-type B7-negative EL4 cells develop CTL only to one immunodominant epitope. In contrast, immunization with B7-transduced EL4 cells led to not only the amplification of the CTL response to this immunodominant epitope, but also to the recognition of five otherwise silent subdominant epitopes. The adoptive transfer of a CTL clone against such a subdominant epitope cured mice bearing EL4 lymphoma growing as an ascites tumor. The fact that CTL response can be spread to normally silent epitopes as a result of B7-CD28 costimulation suggests a novel approach to manipulate the hierarchy of CTL epitopes and offers an opportunity to explore novel targets for T cell-mediated cancer therapy.

Animals↗
PubMed1996

Synaptic remodeling revealed by repeated in vivo observations and electron microscopy of identified frog neuromuscular junctions.

This work aimed to examine the mechanism of synaptic remodeling using repeated in vivo observations, followed by electron microscopy, of identified frog neuromuscular junctions (NMJs). Our previous light microscopic studies suggested that extension of synaptic extracellular matrix (ECM) precedes, and may play a role in, nerve terminal (NT) growth during synaptic remodeling. To test this hypothesis, sartorius muscles were double labeled with a fluorescent dye, 4-(4-diethylamino-styryl)-N-methylpyridinium iodide, for NTs and rhodamine-conjugated peanut agglutinin for synaptic ECM. The double-labeled NMJs were observed in vivo with video-enhanced fluorescence microscopy. Two to three months after nerve sprouting was induced by a nerve graft, the same NMJs were restained and reexamined. After the final in vivo observations, the same NMJs were examined with semiserial thin section electron microscopy. Light microscopic observation of NMJs that showed synaptic ECM longer than the NT was confirmed with electron microscopy. At junctional branches where synaptic ECM extended beyond the NT, a Schwann cell process longer than the NT was observed in one example, whereas a Schwann cell with the same length as the NT was seen in other examples. In both cases, junctional folds were absent at the extended ECM region. In contrast, junctional folds were observed at the region vacated by a retracted NT. These results suggest that extension of synaptic ECM and Schwann cell processes may lead, and play a role in, the NT growth during the remodeling of adult synaptic connections.

Animals↗
PubMed1996

Costimulation by CD48 and B7-1 induces immunity against poorly immunogenic tumors.

Genetic modification of many types of mouse tumors to express the B7-1 or B7-2 molecules, natural ligands for the T cell-costimulatory molecule CD28, increases their immunogenicity. However, even after transfection with the B7-1 and/or B7-2 genes, poorly immunogenic tumors fail to elicit and efficient immune response. We report here that two such tumors, the Ag104A sarcoma and the K1735-M2 melanoma, become immunogenic after transfection of the genes encoding murine B7-1 together with CD48, which is the natural ligand for CD2. Tumor-specific CD8+ cytotoxic T lymphocytes were readily generated and were effective for adoptive immunotherapy of metastasis induced by wild-type Ag104A sarcoma cells. A similar approach may be useful for developing therapy for other poorly immunogenic tumors, including those in humans.

Animals↗
PubMed1996

The active site specificity of the Yersinia protein-tyrosine phosphatase.

Yersinia protein-tyrosine phosphatase substrates have been synthesized employing an expedient methodology that incorporates phosphorylated non-amino acid residues into an active site-directed peptide. While the peptidic portion of these compounds serves an enzyme targeting role, the nonpeptidic component provides a critical assessment of the range of functionality that can be accommodated within the active site region. We have found that the Yersinia phosphatase hydrolyzes both L- and D-stereoisomers of phosphotyrosine in active site-directed peptides, with the former serving as a 10-fold more efficient substrate than the latter. In addition, this enzyme catalyzes the hydrolysis of a variety of aromatic and aliphatic phosphates. Indeed, a peptide bearing the achiral phosphotyrosine analog, phosphotyramine, is not only the most efficient substrate described in this study, it is also one of the most efficient substrates ever reported for the Yersinia phosphatase. Straight chain peptide-bound aliphatic phosphates of the general structure, (Glu)4-NH-(CH2)n-OPO3(2-) (n = 2-8), are also hydrolyzed, where the most efficient substrate contains seven methylene groups. Finally, a comparison of the substrate efficacy of the peptide-bound species with that of the corresponding non-peptidic analogs, reveals that the peptide component enhances kcat/Km by up to nearly 3 orders of magnitude.

Binding Sites↗
PubMed1996

A prospective 4-year follow-up study of attention-deficit hyperactivity and related disorders.

BACKGROUND: Previous cross-sectional data showed that children and adolescents with attention-deficit hyperactivity disorder (ADHD) are at increased risk of comorbid conduct, mood, and anxiety disorders as well as impairments in cognitive, social, family, and school functioning. However, longitudinal data were needed to confirm these initial impressions. METHODS: Using DSM-III-R structured diagnostic interviews and raters blinded as to diagnosis, we reexamined psychiatric diagnoses at 1- and 4-year follow-ups in children with ADHD and controls. In addition, subjects were evaluated for cognitive, achievement, social, school and family functioning. RESULTS: Analyses of follow-up findings revealed significant differences between children with ADHD and controls in rates of behavioral, mood, and anxiety disorders, with these disorders increasing markedly from baseline to follow-up assessments. In addition, children with ADHD had significantly more impaired cognitive, family, school, and psychosocial functioning than did controls. Baseline diagnosis of conduct disorder predicted major depression and bipolar disorder at follow-up, and anxiety disorders at baseline predicted anxiety disorders at follow-up. CONCLUSION: These results confirm and extend previous retrospective results indicating that children with ADHD are at high risk of developing a wide range of impairments affecting multiple domains of psychopathology such as cognition, interpersonal, school, and family functioning. These findings provide further support for the value of considering psychiatric comorbidity in both clinical assessment and research protocols involving children with ADHD.

Achievement↗
PubMed1996

Replantation of a completely detached degloved thumb.

Injuries resulting in the degloving of digits, particularly the thumb, offer the reconstructive surgeon a significant challenge, as the restoration of both digital function and appearance is often difficult. This paper is a case report describing the successful microvascular replantation of a completely degloved thumb, the result of an avulsion injury caused by a drill. Aspects of the preoperative assessment, the operative procedure and the appropriate sequence of artery and vein repair are discussed.

Accidents, Occupational↗