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L Shu

Publications and source records attributed to L Shu.

36 records · Page 2Linked to original sources
PubMed1996

Role of cytochrome P450 in DNA damage induced by N-nitrosodialkylamines in cultured rat hepatocytes.

The involvement of cytochrome P450 in the cytotoxicity and DNA damage-repair induced by N-nitrosodipropylamine (NDPA), N-nitroso-n-butyl-n-propylamine (NBPA), and N-nitrosodibutylamine (NDBA) was investigated in cultured hepatocytes isolated from untreated, phenobarbital (PB)- and pyridine (PYR)-pretreated rats. Pretreatment of rats with PB caused a 10-fold increase in the sensitivity of hepatocytes to the cytotoxic actions of NDPA, NBPA and NDBA as measured by trypan blue exclusion, whereas PYR pretreatment increased the sensitivity of hepatocytes to NDPA and NBPA, but not to NDBA. This elevated sensitivity correlated well with increased 7-pentoxyresorufin depentylase activity catalyzed by P450 2B1 in cultures from PB-pretreated rats and enhanced p-nitrophenol hydroxylase activity of P450 2E1 in cultures of hepatocytes from PYR-pretreated rats. Unscheduled DNA synthesis showed that DNA damage-repair was significantly increased in freshly isolated hepatocytes from PB- and PYR-pretreated rats. With increasing time in culture, however, there was a marked reduction in the DNA damage repair response, concomitant with a decrease in the cytotoxicity of NDPA, NBPA and NDBA in primary cultures of hepatocytes. Coincident with this, a rapid loss in the specific activities of P450 2B1 and 2E1 was detected during the first 48 h in all primary cultures. Although N-nitrosodimethylamine (NDMA), used as a positive control, produced high nuclear grain counts in cultures from PYR-pretreated rats, the toxic effect of NDMA in rat hepatocytes was much weaker than that observed with NDPA, NBPA and NDBA. This result suggests that the type of DNA damage or repair efficiently induced by NDPA, NDBA or NDBA might differ from that due to NDMA.

Animals↗
PubMed1996

Identification of the cytochrome P450 isozymes involved in the metabolism of N-nitrosodipropyl-,N-nitrosodibutyl- and N-nitroso-n-butyl-n-propylamine.

The metabolism of N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitroso-n-butyl-n-propylamine (NBPA) was investigated in vitro using liver microsomes and purified isoforms of cytochrome P450 in a reconstituted system. Liver microsomes were prepared from rats pretreated with phenobarbital (PB), pyridine (PYR), beta-naphthoflavone (BNF), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), clofibrate (CLO) or from untreated rats. The purified cytochrome P450s used in the reconstituted system were rat 1A1 and 2B1 and rabbit 2E1. The rates of metabolism and the product profiles for NDPA, NDBA and NBPA changed significantly depending on the pretreatment of the rats or the identity of the purified cytochrome P450 isoforms. Induction by PB dramatically increased cleavage of NDPA, NDBA and NBPA at C-N bonds, leading to substantial increases in formation of the respective aldehydes and the overall metabolic rates. Microsomes from PYR-pretreated rats exhibited increased activities for formation of formaldehyde and propionaldehyde from NDPA and NBPA. Microsomes from BHT-pretreated rats showed a slight increase in activity for N-dealkylation of NDBA and BNPA. Treatment with BHA decreased the overall metabolism of NDBA, but slightly increased N-dealkylation of NBPA. Microsomal metabolism of NDPA, NDBA and NBPA was decreased by pretreatment with BNF and CLO. Results from studies using the reconstituted system with purified cytochrome P450 isoforms demonstrated that cytochrome P450 2B1 specifically catalyzed alpha-hydroxylation of these three long chain nitrosamines with high activity. Cytochrome P450 2E1 catalyzed formation of formaldehyde and propionaldehyde from NDPA and NBPA, but did not catalyze formation of acetaldehyde or butyraldehyde. Cytochrome P450 1A1 exhibited no activity for metabolism of NDPA, NDBA and NBPA. The contributions of cytochrome P450 2B1 and 2E1 to N-dealkylation reactions were determined using inhibitory monoclonal antibodies (mAb). With microsomes from PB-pretreated rats, inhibition by mAb-2B1 indicated a 62% contribution by cytochrome P450 2B1 to debutylation of NDBA and 65% to depropylation of NDPA. In microsomes from PYR-pretreated rats inhibition by mAbs also showed a role for cytochrome P450 2E1 in depropylation of NDPA. These studies provide a better understanding of the role of various forms of cytochrome P450 in metabolic activation of these long chain N-nitrosodialkylamines to potentially toxic, mutagenic and carcinogenic intermediates.

Animals↗
PubMed1996

Artificial neural network to assist psychiatric diagnosis.

BACKGROUND: Artificial Neural Network (ANN), as a potential powerful classifier, was explored to assist psychiatric diagnosis of the Composite International Diagnostic Interview (CIDI). METHOD: Both Back-Propagation (BP) and Kohonen networks were developed to fit psychiatric diagnosis and programmed (using 60 cases) to classify neurosis, schizophrenia and normal people. The programmed networks were cross-tested using another 222 cases. All subjects were randomly selected from two mental hospitals in Beijing. RESULTS: Compared to ICD-10 diagnosis by psychiatrists, the overall kappa of BP network was 0.94 and that of Kohonen was 0.88 (both P < 0.01). In classifying patients who were difficult to diagnose, the kappa of BP was 0.69 (P < 0.01). ANN-assisted CIDI was compared with expert system assisted CIDI (kappa = 0.72-0.76); ANN was more powerful than a traditional expert system. CONCLUSION: ANN might be used to improve psychiatric diagnosis.

Adult↗
PubMed1995

X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase. Implications for the extradiol cleavage mechanism.

The extradiol-cleaving catechol 2,3-dioxygenase (2,3-CTD) isolated from Pseudomonas putida mt-2 and its catechol and ternary E.S.NO complexes are characterized by X-ray absorption spectroscopy (XAS). The intensities of the 1s-->3d transitions in the pre-edge spectra of the uncomplexed enzyme and its substrate complex show that the Fe(II) center is five-coordinate in both complexes, in agreement with earlier magnetic circular dichroism studies [Mabrouk, P. A., Orville, A. M., Lipscomb, J. D., & Solomon, E. I. (1991) J. Am. Chem. Soc. 113, 4053-4061]. Analysis of the EXAFS region of uncomplexed 2,3-CTD shows five N/O ligand atoms 2.09 A from the active site Fe(II). In the 2,3-CTD.catechol complex, one N/O atom is located at 1.93 A and four N/O type ligands are at 2.10 A. By comparison with [FeII-(6TLA)(DBCH)](ClO4), the first well-characterized mononuclear Fe(II).catechol model complex, the 1.93 A scatterer is proposed to be the oxygen from the deprotonated hydroxyl group of the coordinated catecholate monoanion. Nitric oxide binds to the Fe(II) center in the enzyme.catechol complex without displacing the existing ligands, resulting in the formation of a six-coordinate complex, as indicated by the addition of a new N/O type scatterer at 1.74 A. Bond valence sum (BVS) analysis of the bond lengths derived from the EXAFS fits gives values that correspond to the iron oxidation states established for these complexes, thus lending credence to the coordination environment deduced for the iron center in those complexes. The present study provides the first evidence for a monoanionic substrate binding mode in an extradiol dioxygenase, which is distinct from the dianionic binding mode proposed for intradiol dioxygenases. We speculate that this difference in binding mode may have important ramifications for the site of aromatic ring cleavage in the subsequent oxygen insertion reactions.

Binding Sites↗
PubMed1995

Generation and characterization of a single-gene encoded single-chain immunoglobulin-interleukin-2 fusion protein.

BACKGROUND: Interleukin-2 (IL-2), a potent inducer of cellular immune responses, has been used for biological therapy of human cancer; however, the high doses of IL-2 required to mediate patients' immune responses can cause considerable systemic toxicity. The murine monoclonal antibody (MAb) CC49, which reacts with tumor-associated glycoprotein (TAG)-72, expressed on a variety of human carcinomas, has shown excellent tumor localization in recent clinical trials. OBJECTIVES: Development and characterization of a single-chain immunoglobulin-IL-2 (SCIg-IL-2) fusion protein which, by delivering IL-2 selectively to the tumor site, can serve as an effective reagent for CC49/IL-2 combination therapy. STUDY DESIGN: A single-gene encoding the SCIg-IL-2 fusion protein derived from the chimeric (c) CC49 was designed, generated and inserted in an expression vector. The monomeric single-chain protein consisted of the CC49 heavy and light chain variable domains covalently jointed through a (GGGGS)3 linker peptide. The carboxyl end of the variable domain of the light chain was linked to the amino terminus of the human gamma 1 Fc through the hinge region, and the carboxyl end of the CH3 domain was linked to the amino terminus of the human IL-2 through a GGGSGGG linker peptide. The SCIg-IL-2, expressed from the murine myeloma cells transfected with the expression construct, was characterized for its antigen-binding specificity, antibody effector functions and IL-2 biological activity. RESULTS AND CONCLUSION: Transfection of murine myeloma cells with the single-gene expression construct SCIg-IL-2 expressed a single-chain protein of approximately 70 kD, which was secreted into tissue culture fluid as a homodimer of approximately 140 kD. SCIg-IL-2 competed completely with cCC49 for binding to the TAG-72 antigen, but approximately three- to four-fold more of the SCIg-IL-2 was required to achieve levels of competition similar to those observed with the murine or chimeric CC49. With human effector cells, the fusion protein mediated lysis of TAG-72-positive human carcinoma cells. Prior treatment of human effector cells with 100 U/ml of human IL-2 enhanced the fusion protein-mediated cytolysis from 32 to 65%. At doses of > or = 1 ng/ml, the stimulatory effect of SCIg-IL-2 on IL-2 dependent murine HT-2 cell proliferation was comparable to that of the recombinant human IL-2. The single-gene construct may also facilitate inoculation of the gene in animal tissue for in vivo expression of the fusion protein.

Antibodies, Monoclonal↗
PubMed1995

Generation, characterization, and in vivo studies of humanized anticarcinoma antibody CC49.

Monoclonal antibody (MAb) CC49 reacts with tumor-associated glycoprotein (TAG)-72, a human pancarcinoma antigen. In clinical trials, radiolabeled CC49 has shown excellent tumor localization; however, many of the patients receiving MAb CC49 develop a human antimouse antibody response. In an attempt to prevent this antiimmunoglobulin response, we have developed a humanized CC49 (HuCC49) by grafting the MAb CC49 hypervariable regions onto the variable light (VL) and variable heavy (VH) frameworks of the human MAbs LEN and 21/28' CL, respectively, while retaining those murine framework residues that may be required for the integrity of the antigen combining-site structure. The HuCC49 MAb was compared with native murine CC49 (nCC49) and chimeric CC49 (cCC49), using a variety of assays. SDS-PAGE analysis under nonreducing conditions showed that the HuCC49 MAb has virtually identical mobility to that of cCC49. Under reducing conditions, the HuCC49 yielded two bands of approximately 25-28 and approximately 50-55 kDa, characteristic of heavy and light immunoglobulin chains. In competition radioimmunoassays, HuCC49 completely inhibited the binding of 125I-labeled nCC49 to TAG-72, although 23- to 30-fold more HuCC49 was required to achieve a level of competition similar to those of cCC49 and nCC49. The relative affinity of HuCC49 was 2- to 3-fold less than those of the cCC49 and nCC49 MAbs, respectively. The plasma clearance in mice of HuCC49 was virtually identical to that of cCC49. Biodistribution studies demonstrated equivalent tumor-targeting of HuCC49 and cCC49 to human colon carcinoma xenografts. These studies thus suggest that HuCC49 and genetically modified molecules, such as sFv and domain-deleted immunoglobulins developed by using the HuCC49 variable region as a cassette, may be potentially useful in both diagnostic and therapeutic clinical trials in patients with TAG-72-positive tumors.

Amino Acid Sequence↗
PubMed1994

Activation of the leu-500 promoter by adjacent transcription.

The leu-500 mutation is an A-to-G point mutation in the -10 region of the promoter controlling the leuABCD operon of Salmonella typhimurium. Suppression of the leu-500 mutation in an S. typhimurium topA mutant has demonstrated the functional dependency of this mutated promoter on negative supercoiling. A plasmid bearing a minimal leu-500 promoter region (positions -80 to +87) failed to restore its expression in the S. typhimurium topA mutant. We showed that transcription-mediated local negative supercoiling can activate the leu-500 promoter on a plasmid. The coupled transcription and translation process is required for this activation, but peptide-mediated membrane anchorage may not be involved in this activation. Although the effect of negative supercoiling generated during transcription away from the promoter is limited to a short distance of 250 bp, it can activate the negative-supercoiling-dependent leu-500 promoter from positions either 5' or 3' of the leu-500 promoter. In the presence of a parallel-oriented lac promoter which transcribed away from the 3' end of the leu-500 promoter, transcriptional activation of the leu-500 promoter is a strong indication of cooperativity during the transcriptional initiation process.

Base Sequence↗
PubMed1993

Secretion of a single-gene-encoded immunoglobulin from myeloma cells.

We describe construction of a single gene encoding a single-chain immunoglobulin-like molecule. This single-gene approach circumvents inefficiencies inherent in delivering two genes into a mammalian cell and in the assembly of a functional immunoglobulin molecule. It would also facilitate ex vivo transfection of cells for gene-therapy protocols. SP2/0 murine myeloma cells transfected with the single gene SG delta CLCH1 expressed a single-chain protein, SC delta CLCH1, comprising approximately 60 kDa of the anti-carcinoma monoclonal antibody (mAb) CC49. The single-chain protein consisted of the heavy- and light-chain variable (VH and VL) domains of the mAb covalently joined through a short linker peptide, while the carboxyl end of the VL domain was linked to the amino terminus of the human gamma 1 Fc region through the hinge region. The single-chain protein assembled into a dimeric molecule, termed SCA delta CLCH1, of approximately 120 kDa and was secreted into the tissue culture fluid. SDS/PAGE analysis of the secreted immunoglobulin purified by protein G affinity chromatography confirmed the size of the molecule. The native mAb CC49 and SCA delta CLCH1 of CC49 showed similar binding to the tumor-associated glycoprotein TAG-72, and the chimeric mAb CC49 and SCA delta CLCH1 showed similar cytotoxic activity. This single-gene construct approach provides a way of generating an immunoglobulin-like molecule which retains the specificity, binding properties, and cytolytic activity of the chimeric mAb CC49. The immunoglobulin-like molecule SCA delta CLCH1 is potentially a therapeutic and diagnostic reagent against a range of human carcinomas.

Animals↗
PubMed1993

A human T cell line engineered to secrete chimeric monoclonal antibody.

Both monoclonal antibodies (MAbs) and human T cells have been used in human tumor immunotherapy protocols. Tumor-infiltrating lymphocytes (TILs) and MAbs that can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) via human effector cells have shown antitumor effects in both animal models and clinical trials. One potential novel approach would be to combine these two modalities in the creation of a T cell capable of secreting antitumor immunoglobulins (Ig), in essence, creating an antitumor Ig "factory" at the tumor site. In the studies reported here, we have cloned the D612 MAb Ig genes and generated a chimeric D612 IgG1 containing the murine variable region and human constant region. D612 MAb has been shown to mediate lysis of human colon carcinomas via effector cell-mediated ADCC. We have demonstrated that following transfection, chimeric D612 can be expressed and secreted by the human T-cell line MOLT-4 at a rate of 0.25 micrograms/ml per 10(6) cells in 72 hours. The secreted Ig retained its antigen-binding properties as assayed by competition radioimmunoassay and also its ability to mediate ADCC against human tumor cells. To our knowledge, this is the first demonstration of the production of a chimeric IgG by human T cells and opens the possibility of a therapeutic approach in which TILs secrete humanized antitumor MAb capable of mediating ADCC at the tumor site.

Antibodies, Monoclonal↗
PubMed1993

Analysis of the functional and host range-determining regions of the murine ectropic and amphotropic retrovirus envelope proteins.

A series of Moloney murine leukemia virus (Mo-MuLV) envelope gene constructs were analyzed for biological activity. Three classes of recombinant envelopes were examined: insertions, deletions, and chimeras. Insertion (4 to 5 amino acids) and deletion (31 to 62 amino acids) mutants spanned most of the SU (gp70)-coding region and were all biologically inactive. Radioimmunoprecipitation demonstrated that the mutant envelope proteins were incorrectly processed. The Pr80env envelope precursor proteins failed to obtain the proper posttranslational modifications and were not cleaved into SU (gp70) and TM (p15E), suggesting that disruption of Pr80env structure prevents intracellular transport and processing. To analyze the functional domains of the SU portion of the Env protein, we assembled several chimeric constructs. In these constructs, portions of the ecotropic Mo-MuLV envelope gene were replaced with corresponding sequences from the 4070A amphotropic MuLV envelope. Using a retroviral vector pseudotyping assay, 5 of 12 chimeric envelope proteins were shown to be biologically active. Host range was determined by retroviral vector transduction of the appropriate cell, by viral interference studies, and by the productive infection of Chinese hamster ovary cells expressing the murine ecotropic receptor. These results permit assignment of the amino acids responsible for host range determination. Ecotropic host range is determined by the first 88 amino acids of the Mo-MuLV SU, while the amphotropic host range-determining region spans the first 157 amino acids of the 4070A SU.

3T3 Cells↗
PubMed1988

The precise structure and coding capacity of mRNAs from early region 2B of human adenovirus serotype 2.

Replication of human adenovirus (Ad) DNA requires three virus-encoded proteins that are coordinately transcribed from a single promoter at early times after infection. The mRNAs for two of these proteins, the precursor to the terminal protein (pTP) and the Ad DNA polymerase (Ad Pol), share several exons, including one encoded near Ad genome coordinate 39. The positions of the splice points of these mRNAs have been mapped by S1 nuclease mapping, by RNA sequencing, and by cDNA cloning. As a result of RNA splicing events, a short open reading frame (ORF) encoded at genome coordinate 39 is connected to the beginning of both the pTP and Ad Pol coding sequences; inclusion of this upstream ORF is essential for expression of functional pTP and Ad Pol proteins.

Adenoviruses, Human↗
PubMed1984

Haloperidol concentrations elevated in Chinese patients.

After 6 weeks of fixed dose oral haloperidol treatment. Chinese schizophrenic patients (in the People's Republic of China) had 52% higher plasma haloperidol concentrations than U.S. non-Asian schizophrenic patients. Chinese and U.S. patients were matched for sex and body weight. The difference in plasma drug concentrations may explain why Asians are reported to require smaller dosages of neuroleptic drugs than non-Asians, and why they may be more sensitive to neuroleptic-induced side effects. When prescribing haloperidol to persons of Asian ancestry, physicians should consider that higher than expected plasma haloperidol concentrations and an increased sensitivity to haloperidol may occur.

Adult↗