Biomedical subjects

Z Chen

Publications and source records attributed to Z Chen.

At least 379 records · Page 21Linked to original sources

PubMed2000

Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor.

Histone acetylation alters chromatin state by modifying lysines on histone and plays an important role in modulating gene transcription. A dynamic balance of histone acetylation/deacetylation is maintained by histone acetyltransferases and histone deacetylases. Emerging evidence suggests that a family of histone deacetylases may exist to regulate diverse cellular functions, including chromatin structure, gene expression, cell cycle progression, and oncogenesis. We describe here a novel human histone deacetylase, named HDAC8, cloned from human kidney. HDAC8 encodes 377 amino acid residues and shares extensive homology to several known HDACs, in particular a histone deacetylase from Arabidopsis thaliana. Northern blot analyses revealed that HDAC8 expression pattern for HDAC8 is distinct from that for HDAC1 and HDAC3, and expression of HDAC8 mRNA occurs in multiple organs including heart, lung, kidney, and pancreas. HDAC8 mRNA was also observed in several cell lines derived from cancerous tissues. When expressed in HEK293 cells, HDAC8 exhibited deacetylase activity toward acetylated histone, indicating that this protein is a bona fide histone deacetylase. Its histone deacetylase activity was inhibited by trichostatin and other known histone deacetylase inhibitors. Furthermore, active recombinant HDAC8 was expressed and purified from Escherichia coli. When ectopically expressed in cells, HDAC8 was found to be localized to the nucleus. Co-transfection experiments demonstrated that expression of HDAC8 repressed a viral SV40 early promoter activity. These results indicate that HDAC8 is a novel member of the histone deacetylase family, which may play a role in the development of a broad range of tissues and potentially in the etiology of cancer.

3T3 Cells↗

PubMed2000

Mouse mammary tumor virus-Ki-rasB transgenic mice develop mammary carcinomas that can be growth-inhibited by a farnesyl:protein transferase inhibitor.

For Ras oncoproteins to transform mammalian cells, they must be posttranslationally modified with a farnesyl group in a reaction catalyzed by the enzyme farnesyl:protein transferase (FPTase). Inhibitors of FPTase have therefore been developed as potential anticancer agents. These compounds reverse many of the malignant phenotypes of Ras-transformed cells in culture and inhibit the growth of tumor xenografts in nude mice. Furthermore, the FPTase inhibitor (FTI) L-744,832 causes tumor regression in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice and tumor stasis in MMTV-N-ras mice. Although these data support the further development of FTIs, it should be noted that Ki-ras is the ras gene most frequently mutated in human cancers. Moreover, Ki-RasB binds more tightly to FPTase than either Ha- or N-Ras, and thus higher concentrations of FTIs that are competitive with the protein substrate may be required to inhibit Ki-Ras processing. Given the unique biochemical and biological features of Ki-RasB, it is important to evaluate the efficacy of FTIs or any other modulator of oncogenic Ras function in model systems expressing this Ras oncoprotein. We have developed strains of transgenic mice carrying the human Ki-rasB cDNA with an activating mutation (G12V) under the control of the MMTV enhancer/promoter. The predominant pathological feature that develops in these mice is the stochastic appearance of mammary adenocarcinomas. High levels of the Ki-rasB transgene RNA are detected in these tumors. Treatment of MMTV-Ki-rasB mice with L-744,832 caused inhibition of tumor growth in the absence of systemic toxicity. Although FPTase activity was inhibited in tumors from the treated mice, unprocessed Ki-RasB was not detected. These results demonstrate the utility of the MMTV-Ki-rasB transgenic mice for testing potential anticancer agents. Additionally, the data suggest that although the FTI L-744,832 can inhibit tumor growth in this model, Ki-Ras may not be the sole mediator of the biological effects of the FTI.

Alkyl and Aryl Transferases↗

PubMed2000

IL-12- and IL-2-induced tumor regression in a new murine model of oral squamous-cell carcinoma is promoted by expression of the CD80 co-stimulatory molecule and interferon-gamma.

Therapy with IL-12 or IL-2 induces tumor regression in only a few patients with head-and-neck squamous cell carcinoma (SCC), and the factors promoting responsiveness have not been well defined. In this study, we examined whether combined IL-12 and IL-2 therapy can induce tumor regression in a new murine model of oral SCC and determined if the anti-tumor response is promoted by expression of the immune co-stimulatory molecule CD80 and cytokine IFN-gamma. In CD80-positive or -negative subclones of a BALB/c oral SCC line in syngeneic mice, we showed that systemic rIL-12 alone was comparable in effectiveness to combined therapy with IL-12 and peri-tumoral rIL-2, inducing complete regression of the CD80(+) line B7E11-4scid. However, therapy with these cytokines had no effect on growth of the CD80(-) subclone B7E3-4scid and did not induce complete regression of the CD80(+) subclone B7E11-4scid in congenic BALB/c IFN-gamma knockout mice, indicating that expression of the CD80 co-stimulatory molecule and IFN-gamma contributes to tumor regression. In cytokine-treated mice that rejected the CD80(+) SCC line, an increase in infiltrating CD4(+) lymphocytes and apoptotic bodies within the tumor specimens was observed, and resistance to rechallenge with the same tumor was detected in 50% of recipients, consistent with an immune response. Our results provide evidence that regression of oral head-and-neck SCC may be induced by therapy with systemic IL-12 and that expression of the CD80 co-stimulatory molecule by SCC and IFN-gamma by the host promote IL-12 induced regression of SCC.

Angiogenesis Inhibitors↗

PubMed2000

Humoral immune responses in Cr2-/- mice: enhanced affinity maturation but impaired antibody persistence.

Deficiency in CD21/CD35 by disruption of the Cr2 loci leads to impaired humoral immune responses. In this study, we detail the role of CD21/CD35 on Ab responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken gamma-globulin. Surprisingly, Cr2-/- mice generate significant Ab responses and germinal center (GC) reactions to low doses of this Ag in alum, although the magnitude of their responses is much reduced in comparison with those of Cr2+/- and C57BL/6 controls. Increasing Ag dose partially corrected this deficit. In situ study of the somatic genetics of GC B cells demonstrated that VDJ hypermutation does not require CD21/CD35, and Cr2-/- mice exhibited enhanced affinity maturation of serum Ab in the post-GC phase of the primary response. On the other hand, Cr2-/- mice displayed accelerated loss of serum Ab and long-lived Ab-forming cells. These observations suggest that B cell activation/survival signals mediated by CD21 and/or the retention of Ag by CD21/CD35 play important roles in the generation, quality, and maintenance of serum Ab.

Amino Acid Sequence↗

PubMed2000

Reaction rate, activation energy, and detection limit for the reaction of 5-furoylquinoline-3-carboxaldehyde with neurotransmitters in artificial cerebrospinal fluid.

The fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ) has proven valuable in the analysis of proteins and small neurotransmitters. We monitored the reaction rate between this reagent and five neurotransmitters at 40 degrees and 65 degrees C in artificial cerebrospinal fluid. The reactions followed pseudo-first order kinetics. The activation energy for the reaction of FQ was 10.6, 10.7, 22.0, 31.4, and 34.4 kJ mol(-1) for alanine, taurine, gamma-aminobutyric acid (GABA), glutamine, and glutamic acid, respectively. At 65 degrees C, the reaction rate was quite similar for alanine, taurine, glutamine, and glutamic acid (1.8 x 10(-3) s(-1)) but was twice as fast for GABA. A reaction time of nearly 1 h was required to quantitatively convert these neurotransmitters to their fluorescent products at 65 degrees C. Detection limits for the labeled neurotransmitters were 10(-9)-10(-8) M, which corresponded to 0.3-7 amol injected onto the capillary.

Animals↗

PubMed2000

Purification of the newly found selenium-containing proteins in the arterial wall and brain of the rat.

Previously several selenium-containing proteins with different subunit molecular masses (M(r)) were detected in the arterial wall and brain of rats. In continuation of this work, after labeling of rats in vivo with [(75)Se]selenite, the new selenium-containing proteins of interest were purified on a Sephadex G-200 column followed by preparative isoelectric focusing. Nuclear analytical methods (gamma-counter and gamma-detector) were applied in the detection and identification of the (75)Se-labeled proteins. The two (75)Se-containing proteins from the arterial wall migrated as 15.0- and 67.0-kDa species on SDS-PAGE gels with pI values of 4.5 and 5.1, respectively. The three (75)Se-containing proteins from brain purified to homogeneity had M(r) values of 18.0, 30.0, and 42.9 kDa and pI values of 6.3, 6.5, and 6.0, respectively. Of these proteins, the 67.0-, 42.9-, and 30.0-kDa species may be yet not characterized selenoproteins with important biological functions.

Animals↗

PubMed2000

Identification of a urokinase receptor-integrin interaction site. Promiscuous regulator of integrin function.

Adhesion and signaling by integrins require their dynamic association with nonintegrin membrane proteins. One such protein, the glycolipid-anchored urokinase receptor (uPAR), associates with and modifies the function of the beta(2)-integrin Mac-1 (CD11b/CD18). In this study, a critical non-I-domain binding site for uPAR on CD11b (M25; residues 424-440) is identified by homology with a phage display peptide known to bind uPAR. Recombinant soluble uPAR and cells expressing uPAR bound to immobilized M25, binding being promoted by urokinase and blocked by soluble M25, but not a scrambled control or homologous peptides from other beta(2)-associated alpha-chains. Mac-1, but not a mutated Mac-1 in which M25 was replaced with the homologous sequence of CD11c, co-precipitated with uPAR. In the beta-propeller model of alpha-chain folding, M25 spans an exposed loop on the ligand-binding, upper surface of alphaM, identifying uPAR as an atypical alphaM ligand. Although not blocking ligand binding to Mac-1, M25 (25-100 microM) inhibited leukocyte adhesion to fibrinogen, vitronectin, and cytokine-stimulated endothelial cells. M25 also blocked the association of uPAR with beta(1)-integrins and impaired beta(1)-integrin-dependent spreading and migration of human vascular smooth muscle cells on fibronectin and collagen. These observations indicate that uPAR associates with integrins directly and that disruption of this association broadly impairs integrin function, suggesting a novel strategy for regulation of integrins in the settings of inflammation and tumor progression.

Amino Acid Sequence↗

PubMed2000

Lidocaine induces a slow inactivated state in rat skeletal muscle sodium channels.

1. Local anaesthetics such as lidocaine (lignocaine) interact with sodium channels in a manner that is exquisitely sensitive to the voltage-dependent conformational state of the ion channel. When depolarized in the presence of lidocaine, sodium channels assume a long-lived quiescent state. Although studies over the last decade have localized the lidocaine receptor to the inner aspect of the aqueous pore, the mechanistic basis of depolarization-induced 'use-dependent' lidocaine block remains uncertain. 2. Recent studies have shown that lowering the extracellular Na+ concentration ([Na+]o) and mutations in the sodium channel outer P-loop modulate occupancy of a quiescent 'slow' inactivated state with intermediate kinetics (termed IM) that involves structural rearrangements in the outer pore. 3. Site-directed mutagenesis and ion-replacement experiments were performed using voltage-clamped Xenopus oocytes and cultured (HEK-293) cells expressing wild-type and mutant rat skeletal muscle (mu1) sodium channels. 4. Our results show that lowering [Na+]o potentiates use-dependent lidocaine block. The effect of [Na+]o is maintained despite a III-IV linker mutation that partially disrupts fast inactivation (F1304Q). In contrast, the effect of lowering [Na+]o on lidocaine block is reduced by a P-loop mutation (W402A) that limits occupancy of IM. 5. Our findings are consistent with a simple allosteric model where lidocaine binding induces channels to occupy a native slow inactivated state that is inhibited by [Na+]o.

Amino Acid Substitution↗

PubMed2000

Role of endothelium in thapsigargin-induced arterial responses in rat aorta.

We assessed the role of endothelium in the arterial response to thapsigargin, the Ca(2+)-ATPase inhibitor of the endoplasmic reticulum, in rat isolated aortic rings. Thapsigargin induced an endothelium-dependent relaxation of phenylephrine-contracted aortic rings with an EC(50) of 2.6+/-0.4 nM and a 75% maximum relaxation, while it was less effective against 30 mM K(+)-induced contraction. Pretreatment of aortic rings with N(G)-nitro-L-arginine methyl ester (30 microM) or methylene blue (1 microM) reduced thapsigargin-induced relaxation by approximately 85%. Thapsigargin failed to relax the endothelium-denuded rings. L-Arginine (3 mM) partially, but significantly, antagonized the effect of 30 microM N(G)-nitro-L-arginine methyl ester. Pretreatment with indomethacin (3 microM), glibenclamide (1 microM) or iberiotoxin (100 nM) did not alter the thapsigargin-induced relaxation. In contrast, pretreatment with tetrapentylammonium ions (TPA(+), 1-3 microM) or with 300 microM Ba(2+) suppressed the relaxant response to thapsigargin. TPA(+) (3 microM) also attenuated acetylcholine-induced relaxation. Thapsigargin-induced endothelium-dependent relaxation was primarily dependent on the presence of extracellular Ca(2+). Interestingly, when the tissues were exposed to very low concentrations of thapsigargin (1-3 nM) the nitric oxide-dependent relaxation induced by acetylcholine or A23187 was markedly reduced. While thapsigargin (3 nM) did not influence the relaxation induced by endothelium-independent dilators, sodium nitroprusside and verapamil. These results indicate that thapsigargin produced complex vascular effects primarily by acting on the endothelial cells. Thapsigargin causes an endothelial nitric oxide-dependent relaxation; on the other hand, it inhibits nitric oxide-mediated relaxation at the similar concentrations. Activation of TPA(+)- and Ba(2+)-sensitive but not Ca(2+)-activated or ATP-sensitive K(+) channels may be also involved in thapsigargin-induced relaxation of rat isolated aortic rings.

Animals↗

PubMed2000

Effects of metabolic fragments of [Arg(8)]-vasopressin on nerve growth in cultured hippocampal neurons.

The effects of metabolic fragments of [Arg(8)]-vasopressin (AVP), [pGlu(4), Cyt(6)]AVP (AVP(4-9)), and desglycinamide-[pGlu(4), Cyt(6)]AVP (AVP(4-8)) on the growth of hippocampal neurons in culture were investigated in comparison with those of AVP. AVP(4-9) caused a significant increase in filopodial length following 96 h of exposure at concentrations higher than 300 nM. AVP(4-9) was more potent than AVP. AVP(4-8) also induced an increase in filopodial length, but this effect was less than that of AVP. The selective V(1) agonist [Phe(2), Ile(3), Orn(8)]-vasopressin caused a significant increase in filopodial length, whereas the selective V(2) agonist [deamino-Cys(1), D-Arg(8)]-vasopressin showed no such effect. OPC-21268, a vasopressin V(1) antagonist, blocked AVP and AVP fragment-induced increases in filopodial length. However, the V(2) antagonist OPC-31260 showed no such effect. A23187, a representative Ca ionophore, also increased filopodial length, and the A23187-induced increase in filopodial length was potentiated by AVP and AVP fragments. These results indicated that AVP(4-9) and AVP(4-8) increased filopodial length in cultured hippocampal neurons by activating V(1) receptors. Both phenomena induced by AVP(4-9) and AVP(4-8) were associated with intracellular calcium mobilization.

Animals↗

PubMed2000

Bright spatial solitons on a partially incoherent background

We present the first observation of incoherent antidark spatial solitons in noninstantaneous nonlinear media. This new class of soliton states involves bright solitons on a partially incoherent background of infinite extent. In the case where the nonlinearity is of the Kerr type, their existence is demonstrated analytically by means of an exact solution. Computer simulations and experiments indicate that these incoherent antidark solitons can propagate in a stable fashion provided that the spatial coherence of their background is reduced below the incoherent modulation instability threshold.

Journal Article↗

PubMed2000

Comparison of three treatment options for single brain metastasis from lung cancer.

Whole brain radiotherapy (WBRT), stereotactic radiosurgery (SRS), and the combination of both treatment methods were used for the management of single brain metastasis from lung cancer. The purpose of this study is to compare these three different treatment options in terms of local response, survival, and quality of life. From June 1995 to July 1998, 70 lung cancer patients with new diagnosed single brain metastasis were treated with either WBRT alone (n = 29), or SRS alone (n = 23), or the combination of both methods (n = 18). Multiple endpoints, including survival, freedom from local progression (FFLP), freedom from new brain metastasis (FFNBM), local control, Karnofsky performance status (KPS), and causes of death, were measured from the date of treatment completion and compared using univariate and multivariate analyses. For patients treated with WBRT-alone, SRS-alone, and SRS+WBRT, the median survivals were 5.7, 9.3, and 10.6 months, the median FFLP were 4.0, 6.9, and 8.6 months, the median FFNBM were 4.1, 6.7, and 8.6 months, and the local response rates were 55.6, 87.0, and 88.9%, respectively. Four of the 29 patients treated with WBRT-alone continued with progression of disease. The post treatment KPS showed improvement in 41.4, 82.6, and 88.9% of patients treated with WBRT-alone, SRS-alone, and SRS+WBRT, respectively. The progression of new and/or recurred metastatic brain tumor as the cause of death accounted for 51.7%, 50. 0%, and 28.3% of the patients treated with WBRT-alone, SRS-alone, and SRS+WBRT, respectively. Univariate analyses showed that the significant differences among the three treatment arms were observed based on all of the above mentioned endpoints. However, the comparison between SRS-alone and SRS+WBRT groups indicated that adding WBRT only improves FFNBM (P = 0.0392). Cox regression analyses revealed no significant difference in both of the KPS (P = 0.1082) and causes of death (P = 0.081) among the three arms. Both SRS alone and SRS+WBRT seem better in prolonging life and improving quality of life than WBRT alone for patients with single brain metastasis from lung cancer. But the combined therapy did not show significant advantage over SRS alone in improving survival, enhancing local control, and quality of life except for a more favorable FFNBM. Further investigation via a randomized trial is needed to access the value of adding WBRT to SRS in the management of this group of patients. Int. J. Cancer (Radiat. Oncol. Invest.) 90, 37-45 (2000).

Adult↗

PubMed2000

X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293)--an initial glance of the viral DNA binding platform.

The crystal structure of simian immunodeficiency virus (SIV) integrase that contains in a single polypeptide the core and the C-terminal deoxyoligonucleotide binding domain has been determined at 3 A resolution with an R-value of 0.203 in the space group P2(1)2(1)2(1). Four integrase core domains and one C-terminal domain are found to be well defined in the asymmetric unit. The segment extending from residues 114 to 121 assumes the same position as seen in the integrase core domain of avian sarcoma virus as well as human immunodeficiency virus type-1 (HIV-1) crystallized in the absence of sodium cacodylate. The flexible loop in the active site, composed of residues 141-151, remains incompletely defined, but the location of the essential Glu152 residue is unambiguous. The residues from 210-218 that link the core and C-terminal domains can be traced as an extension from the core with a short gap at residues 214-215. The C(alpha) folding of the C-terminal domain is similar to the solution structure of this domain from HIV-1 integrase. However, the dimeric form seen in the NMR structure cannot exist as related by the non-crystallographic symmetry in the SIV integrase crystal. The two flexible loops of the C-terminal domain, residues 228-236 and residues 244-249, are much better fixed in the crystal structure than in the NMR structure with the former in the immediate vicinity of the flexible loop of the core domain. The interface between the two domains encompasses a solvent-exclusion area of 1500 A(2). Residues from both domains purportedly involved in DNA binding are narrowly distributed on the same face of the molecule. They include Asp64, Asp116, Glu152 and Lys159 from the core and Arg231, Leu234, Arg262, Arg263 and Lys264 from the C-terminal domain. A model for DNA binding is proposed to bridge the two domains by tethering the 228-236 loop of the C-terminal domain and the flexible loop of the core.

Amino Acid Sequence↗

PubMed2000

Isolation of a non-covalent aldose reductase-nucleotide-inhibitor complex.

A method for the isolation of an intact, non-covalent complex formed by the interaction of aldose reductase, NADP(H) nucleotide, and inhibitor has been developed to aid in the discovery and development of novel aldose reductase inhibitors. In the complexes isolated, both the carboxylic acid-containing inhibitor tolrestat and the spirohydantoin-containing inhibitor AL1576 (2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione) tightly bound in a 1:1 ratio to aldose reductase complexed with either NADPH or NADP+. Inhibitor binding to either the enzyme-NADP+ or enzyme-NADPH complex appeared to be equal and pH-dependent, with maximum binding observed at a pH range of 7 to 8.5 where the inhibitors are ionized. These results indicated that the charge state of the cofactor (NADPH vs NADP+) is not critical for inhibitor binding to aldose reductase. Molecular modeling studies suggested that His110 plays a crucial role in directing charged inhibitors containing either a carboxylate or an ionizable hydantoin group to the active site of aldose reductase by providing charge interaction.

Aldehyde Reductase↗

PubMed2000

Cytokine mRNAs in the nasal-associated lymphoid tissue during influenza virus infection and nasal vaccination.

Intranasal immunization with a current inactivated influenza vaccine together with an adjuvant (cholera toxin B subunit supplemented with a trace amount of whole toxin, CTB*) was confirmed in BALB/c mice to mimic influenza virus (A/PR/8/34, H1N1) infection with respect to mucosal IgA antibody responses, in which IgA antibody-forming cell responses in the nasal-associated lymphoid tissue (NALT) were involved with a peak around 7 days after infection or vaccination. Next, the expression of various cytokine mRNAs in the NALT was compared in mice either infected with viruses or immunized with CTB*-combined vaccine, to examine Th cell and cytokine regulation of mucosal IgA antibody responses. In infected mice, strong IL-2, weak IL-4, strong IL-6 and strong IFN-gamma mRNA expressions were induced during early days of infection; especially, IFN-gamma mRNA was expressed by both CD4(+) and CD8(+) T cells around 7 days after infection. In mice given CTB*-combined vaccine, weak IL-2, strong IL-4, strong IL-6 and weak IFN-gamma mRNA expressions were induced during early days of vaccination; especially, IL-4 mRNA was expressed by CD4(+) T cells. Thus, IL-6 mRNAs were expressed strongly in both infected and vaccinated mice. The IFN-gamma-rich cytokine mRNA profiles in the infected mice were reflected upon serum IgG2a-rich Ab responses, while the IL-4-rich profiles in the vaccinated mice were reflected upon the IgG1-rich Ab responses. Thus, influenza virus infection and CTB*-combined nasal vaccine induced Th1 dominant and Th2 dominant cytokine profiles, respectively, while the similarity of mucosal IgA antibody responses between infection and vaccination could be explained by the appearance of IL-6 mRNAs.

Administration, Intranasal↗

PubMed2000

Detection of circulating gastric carcinoma-associated antigen MG7-Ag in human sera using an established single determinant immuno-polymerase chain reaction technique.

BACKGROUND: In 1994, a novel sensitive method termed immuno-polymerase chain reaction (PCR) for the detection of the gastric carcinoma-associated antigen MG7-Ag in the gastric carcinoma cell line KATO III was reported. Compared with the enzyme-linked immunoadsorbent assay, the single determinant immuno-PCR technique could allow for as few as 20 cells to be detected and was found to show an approximately 10,000-fold enhancement in sensitivity of the detection limit. The current study clinically evaluated the significance of serum MG7-Ag detection in gastric carcinoma patients. METHODS: The sera of patients were immobilized on wells and a specific DNA molecule, which could be amplified by PCR, was employed as a marker. The biotinylated monoclonal antibody against gastric carcinoma was added to bind the antigen immobilized on the wells. After the biotinylated antibody was bound to the antigen, free avidin was used to attach a biotinylated monoclonal antibody and biotinylated DNA molecule. The biotinylated DNA complexed with antigen-antibody-avidin was amplified by PCR and the PCR products were analyzed by agarose gel electrophoresis. In the current study this method was used to detect circulating MG7-Ag in the sera of patients with gastric carcinoma and other various malignancies. For comparison, carcinoembryonic antigen, CA 50, CA 19-9, and TAG-72 were quantitated by radioimmunoassay and immunoradiometric assay using the relevant commercial kits in the same sera samples from 86 patients with pathologically confirmed gastric carcinoma and 83 patients with relevant benign diseases of the stomach. In addition, the semiquantitative analysis of PCR products among gastric carcinoma patients with or without metastasis was performed to compare the intensity of DNA band amplification. RESULTS: Using the immuno-PCR assay, positive results were obtained in 164 of 198 patients with gastric carcinoma (82.8%). The rates of positivity in other malignancies were 17.4% for esophageal carcinoma (15 of 86 patients), 44.4% for colonic carcinoma (40 of 90 patients), 0% for liver carcinoma (none of 84 patients), 2.2% for ovarian carcinoma (1 of 45 patients), 0% for uterine carcinoma (none of 27 patients), and 6.1% for lung carcinoma (4 of 66 patients). The positive results obtained from those patients with benign diseases were: 7.7% for peptic ulcer (6 of 78 patients), 5.9% for chronic gastritis (7 of 118 patients), 3.3% for chronic colitis (2 of 60 patients), and 0.8% for healthy blood donors (2 of 236 patients). In addition, the semiquantitative analysis of PCR products showed that the intensity of DNA band amplified from the PCR products of those patients with metastasis was much higher than that of patients without metastasis or those with early stage tumors (1.94 +/- 0.03 vs. 1.28 +/- 0.02). In comparative studies of immuno-PCR and commercial assays for tumor-associated antigens the sensitivity of immuno-PCR was 81.4% and pseudopositivity was lower (8.4% vs. 7.2-12.0% with radioimmunoassay or immunoradiometric assay). CONCLUSIONS: The results of the current study demonstrate that introducing PCR into the indirect determination of tumor-associated antigen in the serum can improve the sensitivity of detection greatly. This novel assay also might be used to monitor the circulating amount of tumor-associated antigen after gastrectomy and provide information regarding recurrence or metastasis, as well as for screening elderly patients who have no indications for endoscopy and those with precancerous conditions. The application of immuno-PCR in the serologic diagnosis of carcinoma has significant advantages including ready application in the clinical setting as well as use as a potential screening tool in mass surveys of high risk populations with gastric carcinoma. (c) 2000 American Cancer Society.

Antigens, Neoplasm↗

PubMed2000

Neuropathogenicity and sensitivity to antibody neutralization of lactate dehydrogenase-elevating virus are determined by polylactosaminoglycan chains on the primary envelope glycoprotein.

Common strains of lactate dehydrogenase-elevating virus (LDV, an arterivirus), such as LDV-P and LDV-vx, are highly resistant to antibody neutralization and invariably establish a viremic, persistent, yet asymptomatic, infection in mice. Other LDV strains, LDV-C and LDV-v, have been identified that, in contrast, are highly susceptible to antibody neutralization and are incapable of a high viremic persistent infection, but at the same time have gained the ability to cause paralytic disease in immunosuppressed C58 and AKR mice. Our present results further indicate that these phenotypic differences represent linked properties that correlate with the number of N-glycosylation sites associated with the single neutralization epitope on the short ectodomain of the primary envelope glycoprotein, VP-3P. The VP-3P ectodomains of LDV-P/vx possess three N-glycosylation sites, whereas those of LDV-C/v lack the two N-terminal sites. We have now isolated four independent neutralization escape variants of neuropathogenic LDV-C and LDV-v on the basis of their ability to establish a high viremic persistent infection in mice. The VP-3P ectodomains of all four variants had specifically regained two N-glycosylation sites concomitant with decreased immunogenicity of the neutralization eptitope and decreased sensitivity to antibody neutralization as well as loss of neuropathogenicity.

Amino Acid Sequence↗

PubMed2000

Enrichment and functional reconstitution of glutathione transport activity from rabbit kidney mitochondria: further evidence for the role of the dicarboxylate and 2-oxoglutarate carriers in mitochondrial glutathione transport.

In previous studies, we provided evidence for uptake of glutathione (GSH) by the dicarboxylate and the 2-oxoglutarate carriers in rat kidney mitochondria. To investigate further the role of these two carriers, GSH transport activity was enriched from rabbit kidney mitochondria and functionally reconstituted into phospholipid vesicles. Starting with 200 mg of mitoplast protein, 2 mg of partially enriched proteins were obtained after Triton X-114 solubilization and hydroxyapatite chromatography. The reconstituted proteoliposomes catalyzed butylmalonate-sensitive uptake of [(14)C]malonate, phenylsuccinate-sensitive uptake of [(14)C]2-oxoglutarate, and transport activity with [(3)H]GSH. The initial rate of uptake of 5 mM GSH was approximately 170 nmol/min per mg protein, with a first-order rate constant of 0.3 min(-1), which is very close to that previously determined in freshly isolated rat kidney mitochondria. The enrichment procedure resulted in an approximately 60-fold increase in the specific activity of GSH transport. Substrates and inhibitors for the dicarboxylate and the 2-oxoglutarate carriers (i.e., malate, malonate, 2-oxoglutarate, butylmalonate, phenylsuccinate) significantly inhibited the uptake of [(3)H]GSH, whereas most substrates for the tricarboxylate and monocarboxylate carriers had no effect. GSH uptake exhibited an apparent K(m) of 2.8 mM and a V(max) of 260 nmol/min per mg protein. Analysis of mutual inhibition between GSH and the dicarboxylates suggested that the dicarboxylate carrier contributes a somewhat higher proportion to overall GSH uptake and that both carriers account for 70 to 80% of total GSH uptake. These results provide further evidence for the function of the dicarboxylate and 2-oxoglutarate carriers in the mitochondrial transport of GSH.

Animals↗