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Biomedical subjects

J Cheng

Publications and source records attributed to J Cheng.

At least 109 records · Page 6Linked to original sources
PubMed2000

Novel function of human RLIP76: ATP-dependent transport of glutathione conjugates and doxorubicin.

Active transport of conjugated and unconjugated electrophiles out of cells is essential for cellular homeostasis. We have previously identified in human tissues a transporter, DNP-SG [S-(2, 4-dinitrophenyl)glutathione] ATPase, capable of carrying out this function [Awasthi et al. (1998) Biochemistry 37, 5231-5238, 5239-5248]. We now report the cloning of DNP-SG ATPase. The sequence of the cDNA clone was identical to that of human RLIP76, a known Ral-binding protein. RLIP76 expressed in E. coli was purified by DNP-SG affinity chromatography. Purified recombinant RLIP76: (1) had ATPase activity stimulated by DNP-SG or doxorubicin (DOX), and the K(m) values of RLIP76 for ATP, DOX, and DNP-SG were similar to those reported for DNP-SG ATPase; (2) upon reconstitution with asolectin as well as with defined lipids, catalyzed ATP-dependent transport of DNP-SG and DOX with kinetic parameters similar to those of DNP-SG ATPase; (3) when transfected into K562 cells, resulted in increased resistance to DOX, and increased ATP-dependent transport of DNP-SG and DOX by inside-out membrane vesicles from transfected cells; (4) direct uptake of purified RLIP76 protein into mammalian cells from donor proteoliposomes confers DOX resistance. These results indicate that RLIP76, in addition to its role in signal transduction, can catalyze transport of glutathione conjugates and xenobiotics, and may contribute to the multidrug resistance phenomenon.

ATP-Binding Cassette Transporters↗
PubMed2000

Synthesis of Optically Active beta-Amino Acid N-Carboxyanhydrides.

Methodology has been developed for the general synthesis of optically active beta-amino acid N-carboxyanhydrides (beta-NCAs) through cyclization of N(beta)-Boc or N(beta)-Cbz beta-amino acids using phosphorus tribromide. The formation of beta-NCAs was confirmed by spectroscopy as well as an X-ray structural determination of beta-homoalanine-N-carboxyanhydride. The beta-NCA molecules could be polymerized in good yield to give optically active poly(beta-peptides) that adopt stable chiral conformations in solution. For example, helical oligo(L-beta-homophenylalanine) was synthesized by polymerization of L-beta-homophenylalanine-N-carboxyanhydride.

Journal Article↗
PubMed2000

Maturation of NK1 receptor involvement in the nociceptive response to formalin.

Administration of NK1 antagonists in adult animals attenuates the nociceptive response in the formalin test, indicating that the neurokinins and the NK1 receptor play a role in mediating this pain response. The number and distribution of NK1 receptors change dramatically during development, and the age at which they become involved in pain processing is not known. We examined the role of NK1 receptors in the formalin model in rats ranging in age between 3- and 21-days old. An NK1 antagonist, CP99,994, and its less active enantiomer CP100,263 were administered to the spinal cord (intrathecal), systemically (subcutaneous), or locally (intraplantar). Intrathecal administration of CP99,994, but not CP100,263, attenuated pain behaviors in the second phase of the formalin response in 14-day and 21-day old rats, but did not alter the pain response in 3-day or 10-day old rats. CP99,994 also reduced the expression of the c-fos protein in the superficial dorsal horn of 21-day old rats. Systemic and intraplantar injection of either CP99,994 or CP100,263 reduced the pain response to formalin in 3-day and 21-day old rats, suggesting a non-NK1 mediated mechanism of action. These results indicate that, within the spinal cord, NK1 receptors start to play a role in the pain response to formalin between 10 and 21 days. Moreover, analgesia induced by systemic or local injection of NK1 antagonists involves mechanisms other than, or in addition to, the NK1 receptor.

Animals↗
PubMed2000

ADP-Ribosyl cyclase in rat vascular smooth muscle cells: properties and regulation.

We investigated whether ADP-ribosyl cyclase (ADPR-cyclase) in rat vascular smooth muscle cells (VSMCs) has enzymatic properties that differ from the well-characterized CD38-antigen ADPR-cyclase, expressed in HL-60 cells. ADPR-cyclase from VSMCs, but not CD38 ADPR-cyclase from HL-60 cells, was inhibited by gangliosides (10 micromol/L) GT(1B), GD(1), and GM(3). Preincubation of membranes from CD38 HL-60 cells, but not from VSMCs, with anti-CD38 antibodies increased ADPR-cyclase activity; CD38 antigen was detected both in VSMCs and in HL-60 cells. ADPR-cyclase in VSMC membranes was more sensitive than CD38 HL-60 ADPR-cyclase to inactivation by N-endoglycosidase F and to thermal inactivation at 45 degrees C. The specific activity of ADPR-cyclase in membranes from VSMCs was >20-fold higher than in membranes from CD38 HL-60 cells. Most importantly, VSMC ADPR-cyclase was inhibited by Zn(2+) and Cu(2+) ions; the inhibition by Zn(2+) was dose dependent, noncompetitive, and reversible by EDTA. In contrast, Zn(2+) stimulated the activity of CD38 HL-60 ADPR-cyclase and other known types of ADPR-cyclases. Retinoids act either via the nuclear receptor retinoic acid receptor or retinoid X receptor, including all-trans retinoic acid (atRA), and panagonist 9-cis-retinoic acid-upregulated VSMC ADPR-cyclase; the stimulatory effect of atRA was blocked by actinomycin D and cycloheximide. 1,25(OH)(2)-Vitamin D(3) (calciferol) stimulated VSMC ADPR-cyclase dose dependently at subnanomolar concentrations (ED(50) congruent with 56 pmol/L). Oral administration of atRA to rats resulted in an increase of ADPR-cyclase activity in aorta ( congruent with+60%) and, to a lesser degree, in myocardium of left ventricle (+18%), but atRA had no effect on ADPR-cyclases in lungs, spleen, intestinal smooth muscle, skeletal muscle, liver, or testis. Administration of 3,5,3'-triiodothyronine (T(3)) to rats resulted in an increase of ADPR-cyclase activity in aorta ( congruent with+89%), but not in liver or brain. We conclude the following: (1) ADPR-cyclase in VSMCs has enzymatic properties distinct from "classic" CD38 ADPR-cyclase, especially sensitivity to inhibition by Zn(2+) and Cu(2+); (2) ADPR-cyclase in VSMCs is upregulated by various retinoids, calcitriol, and T(3) in vitro; and (3) administration of atRA and T(3) increases ADPR-cyclase in aorta in vivo. We suggest that the cADPR signaling system plays an important role in the regulation of VSMC functions in response to steroid superfamily hormones.

ADP-ribosyl Cyclase↗
PubMed2000

Viral recombinant vaccines to the E6 and E7 antigens of HPV-16.

Most cancerous lesions of the uterine cervix are linked to persistent infections with human papillomaviruses (HPV), most notably HPV-16 or -18. Vaccine-induced immune responses to the HPV early antigens E6 and E7, which contribute to cell transformation and are thus expressed in these cervical cancers, could potentially eradicate malignant cells. We generated recombinant vaccines based on E1-deleted adenovirus human strain 5 or on vaccinia virus strain Copenhagen expressing either the E6 or E7 oncoproteins of HPV-16. The different vaccines were compared in two experimental mouse tumor models employing Balb/c or C57Bl/6 mice. Data presented here demonstrate that depending on the model either CD4(+) or CD8(+) T cells provide protection to tumor cell challenge, resulting in striking differences in the efficacy of the four vaccines under investigation.

Adenoviruses, Human↗
PubMed2000

Quantitative effects of functional bundle branch block in patients with atrioventricular reentrant tachycardia.

Changes in the retrograde conduction time (ventriculoatrial [VA]) interval during functional bundle branch block (BBB) have been used to separate septal from free wall accessory pathways (APs), but different values of the VA interval prolongation (deltaVA) have been described in different reports. A total of 95 patients with single nondecremental APs who developed BBB during atrioventricular reentrant tachycardia were studied. Free wall APs were found in 60 patients, and 35 had septal APs. For patients with free wall APs, complete and incomplete BBB ipsilateral to the atrial insertion site of APs were observed in 39 of 60 patients (65%) and 31 of 60 patients (52%), respectively. For patients who had both complete (QRS > or = 120 ms) and incomplete (QRS <120 ms) BBB during atrioventricular reentrant tachycardia, deltaVA for patients with complete BBB was significantly greater than in those with incomplete BBB, 59 +/- 19 ms versus 30 +/- 11 ms, p <0.001. For patients with septal APs and complete and incomplete BBB during tachycardia, the mean deltaVA for those with complete BBB was 31 +/- 20 ms and was significantly longer than in patients with incomplete BBB (14 +/- 6 ms), p <0.001. There was no significant difference in deltaVA between those with free wall APs and incomplete BBB compared with those with septal APs and complete BBB. The criteria of QRS > or = 120 ms associated with deltaVA > or =40 ms served to best separate free wall from septal APs with a sensitivity of 88% and a specificity of 89%. Left anterior fascicular block was associated with marked lengthening of deltaVA for those with left free wall APs, whereas a left posterior fascicular block pattern resulted in a marked increase in the deltaVA for patients with posteroseptal APs. In the absence of fascicular block patterns, a deltaVA > or =40 ms provides strong evidence of a free wall AP, with a sensitivity of 95% and a specificity of 100%. The left posterior fascicle appears to provide predominant innervation of the posterior septum.

Adolescent↗
PubMed2000

A novel covalent modification of nitrogenase in a cyanobacterium.

In extracts of the unicellular cyanobacterium Gloeothece, the Fe-protein of nitrogenase can be separated by SDS-PAGE into two antigenically identifiable components. Unlike the situation in photosynthetic bacteria such as Rhodospirillum rubrum, these two forms do not arise from covalent modification of the protein by ADP-ribosylation. Rather, the Fe-protein of Gloeothece nitrogenase is subjected to modification by palmitoylation.

Cyanobacteria↗
PubMed2000

Novel myosin VI isoform is abundantly expressed in retina.

Several forms of sensory deficit have been associated with unconventional myosin defects in humans and other animals. Normal hearing in mammals has been shown to require functional myosin VI (Avraham et al., 1995) and myosin VIIA (Gibson et al., 1995; Liu et al., 1997), and the combined blindness and deafness of Usher syndrome type IB has been shown to be produced by specific defects in myosin VIIA (Weil et al., 1997). Here we report the cloning and characterization of two distinct myosin VI isoforms (FMVIA and FMVIB) initially identified in a degenerate PCR screen of retinal cDNA from the striped bass, Morone saxatilis. Open reading frames for FMVIA and FMVIB encode predicted proteins of 1304 and 1270 amino acids respectively, which are 83% identical at the amino acid level. Both fish isoforms are likewise approximately 83-86% identical to mammalian class VI myosins (Hasson and Mooseker, 1994). Northern blot analysis revealed that FMVIA mRNA is broadly expressed and most abundant in kidney, a pattern similar to that previously reported for mammalian myosin VI. FMVIB expression is dramatically more abundant in retina than in any other tissue examined. Antibodies directed against pig myosin VI (Hasson and Mooseker, 1994) detect a doublet at approximately 150 kDa in bass retina and RPE. Since both fish VIA and VIB isoforms share high sequence identity with pig myosin VI within the domain used for antibody production, it seems likely that this antibody crossreacts with both FMVIA and FMVIB. Immunocytochemistry with this same affinity-purified rabbit anti-myosin VI antibody shows that myosin VI isoforms are primarily localized in photoreceptors, horizontal cells and Müller cells in both fish and primate retinas. This report is the first demonstration that two myosin VI genes are expressed in the same organism and the same cell type (RPE). The relatively high abundance of FMVIB expression in retina suggests that it may play an important role in retinal motility events.

Amino Acid Sequence↗
PubMed2000

Perlecan (heparan sulfate proteoglycan) gene expression reflected in the characteristic histological architecture of salivary adenoid cystic carcinoma.

In order to determine the role of the basement membrane-type heparan sulfate proteoglycan (HSPG), known as perlecan, in the formation of the characteristic cribriform 'structures of salivary adenoid cystic carcinomas, the mode of expression of mRNA for the core protein of HSPG was investigated by using in situ hybridization (ISH) both in surgical specimens and in a cell system (ACC3) of adenoid cystic carcinomas. In the surgical specimens, the mRNA for the HSPG core was more intensely expressed in solid tumor cell nests, especially in smaller ones. Within the nests, the signals were detected almost exclusively in cuboidal cells forming small pseudocysts. In contrast, signals were absent in flat cells forming large pseudocysts or in carcinoma cell nests attaching to the peripheral nerves or blood vessels. In normal salivary gland tissues, myoepithelial cells expressed the mRNA at a high level, but acinar and duct epithelial cells did not. In the time-course experiment of ACC3 cells, signals for HSPG core increased with time and reached the maximum on day 4, decreasing thereafter in a culture condition in which cells reached confluence in a week. The results indicate that HSPG is biosynthesized by adenoid cystic carcinoma cells which are in the proliferation phase, and that tumor cells producing HSPG tend to form initial structures of stromal pseudocysts.

Carcinoma, Adenoid Cystic↗
PubMed2000

Surface-enhanced raman spectra of dyes and organic acids in silver solutions: chloride ion effect.

We have recorded surface-enhanced Raman (SER) spectra of two different classes of compounds, cationic dyes and organic acids, and studied their chloride ion effects on the surface-enhanced Raman scattering (SERS) activities of the silver solution. For the positive charge dyes, rhodamine 6G (R6G) and 1,1'-dimethyl-2,2'-cyanine iodide (DECI), no SERS could be observed without the addition of chloride ions because of lack of the electrostatic interaction between the dye species and the silver particles in the silver solution. The chloride ions served to enlarge silver particles and to contribute the existence of the surface active sites, making the silver solution SERS active to the dye samples. Surface-enhanced resonance Raman scattering (SERRS) intensity of the dye molecules increased with the chloride ion concentration. After reaching a maximum intensity, a Cl- quenching effect on the intensity took place. For the organic acids, benzoic acid and p-aminobenzoic acid (PABA), SERS could be observed without the coexistence of chloride ions. The intensity of the Raman scattering did not vary significantly in the presence of small amount of chloride ion. At high Cl- concentration, quenching SERS intensity began to take effect.

4-Aminobenzoic Acid↗
PubMed2000

Investigation of TTV by in situ hybridization in patients with chronic hepatitis.

To clarify whether TT virus (TTV) was present in liver tissues, 12 liver tissue samples from patients with chronic hepatitis positive for TTV in their serum and 11 samples from serum-negative patients were obtained by needle biopsies and investigated using in situ hybridization. Positive staining was observed in nine (75%) of 12 cases positive for TTV (serum-positive group) and three (27.3%) of 11 cases negative for TTV (serum-negative group) (P=0.061). Three kinds of staining patterns were observed: nuclear, cytoplasmic and both. In 58.3% (7/12) of the patients positive for TTV staining, the stained areas were found in both the nucleus and cytoplasm. Only cytoplasmic staining was observed in three cases from the serum-positive group. Only nuclear staining was observed in two cases from the serum-negative group. No significant differences were found in the clinical background between the in situ hybridization-positive and -negative groups, and between the serum-positive and -negative groups. The present study shows that TTV exists in the liver tissue, especially in hepatocytes, of chronic hepatitis patients and that the localization of TTV in the cell is different from case to case, although why this is so remains to be clarified.

Journal Article↗
PubMed2000

Enamel proteins and extracellular matrix molecules are co-localized in the pseudocystic stromal space of adenomatoid odontogenic tumor.

In order to examine the functional differentiation of tumor cells of adenomatoid odontogenic tumor (AOT) as ameloblasts and to determine the participation of the extracellular matrix (ECM) in the formation of its characteristic histologic architecture, tissue samples from five cases of adenomatoid odontogenic tumor were examined by immunohistochemical staining for enamel proteins and ECM molecules. Amelogenin, enamelin, laminin, heparan sulfate proteoglycan, fibronectin, collagen type IV and type V were immunolocalized within the luminal space and along the inner rim of duct-like structures. Eosinophilic hyaline droplets within the whorled or rosette masses of tumor cells showed basically the same staining pattern as the luminal contents. High columnar tumor cells that formed duct-like structures were immunopositive for amelogenin, while the staining intensity decreased with flattening of the cells, which was a result of luminal growth. The findings suggest that the constituent cells of duct-like structures are differentiated once to ameloblasts but fail to mature further due instead to increased production of ECM molecules and due to their retention in the lumina. It is possible to regard these special structures in AOT as stromal pseudocysts.

Amelogenin↗
PubMed2000

Inhibition of cell proliferation in HCC-9204 hepatoma cells by a c-myc specific ribozyme.

A ribozyme (RZ) gene targeting c-myc mRNA was synthesized and cloned. Cleavage reaction showed that cleavage of the RZ was efficient and specific. The RZ gene-containing retrovirus vector pDOR-RZ was transfected into HCC-9204 hepatoma cells, which constitutively express high levels of c-myc using Lipofectamine. Positively transfected cells were selected using G418. In situ hybridization showed that both pDOR-RZ and pDOR vectors had been integrated into the chromosome of HCC-9204 cells. Dot blot hybridization indicated that expression of the RZ was only evident in pDOR-RZ-transfected HCC-9204 cells. Avidin-biotin complex enzyme-linked immunosorbent assay showed that c-myc expression was down-regulated. Chromatin aggregation into compact masses, cytoplasmic vacuole degeneration, and blurring of cytoplasm structure were observed by transmission electron microscopy in HCC-9204-RZ cells. These results suggest that the use of a c-myc mRNA cleaving enzyme could be most effective in tumor cells that are highly proliferative and constitutively express high levels of c-myc.

Carcinoma, Hepatocellular↗
PubMed2000

Optimization of TEOAE recording protocols: a linear protocol derived from parameters of a time-frequency analysis: a pilot study on neonatal subjects.

Linear and non-linear TEOAE protocols were compared in terms of nine parameters in order to define the protocol producing recordings with the highest signal quality (lowest noise and highest signal-to-noise ratio). The pilot project acquired data using ILO-92 apparatus from 220 neonates (397 ears) at the second/third day after birth in three European laboratories. A Gabor spectrogram time-frequency representation of the recordings showed considerable frequency dispersion in TEOAE latencies >4.0 ms. The data, analysed with a Wilcoxon test, indicated that a linear TEOAE protocol: (i) generates recordings of a lower noise and a higher signal-to-noise (S/N) ratio in the 2.0, 3.0, 4.0 kHz TEOAE bands; (ii) the increase in the S/N ratio can result in a decrement of the required number of TEOAE sweeps; (iii) the higher values of S/N can be used in the estimation of more robust pass-fail criteria, minimizing the percentage of false positives and negatives.

Acoustic Stimulation↗