Biomedical subjects

J Ding

Publications and source records attributed to J Ding.

At least 55 records · Page 3Linked to original sources

PubMed2001

Orphanin FQ: an endogenous antagonist of rat brain dopamine transporter.

Orphanin FQ, also known as nociceptin (NC),is a well-known ligand for opioid receptor-like ORLI receptor. This heptadecapeptide was identified as potently inhibiting the uptake of rat dopamine transporter (rDAT) which is stably expressed in CHO cells (designated D8 cells). Further kinetic analysis proved that this occurs through competitive inhibition with an IC50 of about 1.9 microM. Orphanin FQ also inhibits [3H]dopamine uptake by rat striatal synaptosomes, which confirmed the effect of orphanin FQ on D8 cells. Orphanin FQ was also found to inhibit GABA transporter type I (GATI) but not the serotonin transporter. These results suggest that orphanin FQ is an endogenous antagonist of dopamine transport and that it affects locomotion and other activities at least partly by inhibiting dopamine transporter and directly affecting dopamine transmission or by inhibiting GABA transporter to indirectly change dopaimne transmission.

Animals↗

PubMed2001

Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA.

We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

Crystallography↗

PubMed2001

Genomic characterization, localization, and functional expression of FGL2, the human gene encoding fibroleukin: a novel human procoagulant.

For diseases in which thrombosis plays a pivotal role, such as virus-induced fulminant hepatitis, fetal loss syndrome, and xenograft rejection, the major procoagulant has remained elusive. Here we describe the isolation and functional expression of a distinct human prothrombinase, termed FGL2. The murine fgl2 gene product has been implicated in the pathophysiology of murine fulminant hepatitis. The predicted ORF corresponds to a 439-amino-acid type II integral membrane protein that contains a carboxy-terminal Fibrinogen-related domain. Functional analysis showed that FGL2-encoded protein is indeed a prothrombinase. This enzyme is a serine protease and directly cleaves prothrombin to thrombin. The FGL2 gene is a single-copy gene in the haploid human genome and has two exons separated by a 2195-bp intron expressing two mRNA transcripts of 1.5 and 5.0 kb. The 5'-flanking region contains putative cis-elements including a TATA box, an AP1 site, CEBP sites, Sp1 site, and Ets binding domains. By both radiation hybrid analyses and fluorescence in situ hybridization, human FGL2 was localized to 7q11.23.

Amino Acid Sequence↗

PubMed2001

Role of C5a in multiorgan failure during sepsis.

In humans with sepsis, the onset of multiorgan failure (MOF), especially involving liver, lungs, and kidneys, is a well known complication that is associated with a high mortality rate. Our previous studies with the cecal ligation/puncture (CLP) model of sepsis in rats have revealed a C5a-induced defect in the respiratory burst of neutrophils. In the current CLP studies, MOF occurred during the first 48 h with development of liver dysfunction and pulmonary dysfunction (falling arterial partial pressure of O(2), rising partial pressure of CO(2)). In this model an early respiratory alkalosis developed, followed by a metabolic acidosis with increased levels of blood lactate. During these events, blood neutrophils lost their chemotactic responsiveness both to C5a and to the bacterial chemotaxin, fMLP. Neutrophil dysfunction was associated with virtually complete loss in binding of C5a, but binding of fMLP remained normal. If CLP animals were treated with anti-C5a, indicators of MOF and lactate acidosis were greatly attenuated. Under the same conditions, C5a binding to blood neutrophils remained intact; in tandem, in vitro chemotactic responses to C5a and fMLP were retained. These data suggest that, in the CLP model of sepsis, treatment with anti-C5a prevents development of MOF and the accompanying onset of blood neutrophil dysfunction. This may explain the protective effects of anti-C5a in the CLP model of sepsis.

Acidosis↗

PubMed2001

Modulation of the promoter region of prepro-hypocretin by alpha-interferon.

Hypocretins 1 and 2 (also called orexins A and B, respectively) are hypothalamic neuropeptides that have recently been shown to be involved in the sleep disorder narcolepsy and possibly in the normal regulation of sleep and wake functions. These two peptides are derived from a single precursor molecule called prepro-hypocretin, also known as prepro-orexin. We have cloned a 450 bp fragment from the 5'-flanking region of the human prepro-hypocretin gene and demonstrated that this fragment has promoter activity in vitro. Deletions at the 5' end from -450 to -188 reduced the promoter activity by approximately 50%. Further deletion from the 5'-end to -69 almost completely abolished promoter activity. The 450 bp fragment contains a number of potential transcription factor binding sites, including an interferon (IFN) response element. Our studies demonstrate that alpha-IFN strongly inhibits the promoter activity of both 450 and 188 bp fragments in a dose-dependent manner. The inhibitory effect of alpha-IFN is consistent with recent studies which suggest that hypocretin 1/orexin A may be involved in modulating arousal states and with the literature indicating involvement of immune-related molecules in sleep regulation.

Animals↗

PubMed2001

Novel antitumor artemisinin derivatives targeting G1 phase of the cell cycle.

Modification of artemisinin structure led us to the discovery of a novel class of antitumor compounds. These artemisinin derivatives containing cyano and aryl groups showed potent antiproliferative effect in vitro against P388 and A549 cells. This activity was reflected in P388 murine leukemia by an accumulation of cells in G1 phase, and induction of apoptosis.

Animals↗

PubMed2001

Effects of lung surfactant proteins, SP-B and SP-C, and palmitic acid on monolayer stability.

Langmuir isotherms and fluorescence and atomic force microscopy images of synthetic model lung surfactants were used to determine the influence of palmitic acid and synthetic peptides based on the surfactant-specific proteins SP-B and SP-C on the morphology and function of surfactant monolayers. Lung surfactant-specific protein SP-C and peptides based on SP-C eliminate the loss to the subphase of unsaturated lipids necessary for good adsorption and respreading by inducing a transition between monolayers and multilayers within the fluid phase domains of the monolayer. The morphology and thickness of the multilayer phase depends on the lipid composition of the monolayer and the concentration of SP-C or SP-C peptide. Lung surfactant protein SP-B and peptides based on SP-B induce a reversible folding transition at monolayer collapse that allows all components of surfactant to be retained at the interface during respreading. Supplementing Survanta, a clinically used replacement lung surfactant, with a peptide based on the first 25 amino acids of SP-B also induces a similar folding transition at monolayer collapse. Palmitic acid makes the monolayer rigid at low surface tension and fluid at high surface tension and modifies SP-C function. Identifying the function of lung surfactant proteins and lipids is essential to the rational design of replacement surfactants for treatment of respiratory distress syndrome.

Adsorption↗

PubMed2001

Peptide derived from insulin with regulatory activity of dopamine transporter.

A nonapeptide derived from the C terminus of the insulin B chain, H(2)N-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala-COOH, was found to strongly inhibit dopamine (DA) uptake by rat dopamine transporter (DAT) stably expressed in CHO cells (designated D8 cells). The kinetic experiments on D8 cells gave a curve typical of competitive inhibition with an IC(50)=6.9 microM. This inhibitory effect was also confirmed by experiments on striatal synaptosomes. The rat administered with the nonapeptide unilaterally into substantia nigra showed dose-dependent velocity and duration of the round movement contralateral to the nonapeptide-injected side. In addition, the nonapeptide dose-dependently reduced the binding of the tritium-labeled cocaine analog (-)-2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (WIN35,428) to DAT of D8 cells, which suggests that the nonapeptide may inhibit the transport activity of DAT in the way as cocaine does. Meanwhile, the peptide DOI (insulin with 8 amino acid residues deleted at the C terminus of the B chain) shows a significantly stimulating effect on DAT uptake activity in D8 cells. So insulin is proposed as a kind of neuropeptide precursor in the brain and insulin-derived peptides may be involved in the process of regulating the DA system, and these peptides may be developed into new medicines for disorders concerning the DA system such as Parkinson's disease and cocaine addiction.

Aminobutyrates↗

PubMed2001

Different effects of toosendanin on perineurially recorded Ca(2+) currents in mouse and frog motor nerve terminals.

By perineurial recording, the effects of toosendanin (TSN), a presynaptic blocker, on nerve terminal calcium currents (I(Ca)) were observed in innervated triangularis sterni of the mouse and cutaneous pectoris of the frog. It was found that TSN blocked the slow component of I(Ca) insensitive to nifedipine and omega-conotoxin-GVIA, and increasing the extracellular Ca(2+) concentration partially antagonized the inhibitory effect in mouse motor nerve terminals. However, in the frog, TSN increased the slow component of I(Ca) and this effect disappeared in the presence of nifedipine in perfusion solution. Based on previous data showing that the slow component of I(Ca) were mediated by different subtypes of calcium channels in mouse and frog motor nerve terminals, we presume that TSN could exercise different effects on various subtypes of calcium channels.

Animals↗

PubMed2001

Synthesis and cytotoxicity of artemisinin derivatives containing cyanoarylmethyl group.

A series of 12alpha-deoxoartemisinyl cyanoarylmethyl dicarboxylates (4a-4o), dicarboxylic acids 12alpha-deoxoartemisinyl ester cyanoarylmethyl amide (5a-5k), and dicarboxylic acids 12alpha-deoxoartemisinyl ester N-methylcyanoarylmethyl amide (6a-6l), showing moderate cytotoxicity against P388 and L1210 cells were prepared. They induced the significant accumulation of L1210 and P388 cells in the G1 phase of the cell cycle. This mechanism of action was quite different from that of the majority of cytotoxic compounds used in the chemotherapy of cancer. Compound 4b possessed better cytotoxicity than the other compounds.

Animals↗

PubMed2001

Low synthesis of retinoic acid due to impaired cytochrome P450 1a1 expression in mouse xeroderma pigmentosum fibroblasts.

New tumor formation was suppressed by retinoic acid (RA) administration in xeroderma pigmentosum (XP) patients who have a defect in nuclear excision repair. However, the inhibition is not due to enhanced removal of UV-damaged DNA. These results prompted us to investigate whether or not RA metabolism is abnormal in XP fibroblasts and what the underlying mechanism is. Compared with wild type fibroblasts, low activities of RA synthesis were determined on HPLC in mouse fibroblasts lacking XP group A (XPA) gene and UV-induced XPA deficient cancer cells. Moreover, we observed an impaired expression of cytochrome P450 1a1 in XPA deficient fibroblasts by RT-PCR and a decreased expression of retinoic acid receptor gamma in XPA deficient cancer cells by Western blotting. Finally, pre-treatment of RA isoforms significantly protected the XPA deficient fibroblasts from UV-induced death. These results suggest that decreased structure activity of RA synthesis, resulting from impaired mRNA expression of cytochrome P450 1a1 may, at least together with UV irradiation, involve in skin carcinogenesis in XP patients.

Alitretinoin↗

PubMed2001

Cytotoxic oxoisoaporphine alkaloids from Menispermum dauricum.

Four new oxoisoaporphine alkaloids, daurioxoisoporphines A-D (1-4), were isolated from the rhizomes of Menispermum dauricum. The structures of these alkaloids were established by spectroscopic methods. The cytotoxic evaluation of 1 and 2 is reported against four cancer cell lines.

Adenocarcinoma↗

PubMed2001

Transgenic zebrafish for studying nervous system development and regeneration.

Alpha1 tubulin gene expression is induced in the developing and regenerating CNS of vertebrates. Therefore, alpha1 tubulin gene expression may serve as a good probe for mechanisms underlying CNS development and regeneration. One approach to identify these mechanisms is to work backwards from the genome. This requires identification of alpha1 tubulin DNA sequences that mediate its developmental and regeneration-dependent expression pattern. Therefore, we generated transgenic zebrafish harboring a fragment of the alpha1 tubulin gene driving green fluorescent protein expression (GFP). In these fish, and similar to the endogenous gene, transgene expression was dramatically induced in the developing and regenerating nervous system. Although transgene expression generally declined during maturation of the nervous system, robust GFP expression was maintained in progenitor cells in the retinal periphery, lining brain ventricles and surrounding the central canal of the spinal cord. When these cells were cultured in vitro they divided and gave rise to new neurons. We also show that optic nerve crush in adult fish re-induced transgene expression in retinal ganglion cells. These studies identified a relatively small region of the alpha1 tubulin promoter that mediates its regulated expression pattern in developing and adult fish. This promoter will be extremely useful to investigators interested in targeting gene expression to the developing or regenerating nervous system. As adult transgenic fish maintain transgene expression in neural progenitors, these fish also provide a valuable resource of labeled adult neural progenitor cells that can be studied in vivo or in vitro. Finally, these fish should provide a unique in vivo system for investigating mechanisms mediating CNS development and regeneration.

Animals↗

PubMed2001

Requirement of Syk-phospholipase C-gamma2 pathway for phorbol ester-induced phospholipase D activation in DT40 cells.

BACKGROUND: Treatment of many cell types with phorbol esters stimulates phospholipase D (PLD) activity implying regulation of the enzyme by protein kinase C. Studies of the effects of several protein-tyrosine kinase (PTK) inhibitors have suggested that PTK(s) play some roles in the phorbol ester-induced PLD activation, but it remains unclear how and which PTK(s) is involved in this pathway. In this study, we investigated the roles of Syk and other PTKs for the phorbol esters, 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced PLD activation in K562 and DT40 cells. RESULTS: TPA-induced PLD activation was remarkably reduced in both Syk dominant negative mutant K562 cells and Syk deficient DT40 B cells. Mutational analysis further indicated that two major autophosphorylation sites (Tyr-518 and Tyr-519) of Syk are critical for PLD activation. Similarly, TPA-induced PLD activation was reduced in Btk deficient cells, but unaffected in Lyn deficient cells. Finally, in cells deficient in the PLC-gamma2, one of the phosphorylated substrates regulated by Syk and Btk, TPA-induced PLD activation, as well as phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis was remarkably reduced. CONCLUSIONS: We demonstrated that the Syk, Btk and PLC-gamma2 pathways are required for TPA-induced PLD activation in DT40 cells.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed2001

Immunogenicity and specificity of the candidate multi-epitope-vaccines against HIV-1.

The failure of some candidate HIV-1 vaccines may result from inducing very weak neutralization activity against representative primary viral isolates. Based on our hypothesis that epitope-vaccine may be a new strategy to induce high levels of neutralizing antibodies against HIV-1, we designed two candidate multi-epitope-vaccines, EP1 [C-G-(ELDKWA-GPGRAFY)2-K] and EP2 (CG-GPGRAFY-G-ELDKWA-G-RILAVERYLKD), containing three neutralizing epitopes (GPGRAFY, ELDKWA and RILAVERYLKD) on HIV-1 envelope protein, and expected them to induce epitope-specific antibodies of predefined epitope-specificity. The two peptides were conjugated to carrier protein bovine serum albumin (BSA) and used for immunization of rabbits. Proteins were purified from the rabbit sera induced by both candidate multi-epitope-vaccines (EP1-BSA and EP2-BSA) through affinity chromatography with epitope-peptide-conjugated sepharose-column, and identified as antibodies in silver-staining and immunoblotting. These antibodies were demonstrated to recognize three neutralizing epitopes on peptides and the recombinant gp41 in ELISA-assay and immunoblotting. These results indicated that both candidate multi-epitope-vaccines could induce high levels of antibodies of predefined epitope-specificity which recognized a few of neutralizing epitopes on peptides and protein, providing experimental evidence for the new strategy to develop an effective neutralizing-antibody-based multi-epitope-vaccine against HIV-1.

AIDS Vaccines↗

PubMed2001

Role of BLNK in oxidative stress signaling in B cells.

BLNK (B cell linker protein) represents a central linker protein that bridges the B cell receptor-associated kinases with a multitude of signaling pathways. In this study, we have investigated the role of BLNK in oxidative stress signaling in B cells. H2O2 treatment of B cells induced a rapid tyrosine phosphorylation of BLNK in a H2O2 dose-dependent manner, which was inhibited in Syk-deficient DT40 cells. Calcium mobilization in BLNK-deficient as well as Syk-deficient and phospholipase C (PLC)-gamma2-deficient cells after H2O2 treatment was completely abolished. These were derived from decreased inositol 1,4,5-trisphosphate generation through PLC-gamma2 in BLNK-deficient cells. Moreover, viability of BLNK-deficient as well as PLC-gamma2-deficient cells after exposure to low doses of H2O2 was dramatically enhanced compared with that of the wild-type cells. Furthermore, c-Jun N-terminal kinase activation following high doses of H2O2 stimulation, but not low doses of H2O2 stimulation, was abrogated in BLNK-deficient as well as Syk-deficient cells. These findings have led to the suggestion that BLNK is required for coupling Syk to PLC-gamma2, thereby accelerating cell apoptosis in B cells exposed to low doses of H2O2.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Apoptosis in endometrial glandular and stromal cells in women with and without endometriosis.

BACKGROUND: The aetiology of endometriosis is unknown. Ectopic dissemination of the endometrial cells gives origin to endometriotic lesions, but occurs in women with and without endometriosis. It has been suggested that increased ectopic cell survival facilitates their implantation. The objectives of this study were to evaluate endometrial apoptosis in women with endometriosis according to: (i) cyclic changes, (ii) glandular and stromal contribution, and (iii) stage of the disease. METHODS: The subjects were women undergoing diagnostic laparoscopy and endometrial biopsies for suspected endometriosis. Spontaneous apoptosis was evaluated using TdT-mediated dUTP-biotin nick end-labelling (TUNEL) assay. Apoptotic cells per 10 mm(2) (apoptotic index) in an area of 10-50 mm(2) in 5 microm endometrial tissue sections were counted and location of these cells was recorded. RESULTS: The apoptotic index in glandular epithelium was lower in endometriosis than controls (26.0 +/- 5.5 versus 51.2 +/- 9.7, P = 0.03) but not in the stroma (36.3 +/- 6.4 versus 48.4 +/- 11.3, NS). In controls, apoptosis was highest during the late secretory/menstrual and early proliferative phases and cyclic variability was apparent. In endometriosis, this cyclic variability was lost. There was a trend toward decreased apoptosis with increasing stage of the disease, but the differences lacked statistical significance. CONCLUSIONS: Spontaneous apoptosis is decreased in the endometrial glands in women with endometriosis, especially during late secretory/menstrual and early proliferative phases of the cycle. This may indicate increased viability of endometrial cells shed during menses, facilitating their ectopic survival and implantation.

Adult↗

PubMed2001

Identification of disease-specific genes in chronic pancreatitis using DNA array technology.

OBJECTIVE: To use DNA arrays to analyze the differential gene expression patterns in the normal pancreas and in pancreatic diseases. SUMMARY BACKGROUND DATA: Genome-wide gene expression analysis will provide new insights into gene function and cause of disease. METHODS: RNA was extracted from eight normal pancreatic specimens, eight specimens with chronic pancreatitis (CP), and eight pancreatic cancer (PCa) tissues. Poly A(+) RNA was purified, reverse-transcribed, and converted into cRNA using biotinylated nucleotides. The HuGeneFL DNA array containing 5,600 full-length human genes was used for analysis. RESULTS: First, normal pancreatic tissues were analyzed in comparison with a panel of other normal tissues (colon, liver, prostate, lung, lymph node). This analysis revealed 11 signature genes that were selectively expressed in the pancreas (e.g., pancreatic elastase-IIA). Comparison of the expression of 5,600 genes between the normal pancreas, CP, and PCa specimens showed that the expression of 34 genes was decreased in CP tissues compared with normal pancreatic tissues, and that the expression of all of these genes was simultaneously decreased in PCa. In addition, the expression of 157 genes was increased in CP tissues compared with the normal pancreas. Of those, 152 genes were simultaneously increased in PCa. Thus, only 5 of 5,600 genes were significantly overexpressed in CP compared with both normal pancreas and PCa. CONCLUSIONS: The majority of alterations observed in CP are present in PCa, and the number of genes whose expression is selectively deregulated in CP is surprisingly small. These results may provide new insight into the pathobiology of CP and help identify certain molecular alterations that might serve as targets for new diagnostic tools and disease-specific therapy.

Adult↗