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Biomedical subjects

W Dai

Publications and source records attributed to W Dai.

At least 55 records · Page 3Linked to original sources
PubMed2000

Difference of rejection in single versus combined pancreas and kidney transplantation in rats.

OBJECTIVE: To investigate the difference of rejection in single versus combined pancreas and kidney transplantation in rats. METHODS: Allograft models including simultaneous pancreas and kidney (SPK) transplant and pancreas or kidney transplant alone were established in SD-Wistar rats, rejections of pancreas and kidney in different models were compared morphologically and functionally. RESULTS: Mean survival time (MST) of pancreas was significantly prolonged in SPK than in pancreas transplant alone (PTA) (11.5 days vs. 9.2 days, P < 0.05). Incidence of interstitial pancreatic rejection at gade II and grade III was much obvious in PTA than in SPK (42.9% vs. 12.5% at grade II and 28.6% vs 6.3% at grade II , P < 0.05). No significant difference was found in MST between SPK and kidney transplant alone( KTA). Administration of cyclosporine A prolonged the MST of pancreas and kidney, without altering the tendency stated above. CONCLUSIONS: In SPK, the function of pancreas is protected by kidney hence the severity of rejection is reduced, whereas the function of kidney is not protected by pancreas. It suggests that different organs differ in immunoallergization and immunoregulation, and immune response tend to attack organs with greater immunoactivity, those organs with minor one could be protected. Cyclosporine A is effective on prolonging the MST of pancreas and kidney.

Animals↗
PubMed2000

[Resources of spectroscopy and spectrometry on the World Wide Web--I].

Some resources of spectroscopy and spectrometry on the world wide web, including spectra databases, journals, societies, associations and organizations, conferences and syposia, institutes and companies, and spectroscopists, are introduced. A lot of useful websites are given in this paper.

Databases as Topic↗
PubMed1999

The physical association and phosphorylation of Cdc25C protein phosphatase by Prk.

prk encodes a protein serine/threonine kinase involved in regulating M phase functions during the cell cycle. We have expressed His6-Prk and His6-Cdc25C proteins using the baculoviral vector expression system. Purified recombinant His6-Prk, but not a kinase-defective mutant His6-PrkK52R, is capable of strongly phosphorylating His6-Cdc25C in vitro. Co-immunoprecipitation and affinity column chromatography experiments demonstrate that GST-Prk and native Cdc25C interact. When co-infected with His6-Prk and His6-Cdc25C recombinant baculoviruses, sf-9 cells produce His6-Cdc25C antigen with an additional slower mobility band on denaturing polyacrylamide gels compared with cells infected with His6-Cdc25C baculovirus alone. In addition, His6-Cdc25C immunoprecipitated from sf-9 cells co-infected with His6-Prk and His6-Cdc25C baculoviruses, but not with His6-PrkK52R and His6-Cdc25C baculoviruses, contains a greatly enhanced kinase activity that phosphorylates His6-Cdc25C in vitro. Moreover, phosphopeptide mapping shows that His6-Prk phosphorylates His6-Cdc25C at two sites in vitro and that the major phosphorylation site co-migrates with the one that is phosphorylated in vivo in asynchonized cells. Further studies reveal that His6-Prk phosphorylates Cdc25C on serine216, a residue also phosphorylated by Chk1 and Chk2. Together, these observations strongly suggest that Prk's role in mitosis is at least partly mediated through direct regulation of Cdc25C.

Animals↗
PubMed1999

The homeobox gene NKX3.2 is a target of left-right signalling and is expressed on opposite sides in chick and mouse embryos.

Vertebrate internal organs display invariant left-right (L-R) asymmetry. A signalling cascade that sets up L-R asymmetry has recently been identified (reviewed in [1]). On the right side of Hensen's node, activin represses Sonic hedgehog (Shh) expression and induces expression of the genes for the activin receptor (ActRIIa) and fibroblast growth factor-8 (FGF8) [2] [3]. On the left side, Shh induces nodal expression in lateral plate mesoderm (LPM); nodal in turn upregulates left-sided expression of the bicoid-like homeobox gene Pitx2 [4] [5] [6]. Here, we found that the homeobox gene NKX3.2 is asymmetrically expressed in the anterior left LPM and in head mesoderm in the chick embryo. Misexpression of the normally left-sided signals Nodal, Lefty2 and Shh on the right side, or ectopic application of retinoic acid (RA), resulted in upregulation of NKX3.2 contralateral to its normal expression in left LPM. Ectopic application of FGF8 on the left side blocked NKX3.2 expression, whereas the FGF receptor-1 (FGFR-1) antagonist SU5402, implanted on the right side, resulted in bilateral NKX3.2 expression in the LPM, suggesting that FGF8 is an important negative determinant of asymmetric NKX3.2 expression. NKX3.2 expression was also found to be asymmetric in the mouse LPM but, unlike in the chick, it was expressed in the right LPM. In the inversion of embryonic turning (inv) mouse mutant, which has aberrant L-R development, NKX3.2 was expressed predominantly on the left side. Thus, NKX3.2 transcripts accumulate on opposite sides of mouse and chick embryos although, in both the mouse and chick, NKX3.2 expression is controlled by the L-R signalling pathways.

Animals↗
PubMed1999

A requirement for K+-channel activity in growth factor-mediated extracellular signal-regulated kinase activation in human myeloblastic leukemia ML-1 cells.

Voltage-gated K+ channels have been shown to be required for proliferation of various types of cells. Much evidence indicates that K+-channel activity is required for G1 progression of the cell cycle in different cell backgrounds, suggesting that K+-channel activity is required for early-stage cell proliferation in these cells. However, little is known about the molecular mechanisms that underlie this phenomenon. We have shown in human myeloblastic leukemia ML-1 cells that K+ channels are activated by epidermal growth factor (EGF), whereas serum starvation deprivation suppressed their activity. In addition, voltage-gated K+ channels are required for G1/S-phase transition of the cell cycle. We report here that suppression of K+ channels prevented the activation of extracellular signal-regulated protein kinase 2 (ERK-2) in response to EGF and serum. However, blockade of K+ channels did not prevent ERK-2 activation induced by 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Elimination of extracellular Ca2+ did not alter either ERK-2 activation or the effect of K+-channel blockade on ERK-2 activation. Our data demonstrate that the K+ channel is a part of the EGF-mediated mitogenic signal-transduction process and is required for initiation of the EGF-mediated mitogen-activated protein kinase (MAPK) pathways. Our findings may thus explain why an increase in K+-channel activity is associated with cell proliferation in many types of cells, including ML-1 cells.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1999

Specific changes in beta2-adrenoceptor trafficking kinetics and intracellular sorting during downregulation.

Agonist-activated beta2-adrenoceptors rapidly internalize and then recycle to the cell surface, however chronic agonist eventually causes receptor downregulation. To characterize beta2-adrenoceptor trafficking kinetics and intracellular sorting during downregulation, human embryonic kidney cells expressing epitope-tagged receptors were examined by radioligand binding with (+/-)-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole- 2-on hydrochloride ([3H]CGP12177) and immunofluorescence microscopy. The first-order receptor recycling rate constant declined after 18 h of agonist compared with 15 min (0.05 min(-1) vs. 0.12 min(-1)), thus increasing the intracellular transit time (20.0 min vs. 8.3 min). There was also a reduction in the rate of receptor endocytosis and a decline in the total number of receptors. Although the intracellular receptor fraction did not increase between 15 min and 18 h of agonist, some receptors moved irreversibly into a protease-containing compartment while retaining radioligand binding activity. Our results indicate that beta2-adrenoceptor downregulation is associated principally with an increased intracellular transit time during recycling. This could promote the diversion of receptors into protease-containing compartments, where there is an irreversible commitment to downregulation prior to loss of radioligand binding activity.

Adrenergic beta-Agonists↗
PubMed1999

An ultraviolet-activated K+ channel mediates apoptosis of myeloblastic leukemia cells.

Exposure of mammalian cells to UV light causes initial changes in the cell membrane, induces phosphorylation and clustering of growth factor/cytokine receptors, and activates the Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathway leading to programmed cell death (apoptosis). In this study, we found that an early event in the cell membrane of myeloblastic leukemia (ML-1) cells was the vigorous activation of the voltage-gated K+ channel by UV irradiation. The strong enhancement by UV irradiation of K+ channel activity in the cell membrane subsequently activated the JNK/SAPK signaling pathway and resulted in myeloblastic leukemia cell apoptosis. Suppression of UV-induced K+ channel activation with specific channel blockers prevented UV-induced apoptosis through inhibition of UV-induced activation of the proteins SEK (SPAK kinase) and JNK. However, suppression of K+ channel activity could not protect cells from etoposide-induced apoptosis, which bypasses the membrane event. Elimination of extracellular Ca2+ had no effect on the UV-induced and K+ channel-mediated JNK/SAPK activation. Thus, we have identified a novel mechanism in which activation of K+ channels by UV-irradiation upstream of SEK and SAPK/JNK mediates UV-induced myeloblastic cell apoptosis.

Apoptosis↗
PubMed1999

Molecular characterization of the human CRT-1 creatine transporter expressed in Xenopus oocytes.

The protein sequence encoded by a creatine transporter cDNA cloned from a human heart library was identical to that cloned from a human kidney library (Nash et al., Receptors Channels 2, 165-174, 1994), except that at position 285 the former contained an Ala residue and the latter contained a Pro residue. Expression of this human heart cDNA clone in Xenopus laevis oocytes induced a Na+- and Cl--dependent creatine uptake activity that saturated with a Km of approximately 20 microM for creatine. The induced uptake was inhibited by beta-guanidinopropionic acid (IC50 approximately 44.4 microM), 2-amino-1-imidazolidineacetic acid (cyclocreatine; IC50 approximately 369.8 microM), gamma-guanidinobutyric acid (IC50 approximately 697.9 microM), gamma-aminobutyric acid (IC50 approximately 6.47 mM), and amiloride (IC50 approximately 2.46 mM). The inhibitors beta-guanidinopropionic acid, cyclocreatine, and gamma-guanidinobutyric acid also inhibited the uptake activity of the Ala285 to Pro285 (A285P) mutant as effectively as that of the wild type. In contrast, guanidinoethane sulfonic acid, a potent inhibitor of taurine transport, inhibited the uptake activity of the A285P mutant approx. two times more effectively than that of the wild type. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), but not its inactive analog, 4alpha-phorbol 12, 13-didecanoate, inhibited the creatine uptake, and the inhibitory effect of PMA was both time and concentration dependent. The protein kinase A activator 8-bromo-cyclic AMP, however, had no effect on the creatine uptake. The rate of uptake increased hyperbolically with the increasing concentration of the external Cl- (equilibrium constant KCl- approximately 5 mM) and sigmoidally with the increasing concentration of the external Na+ (equilibrium constant KNa+ approximately 56 mM). Further analyses of the Na+ and Cl- concentration dependence data suggested that at least two Na+ and one Cl- were required to transport one creatine molecule via the creatine transporter.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Involvement of capsaicin-sensitive sensory nerves in early and delayed cardioprotection induced by a brief ischaemia of the small intestine.

Early cardioprotection can be achieved by a brief ischaemia of noncardiac tissues. Our study examined whether a brief ischaemia of the small intestine induces both early and delayed cardioprotection in the rabbit and assessed the possible mechanism involved in the activation of capsaicin-sensitive sensory nerves. The plasma concentration of creatine kinase (CK) and infarct size (necrotic zone/left ventricular zone) after 30 min coronary artery occlusion and 180 min reperfusion were determined in rabbits. Infarct size was 35.5+/-6.8% in the control non-preconditioned group. Preconditioning induced by a brief period of 10-min small intestine ischaemia significantly reduced infarct size (6.5+/-1.9%, P<0.01 vs. the control non-preconditioned group) and decreased CK release (3092+/-236 and 1094+/-117 U/l for myocardial ischaemia-reperfusion and preconditioning plus myocardial ischemia-reperfusion, respectively, P<0.01), and the protection was partly abolished by pretreatment with capsaicin (50 mg/kg, s.c.) 4 days before the experiments. A brief period of anterior mesenteric artery occlusion caused an increase in the plasma level of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI), an effect which was abolished by pretreatment with capsaicin. Similar protection was shown in the animals subjected to a brief period of anterior mesenteric artery occlusion 24 h before coronary artery occlusion, and this delayed protection was also abolished partly by pretreatment with capsaicin. Capsaicin treatment (50 mg/kg, s.c.) alone also protected the ischaemic myocardium. The results suggest that brief ischaemia of the small intestine induces both early and delayed protection against reperfusion-induced myocardial injury, and the effects are, at least partly, related to the activation of capsaicin-sensitive sensory nerves.

Animals↗
PubMed1999

Agonist-induced sorting of human beta2-adrenergic receptors to lysosomes during downregulation.

During prolonged exposure to agonist, beta2-adrenergic receptors undergo downregulation, defined by the loss of radioligand binding sites. To determine the cellular basis for beta2-adrenergic receptor downregulation, we examined HEK293 cells stably expressing beta2-adrenergic receptors with an N-terminal epitope tag. Downregulation was blocked by leupeptin, a cysteine protease inhibitor, but not by pepstatin, an inhibitor of aspartate proteases. Immunofluorescence microscopy of cells treated with agonist for 3-6 hours in the presence of leupeptin showed beta2-adrenergic receptors, but not transferrin receptors, localizing with the lysosomal protease cathepsin D, and with lysosomes labeled by uptake of a fluorescent fluid-phase marker. No localization of beta2-adrenergic receptors with lysosomal markers was observed in the absence of leupeptin, most likely due to proteolysis of the epitope. The proton pump inhibitor, bafilomycin A1, significantly inhibited this agonist-induced redistribution of beta2-adrenergic receptors into lysosomes, causing receptors to accumulate in the rab11-positive perinuclear recycling compartment and slowing the rate of beta2-adrenergic receptor recycling. Control experiments showed that leupeptin had no nonspecific effects on the cellular trafficking of either beta2-adrenergic receptors or transferrin receptors. Although cAMP alone caused a small decline in receptor levels without redistributing beta2-adrenergic receptors from the plasma membrane, this effect was additive to that seen with agonist alone, suggesting that agonist-induced beta2-adrenergic receptor downregulation resulted largely from cAMP-independent mechanisms. These results indicate that during agonist-induced downregulation, a significant fraction of beta2-adrenergic receptors are specifically sorted to lysosomes via the endosomal pathway, where receptor degradation by cysteine proteases occurs. These results provide a cellular explanation for the loss of radioligand binding sites that occurs during prolonged exposure to agonist.

Adrenergic beta-2 Receptor Agonists↗
PubMed1999

[Effect of mutation (T1762A1764) on hepatitis B virus core promoter activity].

OBJECTIVE: With the knowledge that the substituted mutation of T1762A1764 in hepatitis B virus (HBV) core promoter (CP) region was the most common variation during chronic HBV infection, we proceed to study the role that the mutation T1762A1764 plays in effecting the core promoter. METHODS: Seven patients with fulminant hepatitis and one asymptomatic HBV carrier were investigated for screening T1762A1764 mutation by cloning and PCR products direct sequencing. PCR products containing HBV precore/core gene regulatory sequences were directly cloned into chloramphenicol acetyltransferase (CAT) expressing plasmid. CAT assay was done to compare CAT expressing level after transfecting into HepG2 cell line and transient expression. RESULTS: Results reveal that T1762A1764 mutation was the main factor that represses CAT expression in vitro. CONCLUSION: Among the several other variations within hepatitis B virus' core promoter region, T1762A1764 mutation has mainly affected the down-regulating activity of core promoter.

Adult↗
PubMed1999

BUBR1 phosphorylation is regulated during mitotic checkpoint activation.

Eukaryotic cells have evolved a mechanism that delays the progression of mitosis until condensed chromosomes are properly positioned on the mitotic spindle. To understand the molecular basis of such monitoring mechanism in human cells, we have been studying genes that regulate the mitotic checkpoint. Our early studies have led to the cloning of a full-length cDNA encoding MAD3-like protein (also termed BUBR1/MAD3/SSK1). Dot blot analyses show that BUBR1 mRNA is expressed in tissues with a high mitotic index but not in differentiated tissues. Western blot analyses show that in asynchronous cells, BUBR1 protein primarily exhibits a molecular mass of 120 kDa, and its expression is detected in most cell lines examined. In addition, BUBR1 is present during various stages of the cell cycle. As cells enter later S and G2, BUBR1 levels are increased significantly. Nocodazole-arrested mitotic cells obtained by mechanical shake-off contain BUBR1 antigen with a slower mobility on denaturing SDS gels. Phosphatase treatment restores the slowly migrating band to the interphase state, indicating that the slow mobility of the BUBR1 antigen is attributable to phosphorylation. Furthermore, purified recombinant His6-BUBR1 is capable of autophosphorylation. Our studies indicate that BUBR1 phosphorylation status is regulated during spindle disruption. Considering its strong homology to BUB1 protein kinase, BUBR1 may also play an important role in mitotic checkpoint control by phosphorylation of a critical cellular component(s) of the mitotic checkpoint pathway.

Cell Cycle Proteins↗
PubMed1999

Experimental study of growth of trabecular cells on the filters and hydraulic conductivity.

OBJECTIVES: To search the method of culturing human trabecular cells (HTC) on a filter support so as to provide a model to study the hydraulic conductivity of HTC in vivo. METHODS: The third passage of HTC was cultured on a nylon filter; after that we measured the rate of different irrigations through the filter with HTC [Lp, microliter/(min.mm Hg.cm2)]. RESULTS: HTC could continuously grow on the filters. The normal Lp was 10.45 microliters/(min.mm Hg.cm2). Irrigated by the solution of epinephrine (EPI) or dexamethasone (DEX), Lp of HTC were higher than that in controls of the same cultural time, while after being exposed to DEX for a few days, Lp was significantly decreased. CONCLUSIONS: (1) More information of hydraulic conductivity and effects of pharmacologic agents on HTC could be got from the dynamic filtery model; (2) EPI could improve the conductivity of HTC while DEX could have the same effect in early period.

Aqueous Humor↗
PubMed1999

Experimental study on preservation of rat fatty liver.

OBJECTIVE: To investigate the effects of cold preservation on rat fatty liver. METHODS: We observed the changes of portal perfusion pressure, endothelin-1, enzymes release in the effluent and mortality of sinusoid lining cell after 0h, 6h, or 12h preservation respectively and a subsequent 30 min reperfusion in rat fatty liver groups and control groups by using isolated perfused rat liver model. And we compared fatty liver groups with control groups by these indices. RESULTS: There was no obvious difference between mildly fatty liver group and control group after long time (12h) preservation, between moderately fatty liver group and control group after short time (6h) preservation, between severely fatty liver group and control group without preservation (0h), while preservation reperfusion injury was more severe in moderately fatty liver group than in control group after long time (12h) preservation and in severely fatty liver group than in control group after short time (6h) preservation. CONCLUSIONS: The authors suggested that a mildly fatty liver donor could be used in the same way as nonfatty liver and a moderately fatty liver donor could be used depending on the time of preservation and the balance of the emergent needs of recipient and donor organ supply, while severely fatty liver donor should be discarded without hesitation.

Animals↗
PubMed1998

Identification of immediate early genes during TPA-induced human myeloblastic leukemia ML-1 cell differentiation.

Human myeloblastic ML-1 can be induced to differentiate into monocytes/macrophages by 12-0-tetradecanoylphorbol-13-acetate (TPA). In order to understand the molecular mechanism regulating ML-1 cell differentiation, we focused on the characterization of immediate early genes activated by TPA using the mRNA differentiation display polymerase chain reaction (DD-PCR) and Northern analyses. A modified procedure, the reverse dot slot, was developed to confirm upregulated genes during the early stages of TPA-induced ML-1 cell differentiation. DNA sequencing analyses of 10 subcloned cDNA fragments, selected on the basis of the outcome of the reverse dot slot procedure, revealed that eight were derived from distinct genes. Among these clones, one was a novel gene (G07-5), another (A02-1) was highly homologous to the sequence of a fetal brain cDNA fragment, and the remaining six corresponded to jun-D, rantes, ssat, CD 14, ferritin heavy chain (fhc) and transposons Tn10-like transcript, respectively. Although these genes were all upregulated by TPA, the peak time of mRNA expression varied. jun-D, ssat and A02-1 expressions were superinduced in the presence of cycloheximide, which indicates that they belong to the immediate early gene family. On the other hand, TPA-induced rantes expression was not superinduced by cycloheximide, suggesting a protein synthesis-dependent process. As there are no previous reports of expression of these genes in TPA-induced ML-1 cells, little or no information is available concerning their function in mediating myeloblastic cell differentiation. Thus, this study illuminates new avenues of research for elucidating the function of genes regulating terminal differentiation of myeloid progenitors. 1998 Elsevier Science B.V.

Cell Differentiation↗
PubMed1998

Megakaryocytic differentiation of HIMeg-1 cells induced by interferon gamma and tumour necrosis factor alpha but not by thrombopoietin.

Activated macrophage-conditioned medium (M-CM) induces megakaryocytic differentiation of HIMeg-1 cells. The megakaryocytic differentiation activity (MDA) is proteinaceous since it is susceptible to treatments by proteinases, heat, and reducing agents. MDA is not thrombopoietin (TPO) since (1) TPO alone or in conjunction with several other recombinant cytokines fails to induce any degree of HIMeg-1 cell differentiation; and (2) a neutralizing antibody against TPO or an antibody against the extracellular domain of c-mpl is unable to abolish M-CM-induced CD41 expression on HIMeg-1 cells. Reverse transcriptase-mediated polymerase chain reaction shows that HIMeg-1 cells express c-mpl but not TPO. Additional neutralizing antibody studies suggest that MDA is not one of the cytokines known to induce some degree of megakaryopoiesis in vitro or in vivo including interleukin 3 (IL-3), IL-6, IL-11, granulocyte-macrophage colony-stimulating factor, erythropoietin, or stem cell factor. On the other hand, MDA appears to be a combination of interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha), since neutralizing antibodies against these two cytokines completely abolish MDA-induced CD41 expression. In addition, either recombinant human IFN-gamma or TNF-alpha alone is capable of inducing CD41 and CD42 expression on HIMeg-1 cells. In combination, IFN-gamma and TNF-alpha induce a maximal level of CD41 and CD42 expression which is also accompanied by an increase in cell size and DNA ploidy level. Thus, our studies indicate that IFN-gamma/TNF-alpha is capable of inducing megakaryocytic differentiation of the HIMeg-1 cell line and that HIMeg-1 is a good system for studying the molecular mechanism mediating megakaryocytic differentiation.

Antibodies↗
PubMed1998

Inversin, a novel gene in the vertebrate left-right axis pathway, is partially deleted in the inv mouse.

Visceral left-right asymmetry occurs in all vertebrates, but the inversion of embryo turning (inv) mouse, which resulted following a random transgene insertion, is the only model in which these asymmetries are consistently reversed. We report positional cloning of the gene underlying this recessive phenotype. Although transgene insertion was accompanied by neighbouring deletion and duplication events, our YAC phenotype rescue studies indicate that the mutant phenotype results from the deletion. After extensively characterizing the 47-kb deleted region and flanking sequences from the wild-type mouse genome, we found evidence for only one gene sequence in the deleted region. We determined the full-length 5.5-kb cDNA sequence and identified 16 exons, of which exons 3-11 were eliminated by the deletion, causing a frameshift. The novel gene specifies a 1062-aa product with tandem ankyrin-like repeat sequences. Characterization of complementing and non-complementing YAC transgenic families revealed that correction of the inv mutant phenotype was concordant with integration and intact expression of this novel gene, which we have named inversin (Invs).

Amino Acid Sequence↗