Biomedical subjects

L Sun

Publications and source records attributed to L Sun.

At least 217 records · Page 12Linked to original sources

PubMed1999

Apoptosis of T cells mediated by Ca2+-induced release of the transcription factor MEF2.

T cell receptor (TCR)-induced apoptosis of thymocytes is mediated by calcium-dependent expression of the steroid receptors Nur77 and Nor1. Nur77 expression is controlled by the transcription factor myocyte enhancer factor 2 (MEF2), but how MEF2 is activated by calcium signaling is still obscure. Cabin1, a calcineurin inhibitor, was found to regulate MEF2. MEF2 was normally sequestered by Cabin1 in a transcriptionally inactive state. TCR engagement led to an increase in intracellular calcium concentration and the dissociation of MEF2 from Cabin1, as a result of competitive binding of activated calmodulin to Cabin1. The interplay between Cabin1, MEF2, and calmodulin defines a distinct signaling pathway from the TCR to the Nur77 promoter during T cell apoptosis.

Adaptor Proteins, Signal Transducing↗

PubMed1999

A soluble transforming growth factor beta type III receptor suppresses tumorigenicity and metastasis of human breast cancer MDA-MB-231 cells.

Transforming growth factor beta (TGF-beta) can promote late stage tumor progression in a number of model systems. In the present study, we have examined whether expression of a truncated soluble extracellular domain of TGF-beta type III receptor (sRIII) in human breast cancer MDA-MB-231 cells can antagonize the tumor-promoting activity of TGF-beta by sequestering active TGF-beta isoforms that are produced by the cancer cells. The secretion of sRIII reduced the amount of active TGF-beta1 and TGF-beta2 in the conditioned medium. This led to a significant reduction of the growth-inhibitory activity of the medium conditioned by sRIII-expressing cells on the growth of mink lung epithelial CCL64 cells in comparison with the medium conditioned by the control cells. The tumor incidence and growth rate of all of the three sRIII-expressing clones studied were significantly lower than those of the control cells in athymic nude mice. Four of five control cell-inoculated mice showed spontaneous metastasis in the lung, whereas none of the sRIII-expressing cell-inoculated mice had any lung metastasis. Thus, our results suggest that the sRIII may be used to antagonize the tumor-promoting activity of TGF-beta.

Animals↗

PubMed1999

A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase.

The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 A and 1.6 A resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when L-leucine p-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces k(cat)/K(m) about 10,000-fold.

Aminopeptidases↗

PubMed1999

Pharmacokinetic determinants of cocaine's differential effects on locomotor and operant behavior.

Dose-response, effect-time and concentration-effect relations of intravenous cocaine (1-4 mg/kg) were investigated on contingency-controlled [fixed-ratio (FR) 70 performance] and unconditioned (locomotor activity) behaviors. Cocaine dose-response curves exhibited decreasing rates of response under the FR 70 schedule but increasing locomotor activity in a dose-related fashion. Effect-time profiles confirmed that these changes were time-dependent and provided additional clarity by mirroring the biexponential decay of cocaine concentrations with time. The duration of action of cocaine was comparatively shorter on locomotor activity than on FR performance. We integrated effect-time profiles of the two behaviors with concentration-time profiles simulated from our previously published pharmacokinetic parameters to derive cocaine's pharmacodynamic parameters. Classical inhibitory Emax and sigmoidal Emax models were used to describe cocaine's effects on FR performance and locomotor activity, respectively. Simultaneous pharmacokinetic-pharmacodynamic modeling reveals evidence of acute tolerance to cocaine in locomotor activity, as indicated by decreasing potency with dose, but not in contingency-controlled behavior.

Animals↗

PubMed1999

CD38/ADP-ribosyl cyclase: A new role in the regulation of osteoclastic bone resorption.

The multifunctional ADP-ribosyl cyclase, CD38, catalyzes the cyclization of NAD(+) to cyclic ADP-ribose (cADPr). The latter gates Ca(2+) release through microsomal membrane-resident ryanodine receptors (RyRs). We first cloned and sequenced full-length CD38 cDNA from a rabbit osteoclast cDNA library. The predicted amino acid sequence displayed 59, 59, and 50% similarity, respectively, to the mouse, rat, and human CD38. In situ RT-PCR revealed intense cytoplasmic staining of osteoclasts, confirming CD38 mRNA expression. Both confocal microscopy and Western blotting confirmed the plasma membrane localization of the CD38 protein. The ADP-ribosyl cyclase activity of osteoclastic CD38 was next demonstrated by its ability to cyclize the NAD(+) surrogate, NGD(+), to its fluorescent derivative cGDP-ribose. We then examined the effects of CD38 on osteoclast function. CD38 activation by an agonist antibody (A10) in the presence of substrate (NAD(+)) triggered a cytosolic Ca(2+) signal. Both ryanodine receptor modulators, ryanodine, and caffeine, markedly attenuated this cytosolic Ca(2+) change. Furthermore, the anti-CD38 agonist antibody expectedly inhibited bone resorption in the pit assay and elevated interleukin-6 (IL-6) secretion. IL-6, in turn, enhanced CD38 mRNA expression. Taken together, the results provide compelling evidence for a new role for CD38/ADP-ribosyl cyclase in the control of bone resorption, most likely exerted via cADPr.

ADP-ribosyl Cyclase↗

PubMed1999

Identification of a cytosolic NADP+-dependent isocitrate dehydrogenase that is preferentially expressed in bovine corneal epithelium. A corneal epithelial crystallin.

Recently, metabolic enzymes have been observed in both the lens and corneal epithelium at levels greatly exceeding what is necessary for normal metabolic functions. These proteins have been termed taxon-specific crystallins and are thought to play a role in maintaining tissue transparency. We report here that cytosolic NADP+-dependent isocitrate dehydrogenase (ICDH) represents a new corneal crystallin. Using suppression subtractive hybridization, we identified a gene (with a deduced amino acid sequence that showed 94% identity to rat cytosolic NADP+-dependent ICDH) that is preferentially expressed in bovine corneal epithelium. Northern blots established that its mRNA level in the corneal epithelium was 31-, 39-, 133-, 230-, and 929-fold more than in the liver, bladder epithelium, stomach epithelium, brain, and heart, respectively. This mRNA was detected primarily in corneal epithelial basal cells by in situ hybridization. SDS-polyacrylamide gel electrophoresis, two-dimensional gel analysis, and Western blotting showed that this protein was overexpressed in the corneal epithelium, constituting approximately 13% of the total soluble bovine corneal epithelial proteins. Enzyme assays showed a corresponding overabundance of this protein in bovine corneal epithelium. Taken together, these data indicate that bovine cytosolic ICDH fulfills the criteria for a corneal epithelial crystallin and may be involved in maintaining corneal epithelial transparency.

Amino Acid Sequence↗

PubMed1999

Control of type II transforming growth factor-beta receptor expression by integrin ligation.

Ectopic expression of the alpha5 integrin subunit in cancer cells with little or no endogenous expression of this integrin often results in reduced proliferation as well as reduced malignancy. We now show that inhibition resulting from ectopic expression of alpha5 integrin is due to induction of autocrine negative transforming growth factor-beta (TGF-beta) activity. MCF-7 breast cancer cells do not express either alpha5 integrin or type II TGF-beta receptor and hence are unable to generate TGF-beta signal transduction. Ectopic expression of alpha5integrin expression enhanced cell adhesion to fibronectin, reduced proliferation, and increased the expression of type II TGF-beta receptor mRNA and cell surface protein. Receptor expression was increased to a higher level in alpha5 transfectants by growth on fibronectin-coated plates. Induction of type II TGF-beta receptor expression also resulted in the generation of autocrine negative TGF-beta activity because colony formation was increased after TGF-beta neutralizing antibody treatment. Transient transfection with a TGF-beta promoter response element in tandem with a luciferase cDNA into cells stably transfected with alpha5 integrin resulted in basal promoter activities 5-10-fold higher than those of control cells. Moreover, when alpha5 transfectants were treated with a neutralizing antibody to either TGF-beta or integrin alpha5, this increased basal promoter activity was blocked. Autocrine TGF-beta activity also induced 3-fold higher endogenous fibronectin expression in alpha5 transfectants relative to that of control cells. Re-expression of type II receptor by alpha5 transfection also restored the ability of the cells to respond to exogenous TGF-beta and led to reduced tumor growth in athymic nude mice. Taken together, these results show for the first time that TGF-beta type II receptor expression can be controlled by alpha5beta1 ligation and integrin signal transduction. Moreover, TGF-beta and integrin signal transduction appear to cooperate in their tumor-suppressive functions.

Animals↗

PubMed1999

The Pex16p homolog SSE1 and storage organelle formation in Arabidopsis seeds.

Mature Arabidopsis seeds are enriched in storage proteins and lipids, but lack starch. In the shrunken seed 1 (sse1) mutant, however, starch is favored over proteins and lipids as the major storage compound. SSE1 has 26 percent identity with Pex16p in Yarrowia lipolytica and complements pex16 mutants defective in the formation of peroxisomes and the transportation of plasma membrane- and cell wall-associated proteins. In Arabidopsis maturing seeds, SSE1 is required for protein and oil body biogenesis, both of which are endoplasmic reticulum-dependent. Starch accumulation in sse1 suggests that starch formation is a default storage deposition pathway.

Amino Acid Sequence↗

PubMed1999

Change in lactate production in Myc-transformed cells precedes apoptosis and can be inhibited by Bcl-2 overexpression.

As a result of Myc-dependent transcription of the LDH-A gene, Myc-transformed cells (Rat1-Myc) exhibit increased lactate production rates (LPR) even under aerobic conditions (the Warburg effect). Recently, the increased susceptibility to stress-induced apoptosis associated with Myc transfection has been linked to the overexpression of the LDH-A gene. In this report we demonstrate that the overexpression of the anti-apoptotic protein Bcl-2 in Rat1-Myc cells (Rat1-Myc-Bcl-2) reduces the molar ratio of lactate production to glucose consumption (Y(L/G)). The Bcl-2 induced reduction in Y(L/G) may be associated with reduced expression of the LDH-A gene, or a decrease in LDH-A activity. Stimulation of apoptosis by staurosporine, a protein kinase C inhibitor, reduces the LPR in Rat1-Myc cells in a dose-dependent manner. The staurosporine effect on the LPR is rapid and precedes the execution phase of apoptosis as defined by caspase activation and PARP cleavage. This effect on LPR is completely blocked by Bcl-2 overexpression. Serum starvation alone does not affect the LPR of Rat1-Myc or Rat1-Myc-Bcl-2 cells; however, the effect of staurosporine on the LPR of Rat1-Myc cells is potentiated by serum starvation. These data demonstrate that Bcl-2 overexpression reduces the Y(L/G) in Rat1-Myc cells, perhaps via a reduction in the activity or expression of the LDH-A gene, and this reduction may desensitize cells to some pro-apoptotic stimuli. The reduction in LPR in response to staurosporine may be an early step in the induction of apoptosis in Rat1-Myc cells. By abolishing the reduction in LPR, Bcl-2 may protect Rat1-Myc cells from staurosporine-induced apoptosis. Moreover, the lack of effect by serum starvation on the LPR supports a model in which serum starvation induces apoptosis through a pathway distinct from that of the staurosporine and glucose-dependent apoptotic pathway(s) in Myc-transformed cells.

Animals↗

PubMed1999

Rate-determining step of Escherichia coli alkaline phosphatase altered by the removal of a positive charge at the active center.

Escherichia coli alkaline phosphatase catalyzes both the nonspecific hydrolysis of phosphomonoesters and a transphosphorylation reaction in which phosphate is transferred to an alcohol via a phosphoseryl intermediate. The rate-determining step for the wild-type enzyme is pH dependent. At alkaline pH, release of the product phosphate from the noncovalent enzyme-phosphate complex determines the reaction rate, whereas at acidic pH hydrolysis of the covalent enzyme-phosphate complex controls the reaction rate. When the lysine at position 328 was substituted with a cysteine (K328C), the rate-determining step at pH 8.0 of the mutant enzyme was altered so that hydrolysis of the covalent intermediate became limiting rather than phosphate release. The transphosphorylation activity of the K328C enzyme was selectively enhanced, while the hydrolysis activity was reduced compared to that of the wild-type enzyme. The ratio of the transphosphorylation to the hydrolysis activities increased 28-fold for the K328C enzyme in comparison with the wild-type enzyme. Several other mutant enzymes for which a positive charge at the active center is removed by site-specific mutagenesis share this characteristic of the K328C enzyme. These results suggest that the positive charge at position 328 is at least partially responsible for maintaining the balance between the hydrolysis and transphosphorylation activities and plays an important role in determining the rate-limiting step of E. coli alkaline phosphatase.

Alanine↗

PubMed1999

Interaction of novel thiocolchicine analogs with the tubulin isoforms from bovine brain.

The antimitotic alkaloid colchicine binds to tubulin and inhibits microtubule assembly. Recently a new series of colchicine derivatives has been synthesized in which the seven-membered B-ring was shortened to a six-membered ring. In an effort to study the role of the B-ring substituents in this new series, we have studied the interaction of two compounds of this series, THC 5 and THC 18, with tubulin isoforms from bovine brain. We find that THC 18, which has a side chain with a pi-bonded SP2 conformation, binds differently to the tubulin isoforms, while THC 5 with a slightly different side chain does not. The results indicate that the conformation of the B-ring domain plays a major role in the differential interaction of a colchicine derivative with different tubulin isoforms. The results will be very important in designing potent antitumor derivatives of colchicine.

Animals↗

PubMed1999

Expression of dominant-negative and mutant isoforms of the antileukemic transcription factor Ikaros in infant acute lymphoblastic leukemia.

Ikaros, a zinc finger-containing DNA-binding protein, is required for normal lymphocyte development, and germline mutant mice that express only non-DNA binding dominant-negative "leukemogenic" Ikaros isoforms lacking critical N-terminal zinc fingers develop an aggressive form of lymphoblastic leukemia 3-6 months after birth. Therefore, we sought to determine whether molecular abnormalities involving the Ikaros gene could contribute to the development of acute lymphoblastic leukemia (ALL) in infants. Primary leukemic cells were freshly obtained from 12 infants (<1 year of age) with newly diagnosed ALL. In leukemic cells from each of the 12 infants with ALL, we found high level expression of dominant-negative isoforms of Ikaros with abnormal subcellular compartmentalization patterns. PCR cloning and nucleotide sequencing were used to identify the specific Ikaros isoforms and detect Ikaros gene mutations in these cells. Leukemic cells from seven of seven infants with ALL, including five of five MLL-AF4(+) infants, expressed dominant-negative Ikaros isoforms Ik-4, Ik-7, and Ik-8 that lack critical N-terminal zinc fingers. In six of seven patients, we detected a specific mutation leading to an in-frame deletion of 10 amino acids (Delta KSSMPQKFLG) upstream of the transcription activation domain adjacent to the C-terminal zinc fingers of Ik-2, Ik-4, Ik-7, and Ik-8. In contrast, only wild-type Ik-1 and Ik-2 isoforms with normal nuclear localization were found in normal infant bone marrow cells and infant thymocytes. These results implicate the expression of dominant-negative Ikaros isoforms and the disruption of normal Ikaros function in the leukemogenesis of ALL in infants.

Amino Acid Sequence↗

PubMed1999

SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types.

SU5416, a novel synthetic compound, is a potent and selective inhibitor of the Flk-1/KDR receptor tyrosine kinase that is presently under evaluation in Phase I clinical studies for the treatment of human cancers. SU5416 was shown to inhibit vascular endothelial growth factor-dependent mitogenesis of human endothelial cells without inhibiting the growth of a variety of tumor cells in vitro. In contrast, systemic administration of SU5416 at nontoxic doses in mice resulted in inhibition of subcutaneous tumor growth of cells derived from various tissue origins. The antitumor effect of SU5416 was accompanied by the appearance of pale white tumors that were resected from drug-treated animals, supporting the antiangiogenic property of this agent. These findings support that pharmacological inhibition of the enzymatic activity of the vascular endothelial growth factor receptor represents a novel strategy for limiting the growth of a wide variety of tumor types.

3T3 Cells↗

PubMed1999

Determination of doping methyltestosterone by capillary electrophoresis immunological analysis with thermally reversible hydrogel and laser-induced fluorescence.

In this paper thermally reversible hydrogel used as a replaceable packed material for capillary electrophoresis was examined. A simple and rapid method of detecting doping methyltestosterone (MTS) was developed, which demonstrated the potential of strong affinity antibodies (k(d) = 10(9)) as a selector for immunologically based separations in serum, not in urine, by capillary electrophoresis. Polyclonal antibodies (Ab) were polymerized on the hydrogel and applied in the separation of fluorescein isothiocyanate (FITC)-labeled antigen and free antigen with laser-induced fluorescence detector (LIF). The results indicated that N-isopropylacrylamide (PNIPA) hydrogel is a steady, replaceable gel. The specific determination of MTS did not require reaching equilibrium of immunological reaction and the PNIPA hydrogel including antibodies can be stockpiled at 4 degrees C, in preparation for determining MTS at any time. It can be used to determine MTS with good precision with a sensitivity lower than 50 ng/mL. The theoretical plate numbers obtained could reach 168,449 for free-antigen. Details of the preparation of hydrogel cross-linked polyclonal antibody and of typical separations of bound and free antigen are presented.

Acrylamides↗

PubMed1999

Clinical implications of the correlation between coenzyme Q10 and vitamin B6 status.

The endogenous biosynthesis of the quinone nucleus of coenzyme Q10 (CoQ10) from tyrosine is dependent on adequate vitamin B6 nutriture. Lowered blood and tissue levels of CoQ10 have been observed in a number of clinical conditions. Many of these clinical conditions are most prevalent among the elderly. Kalen et al. have shown that blood levels of CoQ10 decline with age. Similarly, Kant et al. have shown that indicators of vitamin B6 status also decline with age. Blood samples were collected from 29 patients who were not currently being supplemented with either CoQ10 or vitamin B6. Mean CoQ10 concentrations was 1.1 +/- 0.3 micrograms/ml of blood. Mean specific activities of EGOT was 0.30 +/- 0.13 mumol pyruvate/hr/10(8) erythrocytes and the mean percent saturation of EGOT with PLP was 78.2 +/- 13.9%. Means for all parameters were within normal ranges. Strong positive correlation was found between CoQ10 and the specific activity of EGOT (r = 0.5787, p < 0.001) and between CoQ10 and the percent saturation of EGOT with PLP (r = 0.4174, p < 0.024). Studies are currently in progress to determine the effect of supplementation with vitamin B6 of blood CoQ10 levels. It appears prudent to recommend that patients receiving supplemental CoQ10 be concurrently supplemented with vitamin B6 to provide for better endogenous synthesis of CoQ10 along with the exogenous CoQ10.

Aging↗

PubMed1999

DNA hypermethylation at the D17S5 locus and reduced HIC-1 mRNA expression are associated with hepatocarcinogenesis.

To examine the significance of aberrant DNA methylation in hepatocarcinogenesis, the DNA methylation status at the D17S5 locus and mRNA expression of a candidate tumor suppressor gene, HIC-1 (hypermethylated-in-cancer), which was identified at the D17S5 locus, in primary hepatocellular carcinomas (HCCs) and their corresponding noncancerous liver tissues were assessed. DNA hypermethylation at the D17S5 locus was detected in 44% of the noncancerous liver tissues showing chronic hepatitis or cirrhosis, which are widely considered to be precancerous conditions, but was not observed in noncancerous liver tissues showing no remarkable histological findings. The incidence of DNA hypermethylation at this locus was significantly higher in HCCs (90%) than noncancerous liver tissues (P <.001). Loss of heterozygosity at the D17S5 locus, which was preceded by DNA hypermethylation at the same locus, was detected in 54% of HCCs. The HIC-1 mRNA expression level of noncancerous liver tissues showing chronic hepatitis or cirrhosis was significantly lower than that of noncancerous liver tissues showing no remarkable histological findings (P <.01), and that of HCCs was even lower than that of noncancerous liver tissues (P <.05). Poorly differentiated HCCs showed lower expression levels than well- to moderately differentiated HCCs. Mutation of the p53 gene may be involved in HIC-1 inactivation. Moreover, wild-type p53 did not overcome DNA hypermethylation at the D17S5 locus to activate HIC-1 in HCCs. These data suggest that aberrant DNA methylation at this locus and reduced HIC-1 mRNA expression participate in hepatocarcinogenesis during both early developmental stages and malignant progression of HCCs.

Adult↗

PubMed1999

Liver regeneration after partial hepatectomy is non-uniform: flow cytometric bromodeoxyuridine incorporation and cell cycle studies in a porcine model.

The rate of hepatocyte regeneration at different anatomical locations of the remnant liver after partial hepatectomy was assessed in porcine hepatocytes by bromodeoxyuridine (BrdUr) incorporation and cell cycle kinetics using flow cytometric analysis. Partial hepatectomy was performed in five Yorkshire pigs. A single intravenous injection of BrdUr at 50 mg/kg was administered on the 2nd post-operative day and the animals were sacrificed 1 h later. The remnant liver tissue was harvested and divided into four equal zones, from the liver periphery towards the surgical cut-edge. Biopsy samples were obtained from the centre of each of these zones and similarly from identical anatomical locations in two control pigs that had undergone sham surgery. Hepatocyte nucleus suspension was prepared, double labelled with anti-BrdUr and propidium iodide and analysed by a flow cytometer. The cells in S-phase was used as the parameter to measure the regeneration status. A gradient increase in S-phase from the periphery to the cut edge was observed in all five pigs that had undergone partial hepatectomy. The percentage of S-phase cells in all four zones from the hepatectomy group was significantly higher when compared with that in the controls. Liver regeneration after partial hepatectomy was not uniform but was greatest adjacent to the surgical cut edge and decreased towards the periphery of the liver.

Animals↗

PubMed1999

Platelet anesthesia with nitric oxide with or without eptifibatide during cardiopulmonary bypass in baboons.

OBJECTIVE: This study tested the hypothesis that nitric oxide or nitric oxide and eptifibatide (Integrilin) reversibly inhibit platelet activation and consumption during cardiopulmonary bypass and rapidly restore platelet numbers and function after bypass. METHODS: Nitric oxide, a short-acting, reversible platelet inhibitor, was studied with and without eptifibatide, a short-acting, reversible glycoprotein IIb/IIIa inhibitor, in 21 baboons that underwent 60 minutes of normothermic cardiopulmonary bypass with peripheral cannulas. A control group, a group that received 80 ppm nitric oxide, and a group that received both nitric oxide and eptifibatide were studied. Blood samples were obtained at several time points to determine platelet count, aggregation in response to adenosine diphosphate, and levels of beta-thromboglobulin, prothrombin fragment 1.2, and thrombin-antithrombin complex. Template bleeding times were measured before and at 4 intervals after cardiopulmonary bypass. RESULTS: Both nitric oxide and the combination of the 2 drugs significantly attenuated platelet consumption, improved postbypass function, and reduced plasma beta-thromboglobulin release with respect to values in control animals. Both nitric oxide and the combination restored baseline bleeding times 55 minutes after cardiopulmonary bypass ended. No significant differences between nitric oxide and the combination were found for any measurement. CONCLUSION: Nitric oxide with or without eptifibatide protects platelets during cardiopulmonary bypass and accelerates restoration of normal bleeding times after operation in a baboon model. Although nitric oxide and eptifibatide reversibly inhibit platelets by different mechanisms, in the absence of a wound no synergistic effect was demonstrated.

Animals↗