Search PubMed⌕ Search

Biomedical subjects

J M Li

Publications and source records attributed to J M Li.

At least 55 records · Page 3Linked to original sources
PubMed1998

Transforming growth factor beta stimulates the human immunodeficiency virus 1 enhancer and requires NF-kappaB activity.

Transforming growth factor beta (TGF-beta) is the prototype of a large superfamily of signaling molecules involved in the regulation of cell growth and differentiation. In certain patients infected with human immunodeficiency virus type 1 (HIV-1), increased levels of TGF-beta promoted the production of virus and also impaired the host immune system. In an effort to understand the signaling events linking TGF-beta action and HIV production, we show here that TGF-beta can stimulate transcription from the HIV-1 long terminal repeat (LTR) promoter through NF-kappaB binding sites in both HaCaT and 300.19 pre-B cells. When introduced into a minimal promoter, NF-kappaB binding sites supported nearly 30-fold activation from the luciferase reporter upon TGF-beta treatment. Electrophoretic mobility shift assay indicated that a major factor binding to the NF-kappaB site is the p50-p65 heterodimeric NF-kappaB in HaCaT cells. Coexpression of Gal4-p65 chimeric proteins supported TGF-beta ligand-dependent gene expression from a luciferase reporter gene driven by Gal4 DNA binding sites. NF-kappaB activity present in HaCaT cells was not affected by TGF-beta treatment as judged by the unchanged DNA binding activity and concentrations of p50 and p65 proteins. Consistently, steady-state levels of IkappaB alpha and IkappaB beta proteins were not changed by TGF-beta treatment. Our results demonstrate that TGF-beta is able to stimulate transcription from the HIV-1 LTR promoter by activating NF-kappaB through a mechanism distinct from the classic NF-kappaB activation mechanism involving the degradation of IkappaB proteins.

B-Lymphocytes↗
PubMed1998

Role of G1 phase cyclins and cyclin-dependent kinases during cardiomyocyte hypertrophic growth in rats.

Cell cycle regulatory molecules are implicated in cardiomyocyte hypertrophy. We have investigated protein expression of cyclins A, D1-3, and E and cyclin-dependent kinases (CDKs) 2, 4, 5, and 6 in left ventricular (LV) tissues during the development of LV hypertrophy in rats following aortic constriction (AC). Compared with their expression in sham-operated controls (SH), expression of cyclins D2 and D3 and of CDK4 and CDK6 increased significantly from day 3 to day 21 after AC concomitant with increased LV mass. However, no significant difference was observed for CDK2 or CDK5. Cyclins A, D1, and E were undetectable. In vitro kinase activities of CDK4 and CDK6 increased approximately 70% from day 7 to day 14 in AC myocytes compared with SH myocytes (P < 0.03). Fluorescence-activated cell sorter analysis revealed a G0/G1 to G2/M phase progression in AC myocyte nuclei (22.0 +/- 1.1% in G2/M) by day 7 postoperation compared with progression in SH myocyte nuclei (14.0 +/- 0.8% in G2/M; P < 0.01). Thus an upregulation of certain cell cycle regulators is associated with cardiomyocyte hypertrophy.

3T3 Cells↗
PubMed1998

Effects of N-methyl berbamine on delayed outward potassium current in isolated rat hepatocytes.

AIM: To study the effects of N-methyl berbamine (NMB) on the delayed outward potassium currents (Ik) in isolated rat hepatocytes. METHODS: With patch-clamp techniques and whole-cell recording method, holding potential -50 mV, command potential +30 to +140 mV, duration 900 ms. RESULTS: NMB reduced Ik in a concentration-dependent manner. When the concentrations of NMB were 20, 50, 400 nmol.L-1 and 50 mumol.L-1, the amplitude values of Ik were decreased to 3.6 +/- 0.4 (P > 0.05), 2.1 +/- 1.6 (P > 0.05), 3.7 +/- 1.6 (P < 0.05), 2.3 +/- 1.3 nA (P < 0.01) from 4.4 +/- 1.0 (n = 4), 2.5 +/- 1.8 (n = 4), 5.8 +/- 2.1 (n = 5), 4.6 +/- 1.3 (n = 6) nA of control, respectively. The inhibitory rates were 10%, 15%, 37%, and 51%, respectively. CONCLUSION: NMB was a K+ channel inhibitor.

Alkaloids↗
PubMed1998

[Effects of noradrenaline on delayed outward potassium current in isolated rat hepatocytes].

Heretofore no reports are available on the outward potassium currents in rat hepatocytes. Using whole-cell patch clamp technique, the effects of noradrenaline and other reagents on the delayed outward potassium current (IK) in isolated rat hepatocytes were investigated. The experimental results showed that the magnitude of IK was 2.85 +/- 1.21 nA at a command potential of +140 mV from a holding potential of -50 mV. Noradrenaline decreased IK distinctly. Isoprenaline and acetylcholine showed no effect on IK in isolated rat hepatocytes.

Adrenergic alpha-Agonists↗
PubMed1997

Expression of HGF and cMet in the developing and adult brain.

Hepatocyte growth factor (HGF) was recently recognized as a potential neurotrophic factor in the developing brain. We studied expression of HGF and its receptor using Northern blot analysis and in situ hybridization for mRNA and double immunofluorescent laser confocal microscopy. HGF and cMet messages were abundant in the hippocampus of both human and rat brains. In this region, both messages were localized in the neuronal layer. Segregation of HGF predominantly in the hippocampal CA3-4 and cMet in CA1 supports the hypothesis that HGF may mediate important neurotrophic functions in both developing and adult brains.

Animals↗
PubMed1997

E2F4-RB and E2F4-p107 complexes suppress gene expression by transforming growth factor beta through E2F binding sites.

Transforming growth factor beta (TGF-beta) causes growth arrest in most cell types. TGF-beta induces hypophosphorylation of retinoblastoma susceptibility gene 1 product (RB), which sequesters E2F factors needed for progression into S phase of the cell cycle, thereby leading to cell cycle arrest at G1. It is possible, however, that the E2F-RB complex induced by TGF-beta may bind to E2F sites and suppress expression of specific genes whose promoters contain E2F binding sites. We show here that TGF-beta treatment of HaCaT cells induced the formation of E2F4-RB and E2F4-p107 complexes, which are capable of binding to E2F sites. Disruption of their binding to DNA with mutation in the E2F sites did not change the expression from promoters of E2F1, B-myb, or HsORC1 genes in cycling HaCaT cells. However, the same mutation stimulated 5- to 6-fold higher expression from all three promoters in cells treated with TGF-beta. These results suggest that E2F binding sites play an essential role in the transcription repression of these genes under TGF-beta treatment. Consistent with their repression of TGF-beta-induced gene expression, introduction of E2F sites into the promoter of cyclin-dependent kinase inhibitor p15(INK4B) gene effectively inhibited its induction by TGF-beta. Experiments utilizing Gal4-RB and Gal4-p107 chimeric constructs demonstrated that either RB or p107 could directly repress TGF-beta induction of p15(INK4B) gene when tethered to p15(INK4B) promoter through Gal4 DNA binding sites. Therefore, E2F functions to bring RB and p107 to E2F sites and represses gene expression by TGF-beta. These results define a specific function for E2F4-RB and E2F4-p107 complexes in gene repression under TGF-beta treatment, which may constitute an integral part of the TGF-beta-induced growth arrest program.

Base Sequence↗
PubMed1997

Differential protein expression and subcellular distribution of TGFbeta1, beta2 and beta3 in cardiomyocytes during pressure overload-induced hypertrophy.

The transforming growth factor beta (TGFbeta) superfamily plays an important role in the myocardial response to hypertrophy. We have investigated the protein expression of TGFbeta1, beta2 and beta3 in left ventricular tissue, and determined their subcellular distribution in myocytes by immunoblotting and immunocytochemistry during the development of left ventricular hypertrophy (LVH), using isoform specific antibodies to TGFbeta1, beta2 and beta3. LVH was produced in rats by aortic constriction (AC) and LV tissue was obtained at days (d)0, 1, 3, 7, 14, 21 and 42 following operation. Compared with age matched sham-operated controls (SH), TGFbeta1 levels in LV tissue of AC rats increased significantly from d1-d14 (P<0.03) concomitant with the adaptive growth of LV tissue. In contrast, TGFbeta3 levels decreased in LV tissue of AC rats from d3 post-operation (significant from d14-d42, P<0.03). No significant difference in TGFbeta2 levels were observed from SH and AC rats after operation. Antibodies to TGFbeta1 stained intercalated disks, sarcolemmal membranes and cytoplasm, but not nuclei, of cardiomyocytes on LV sections from untreated and SH rats. However, a trans-localisation of TGFbeta1 to the nuclei of cardiomyocytes was observed in AC hearts. Antibodies to TGFbeta3 stained T tubules, cytoplasm and the nuclei of cardiomyocytes from untreated and SH rats. However, by d7 post-AC operation, TGFbeta3 expression was lost rapidly from nuclei of cardiomyocytes followed by a reduction in total TGFbeta3 immunofluorescence in myocytes. Antibodies to TGFbeta2 stained sarcolemmal membranes of cardiomyocytes from both SH and AC rats without significant difference between groups. Thus, the differential pattern of protein expression and subcellular distribution of TGFbeta1, beta2 and beta3 in myocytes during the development of LVH suggests that these molecules play different roles in the response of cardiomyocytes to LVH.

Animals↗
PubMed1997

Expression and activities of cyclins and cyclin-dependent kinases in developing rat ventricular myocytes.

The molecular mechanisms responsible for the alterations in proliferative capacity of cardiac myocytes during development remain unknown; however, cell cycle dependent molecules may be involved. We have determined the expression of cyclins A, D1-3 and E, and cyclin-dependent kinases (CDKs) 2, 4, 5 and 6 and cdc2 in freshly isolated rat cardiac myocytes from fetal (18 days gestation), neonatal (2 days post-natal) and adult animals by immunoblotting. Our results show a dramatic decrease in expression of these proteins during normal cardiac development, such that levels are highest in fetal myocytes but are significantly down-regulated in adult cells (P<0.05, in each case). We also have determined the in vitro kinase activities of cdc2, CDK2, CDK4, CDK5 and CDK6 immunocomplexes in fetal, neonatal and adult myocytes. There was a consistent and significant loss of cdc2, CDK2, CDK4 and CDK6 kinase activities in adult cardiac cell lysates (5.3-, 10.6-, 1.5- and 1.9-fold decreases, respectively) when compared to neonatal samples (P<0.05); CDK5 activity showed a similar trend but failed to reach significance. In conclusion, our results show that the expression and activities of various positive regulators of the cell cycle are down-regulated significantly during development of the cardiac myocyte, concomitant with the loss of proliferative capacity in adult myocytes. Down-regulation of these proteins may be pivotal in the withdrawal of the cardiac myocyte from the cell cycle.

3T3 Cells↗
PubMed1997

ATP regulates cardiac Ca2+ channel activity via a mechanism independent of protein phosphorylation.

The role of adenosine triphosphate (ATP) in the regulation of L-type Ca2+ channel activity was investigated in inside-out patches from guinea-pig ventricular cells, in which the Ca2+ channel activity had been reprimed by application of cytoplasm from bovine heart. Passing the cytoplasm through a diethylaminoethyl (DEAE)-sepharose column or heating at 60 degrees C for 20 min attenuated the induction Ca2+ channel activity to 6-13% of that in the preceding cell-attached patch. Addition of 10 mM MgATP to the cytoplasm greatly improved the potency of cytoplasm in restoring Ca2+ channel activity (to 83 +/- 22%, mean +/- SE). This effect of MgATP was also produced, although with lower potency, by K2ATP (61 +/- 20%) or 5'-adenylylimidodiphosphate (AMP-PNP, 39 +/- 7%), a non-hydrolyzable ATP analogue, suggesting that hydrolysis of ATP is not required for the stimulatory effect on channel activity. A non-specific protein kinase inhibitor H8 (50-100 microM) did not inhibit the effect of cytoplasm + MgATP on channel activity, suggesting the involvement of a pathway independent of phosphorylation. We conclude that ATP regulates Ca2+ channel activity in dual pathways: one with, and the other without, protein phosphorylation.

Adenosine Triphosphate↗
PubMed1997

Downregulation of cyclin-dependent kinase inhibitors p21 and p27 in pressure-overload hypertrophy.

We postulated that the cyclin-dependent kinase inhibitors p21 and p27 could regulate the alterations in growth potential of cardiomyocytes during left ventricular hypertrophy (LVH). LVH was induced in adult rat hearts by aortic constriction (AC) and was monitored at days 0, 1, 3, 7, 14, 21, and 42 postoperation. Relative to sham-operated controls (SH), left ventricle (LV) weight-to-body weight ratio in AC increased progressively with time without significant differences in body weight or right ventricle weight-to-body weight ratio. Atrial natriuretic factor mRNA increased significantly in AC to 287% at day 42 compared with SH (P < 0.05), whereas p21 and p27 mRNA expression in AC rats decreased significantly by 58% (P < 0.03) and 40% (P < 0.05) at day 7, respectively. p21 and p27 protein expression decreased significantly from days 3 to 21 in AC versus SH, concomitant with LV adaptive growth. Immunocytochemistry showed p21 and p27 expression in cardiomyocyte nuclei. Thus downregulation of p21 and p27 may modulate the adaptive growth of cardiomyocytes during pressure overload-induced LVH.

3T3 Cells↗
PubMed1996

Decreased trkA gene expression within basal forebrain neurons in Alzheimer's disease.

In situ hybridization for TrkA mRNA was combined with quantitative optical densitometry to evaluate whether the expression of this gene is altered within cholinergic basal forebrain neurons (CBF) in Alzheimer's disease (AD). TrkA mRNA within individual nucleus basalis neurons was significantly reduced (66%) in AD cases relative to aged controls. Reverse transcription polymerase chain reaction quantitative analyses confirmed that TrkA mRNA levels decreased markedly in AD. In contrast, expression of the gene coding for for the low affinity p75NTR was not significantly altered in AD relative to aged controls. These data indicate that there is a selective defect in trkA gene expression in AD, supporting the hypothesis that the degeneration of CBF neurons seen in this disease results from impaired nerve growth factor trophic support.

Aged↗
PubMed1996

Functional fetal nigral grafts in a patient with Parkinson's disease: chemoanatomic, ultrastructural, and metabolic studies.

A patient with Parkinson's disease received bilateral fetal human nigral implants from six donors aged 6.5 to 9 weeks post-conception. Eighteen months following a post-operative clinical course characterized by marked improvement in clinical function, this patient died from events unrelated to the grafting procedure. Post-mortem histological analyses revealed the presence of viable grafts in all 12 implant sites, each containing a heterogeneous population of neurons and glia. Approximately 210,146 implanted tyrosine hydroxylase-immunoreactive (TH-ir) neurons were found. A greater number of TH-ir grafted neurons were observed in the right (128,162) than the left (81,905) putamen. Grafted TH-ir neurons were organized in an organotypic fashion. These cells provided extensive TH-ir and dopamine transporter-ir innervation to the host striatum which occurred in a patch-matrix fashion. Quantitative evaluations revealed that fetal nigral grafts reinnervated 53% and 28% of the post-commissural putamen on the right and left side, respectively. Grafts on the left side innervated a lesser area of the striatum, but optical density measurements were similar on both sides. There was no evidence that the implants induced sprouting of host TH-ir systems. Electron microscopic analyses revealed axo-dendritic and occasional axo-axonic synapses between graft and host. In contrast, axo-somatic synapses were not observed. In situ hybridization for TH mRNA revealed intensely hybridized grafted neurons which far exceeded TH mRNA expression within residual host nigral cells. In addition, gamma-amino butyric acid (GABA)-ergic neurons were observed within the graft that formed a dense local neuropil which was confined to the implant site. Serotonergic neurons were not observed within the graft. Cytochrome oxidase activity was increased bilaterally within the grafted post-commissural putamen, suggesting increased metabolic activity. In this regard, a doubling of cytochrome oxidase activity was observed within the grafted post-commissural putamen bilaterally relative to the non-grafted anterior putamen. The grafts were hypovascular relative to the surrounding striatum and host substantia nigra. Blood vessels within the graft stained intensely for GLUT-1, suggesting that this marker of blood--brain barrier function is present within human nigral allografts. Taken together, these data indicate that fetal nigral neurons can survive transplantation, functionally reinnervate the host putamen, establish synaptic contacts with host neurons, and sustain many of the morphological and functional characteristics of normal nigral neurons following grafting into a patient with PD.

Carrier Proteins↗
PubMed1996

A universal tag for recombinant proteins.

Incorporation of tags into recombinant proteins can facilitate their identification and purification. In addition, these tags can also be used to monitor the trafficking or localization of the recombinant proteins inside the cells. Several such tags have been developed. However, the lengths of these tags make it cumber-some to incorporate them into the desired proteins. Typically, one must subclone the desired cDNA into a plasmid containing the tag sequence at a suitable restriction site or ligate a synthetic oligonucleotide containing the tag sequence at a suitable restriction site in the cDNA of the desired protein. These manipulations can be avoided, if one uses a short peptide tag that can be incorporated by PCR. We show here that a short peptide tag, RYIRS can be easily incorporated at the C termini of recombinant proteins by PCR. We also showed that by using a mAb specific for this peptide sequence, the tagged proteins could be easily detected in Western blot analysis, immunofluorescence staining, and immunoprecipitation. The interactions between this tag sequence and the mAb have been well characterized. One can take advantage of this information and control the reactivities between the tagged proteins and the mAb by varying the lengths of the peptide tags. Furthermore, we showed that this tag can be used to monitor whether a recombinant protein is properly translated and terminated because the interactions between this tag sequence and the mAb requires that the tag be at the C-terminus of the protein.

Amino Acid Sequence↗
PubMed1996

Placental and fetal cardiac laminin are targets for cross-reacting autoantibodies from mothers of children with congenital heart block.

The association of congenital heart block (CHB) with maternal autoantibodies to the Ro and La ribonucleoprotein antigens may be due to cross-reactions between maternal anti-La antibodies and fetal cardiac specific antigens. One of the major components of cardiac myocytes, laminin, is accessible for binding by maternal autoantibodies and we have previously reported cross-reactivity of mouse laminin with anti-La antibodies affinity purified from the sera of patients with primary Sjögren's syndrome. Affinity purified anti-La antibodies from ten women who had at some time given birth to a child with CHB were examined for cross-reactivity with human placental laminin, which shares structural similarities with cardiac laminin. All ten anti-La antibodies bound to the surface of cryosections of normal full term placental trophoblasts. Binding could be inhibited by pre-incubation of antibodies with either La or placental laminin. Eight anti-La antibodies also reacted with placental laminin by ELISA and La inhibited up to 82% of binding to laminin while laminin inhibited up to 85% of binding to La in a dose dependent manner. Eight anti-La antibodies also bound to the surface of fetal cardiac myocytes at 10.3 weeks of gestation and five showed lower levels of reactivity with the surface of fetal cardiac myocytes at 16.5 weeks of gestation. None showed any surface staining of normal adult heart. These data confirm the cross-reactivity of anti-La antibodies with laminin and may support a placental role in preventing the majority of potentially pathogenic antibodies from reaching the fetal circulation.

Autoantibodies↗
PubMed1996

Adhesion of activated platelets to venous endothelial cells is mediated via GPIIb/IIIa.

Normal circulating platelets do not adhere to intact, undisturbed endothelium. Studies have shown, however, that platelets will adhere to virally infected or thrombin-stimulated human umbilical vein endothelial cells. Using a novel platelet/endothelial cell adhesion assay we studied the interaction of thrombin-activated platelets to human saphenous vein endothelial cells (HSVEC), and its mechanism(s). Biotinylated platelets were exposed to Hepes-Tyrode buffer, 10E5 or PAC-1 [monoclonal antibodies (Mabs) blocking GPIIb-IIIa], AK4 (Mab blocking P-selectin, 6D1 (Mab blocking vWf binding to GPIb), RGDS (small peptide blocking the fibrinogen binding site), or EDTA (dissociates GPIIb-IIIa complex) and then activated with thrombin. The platelets were subsequently exposed to thrombin-stimulated monolayer HSVEC. Phycoerythrin-streptavidin was added to the wells to fluorescently label the platelets, followed by formaldehyde fixation and washing to remove nonadherent platelets. Adhesion of platelets to HSVEC was assessed using a fluorescent multiwell plate reader. Antibodies which blocked the GPIIb-IIIa receptor and agents which competitively bound the receptor all significantly inhibited activated platelet adhesion to the activated HSVEC. We have found that thrombin significantly increases platelet/HSVEC adhesion, and this event is mediated via the integrin GPIIb-IIIa (fibrinogen receptor). These GPIIb-IIIa receptor blocking Mabs and RGDS may be useful adjuncts for improving patency following angiographic intervention and/or vein grafting in patients with high risk of thrombosis. The assay we have developed is a valuable and relatively simple method for assessing platelet/endothelial cell adhesion and activation.

Amino Acid Sequence↗
PubMed1996

Reduction of bleeding after heart operations through the prophylactic use of epsilon-aminocaproic acid.

UNLABELLED: Excessive postoperative bleeding after heart operations continues to be a source of morbidity. This prospective double-blind study evaluated epsilon-aminocaproic acid as an agent to reduce postoperative bleeding and investigated its mode of action. One hundred three patients were randomly assigned to receive either 30 gm epsilon-aminocaproic acid (51 patients) or an equivalent volume of placebo (52 patients). In a subset of these patients (14 epsilon-aminocaproic acid, 12 placebo), tests of platelet function and fibrinolysis were performed. RESULTS: By multivariate analysis, three factors were associated with decreased blood loss in the first 24 hours after operation: epsilon-aminocaproic acid versus placebo (647 ml versus 839 ml, p = 0.004), surgeon 1 versus all other surgeons (582 ml versus 978 ml, p = 0.002), and no intraaortic balloon versus intraaortic balloon pump use (664 ml versus 1410 ml, p = 0.02). No significant differences in platelet function could be demonstrated between the two groups. Inhibited fibrinolysis, as reflected by less depression of the euglobulin clot lysis and no rise in D-dimer levels, was significant in the epsilon-aminocaproic acid group compared with the placebo group. CONCLUSION: The intraoperative use of epsilon-aminocaproic acid reduces postoperative cardiac surgical bleeding.

Aminocaproic Acid↗
PubMed1996

Common factors direct transcription through the proximal sequence elements (PSEs) of the embryonic sea urchin U1, U2, and U6 genes despite minimal similarity among the PSEs.

The proximal sequence element (PSE) for the sea urchin U6 small nuclear RNA gene has been defined. The most critical nucleotides for expression, located 61 to 64 nucleotides (nt) from the transcription start site, are 4 nt, AACT, at the 5' end of the PSE. Two nucleotide mutations in this region abolish transcription of the sea urchin U6 gene in vitro. The same two nucleotide mutations greatly reduce the binding of specific factors detected by an electrophoretic mobility shift assay. There is also a conserved AC dinucleotide 57 nt from the start site of the sea urchin U1 and U2 PSEs. The sea urchin U1 and U2 PSEs were substituted for the sea urchin U6 PSE, with the conserved AC sequences aligned with those of the U6 PSE. Both of these genes were expressed at levels higher than those observed with the wild-type U6 gene. Similar complexes are formed on the U1 and U2 PSEs, and formation of the complexes is inhibited efficiently by the U6 PSE. In addition, the E-box sequence present upstream of the PSE enhances U6 transcription from both the U1 and U2 PSEs. Finally, depletion of a nuclear extract with a DNA affinity column containing the U6 PSE sequence reduces expression of the U6 genes driven by the U6, U1, or U2 PSE but does not affect expression of the 5S rRNA gene. These data support the possibility that the same factor(s) interacts with the PSE sequences of the U1, U2, and U6 small nuclear RNA genes expressed in early sea urchin embryogenesis.

Animals↗