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M J Jiang

Publications and source records attributed to M J Jiang.

16 recordsLinked to original sources
PubMed1997

Studies on the function of rho A protein in cardiac myofibrillogenesis.

The aim of this study was to provide morphological evidence for the presence of rho A protein in developing cardiomyocytes and to investigate its possible role in myofibrillogenesis. Immunostaining with a monoclonal anti-rho antibody gave a diffuse pattern in the cytosol of cultured cardiomyocytes. Introduction of C3 exoenzyme into the cells by electroporation was used to inactivate rho A protein by ADP-ribosylation. An immunostaining with anti-vinculin, anti-talin, and anti-integrin antibodies showed the focal adhesions in electroporation control cardiomyocytes to be evenly distributed in the ventral sarcolemma; the costameric structure was also detected using these antibodies. In contrast, in C3 exoenzyme treated cells, focal adhesions were disassembled and costamere were absent; in addition, beta-actin-positive, non-striated fibrils were lost and assembly of M-protein, titin, and alpha-actinin into myofibrils was poor, as shown by diffuse and filamentous staining pattern. C3 exoenzyme treatment had a less marked effect on mature cardiomyocytes than on immature cells; in this case, cells became distorted and few myofibrils were seen. The intensity of anti-phosphotyrosine antibody staining of the focal adhesion was also decreased or diffuse in C3 exoenzyme-treated cardiomyocytes, suggesting dephosphorylation of focal adhesion components. We therefore conclude that small G protein rho A plays an important role in myofibril assembly in cardiomyocytes.

ADP Ribose Transferases↗
PubMed1997

Perturbation of platelet adhesion to endothelial cells by plasminogen activation in vitro.

To investigate whether the endothelium-platelet interactions may be altered by plasminogen activation, cultured human umbilical vein endothelial cells (ECs) were treated with tissue-type plasminogen activator (t-PA) in the presence of plasminogen, and platelet adhesion to ECs was subsequently measured by using a tapered flow chamber. Our results demonstrated that platelets adhered more readily to t-PA treated EC monolayer than to the control monolayer at all shear stress levels tested. This phenomenon was treatment time-dependent and dose-dependent, and it could be blocked by adding plasmin inhibitors, such as epsilon-amino caproic acid and aprotinin. Adherent platelets on t-PA treated EC monolayer underwent more severe shape change than those on the control monolayer. While the extracellular matrix directly treated with t-PA attracted less platelets than the control matrix did, platelet adhesion to the matrix that was produced by t-PA-treated ECs was unaltered. These data suggest that t-PA treatment on ECs compromised antiplatelet-adhesion capability on their apical surface without altering the reactivity of their extracellular matrix towards platelets.

Blood Platelets↗
PubMed1996

Increased ferritin gene expression in atherosclerotic lesions.

To identify genes potentially implicated in atherogenesis, a cDNA library was constructed from human atherosclerotic aorta and differentially screened with 32P-labeled-cDNAs prepared from human normal and atherosclerotic aortas. Two cDNA clones exhibiting higher hybridization to the 32P-labeled cDNAs from atherosclerotic vessels were isolated and identified to be genes encoding L-ferritin and H-ferritin, respectively. Northern blot analysis confirmed that the expression of both ferritin genes was notably higher in human and rabbit atherosclerotic aortas than in their normal counterparts. A time-course study illustrated that both L- and H-ferritin mRNAs were markedly increased in aortas of rabbits after feeding with a high cholesterol diet for 6 wk, which was also the time period after which the formation of lesions became evident. In situ hybridization revealed that both L- and H-ferritin mRNAs were induced in endothelial cells and macrophages of human early lesions. The signals were also detected in the smooth muscle cells of advanced lesions. Immunostaining further identified the presence of ferritin protein in atherosclerotic lesions. On the other hand, Prussian blue stain revealed the presence of iron deposits in advanced lesions but not in early human or rabbit lesions. Further experiments with cultured human monocytic THP-1 cells and aortic smooth muscle cells demonstrated that ferritin mRNAs were subjected to up-regulation by treatment with IL-1 or TNF, while TGF, PDGF, and oxidized LDL did not affect the expression of either ferritin gene in both cell lines. Collectively, these results clearly demonstrate that ferritin genes are susceptible to induction in the course of plaque formation.

Animals↗
PubMed1995

Protease-susceptible sites and properties of fragments of aortic smooth-muscle myosin.

We have examined the protease susceptibility of aortic myosin, the thermal unfolding profiles of myosin rod and light meromyosin (LMM) and the solubility properties of the LMM fragments. Two major protease-susceptible sites were found, located at the head-rod junction and the heavy meromyosin (HMM)-LMM junction. Both tryptic and chymotryptic digestion of aortic myosin rod produced the LMM (80-85 kDa) and short subfragment 2 (S-2) (40-45 kDa) segments, which were similar to those of gizzard myosin rod and differed from the short LMM (70 kDa) and long S-2 (58 kDa) segments produced from skeletal-muscle rod. The thermal unfolding profile of aortic myosin rods exhibited three helix-unfolding transitions, at 47.5, 51 and 54 degrees C, similar to those of gizzard rods yet different from those of skeletal-muscle rods. There was a dramatic difference in the solubility of aortic LMM fragments of various molecular mass, as for gizzard smooth-muscle LMM and rabbit skeletal-muscle LMM. LMM fragments of molecular mass 77 kDa or more were completely insoluble in low-ionic-strength buffer, whereas LMM fragments of molecular mass 73 kDa or less were completely soluble in low-ionic-strength buffer. Proteolytic digestion patterns of LMM showed two additional protease-susceptible sites located 13 and 30 kDa from the ends of the LMM molecule. This suggests the existence of flexible regions within the LMM molecule, which may be responsible for the folded form of aortic myosin.

Amino Acid Sequence↗
PubMed1995

Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis.

In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library. Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis. Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme. Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid. Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme. Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme. However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding. These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC.

Amino Acid Sequence↗
PubMed1994

Conformationally altered aortic myosin light chains.

Aorta smooth myosin contains two types of light chain, LC20 and LC17, which fold together with the N-terminal region of each heavy chain to form the globular head region of myosin. We demonstrate an altered conformation of LC20 after its separation from heavy chain by high concentrations of urea, on the basis of the following evidence: 1) A polyclonal antibody against LC20 was not able to recognize this conformationally altered form; 2) Myosin reconstituted from heavy chains and urea-dissociated light chains exhibited extremely low ATPase activity. Circular dichroism unfolding profiles showed that light chains dissociated from heavy chains by SDS appeared to be more stable than those generated by urea dissociation.

Animals↗
PubMed1994

Intracellular calcium and myosin light chain phosphorylation during U46619-activated vascular contraction.

We investigated the relationship between [Ca2+]i, myosin light chain (LC20) phosphorylation and isometric force in guinea pig aortic strips during contractions activated by a thromboxane A2 analogue, (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z, 13E-dienoic acid (U46619). Isometric force and [Ca2+]i were measured simultaneously using preloaded aequorin as the intracellular calcium indicator. LC20 phosphorylation levels were determined by two dimensional polyacrylamide gel electrophoresis in parallel preparations. Contractions induced by U46619 were accompanied by increases in [Ca2+]i and LC20 phosphorylation. The chelation of extracellular calcium with 2.5 mM EGTA significantly inhibited U46619-induced increases in [Ca2+]i, isometric force and LC20 phosphorylation. Steady-state force assumed a similar dependence on LC20 phosphorylation for contractions stimulated by potassium depolarization, alpha 1-adrenergic agonist phenylephrine and U46619 either in the presence or absence of extracellular calcium. On the contrary, the [Ca2+]i/force relation revealed that both U46619 and phenylephrine stimulated greater isometric force at lower [Ca2+]i than did KCl depolarization. The addition of a protein kinase C inhibitor, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), decreases force without significantly affecting either [Ca2+]i or LC20 phosphorylation levels. These results suggest that in guinea pig aortic smooth muscle U46619 increases the calcium sensitivity of the contractile apparatus but does not change the LC20 phosphorylation/force relation in comparison to K+ depolarization. Protein kinase C is activated during U46619-stimulated contraction and might be involved in mechanisms other than LC20 phosphorylation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Acetylpolyamines decrease blood pressure, [Ca++]i and isometric force of vascular smooth muscle.

Polyamines are polycations in cells and acetylation is the first step in their intracellular metabolism. We investigated the effects of the acetylated polyamines on arterial blood pressure and vascular reactivities in rats. Acetylspermine and acetylspermidine, administered at concentrations ranging from 12.5 to 50 mmol/kg b.wt., both induced a transient decrease in mean arterial blood pressure. The magnitudes of the hypotensive effects of these acetylpolyamines and polyamines were in the order of spermine > acetylspermine > acetylspermidine = spermidine. Pretreatment of rats with calcium diminished polyamine-induced hypotensive effects. The effects of spermine and acetylspermine on isolated vascular smooth muscle were examined in rat aortic rings and tail artery strips. Both compounds relaxed precontracted arterial preparations, and this relaxation could be counteracted by increasing extracellular calcium concentration. Tail artery strips were more sensitive to acetylspermine when compared to aortic rings. In tail artery strips preloaded with the bioluminescent protein aequorin, both spermine and acetylspermine caused a concomitant decrease in intracellular calcium and isometric force activated by 36 mM of KCl. These results demonstrate clearly that acetylspermine and spermine alike decrease intracellular calcium concentration of vascular smooth muscle, which is likely to account for the relaxation of vasculature. The relaxation of smooth muscle in the vascular wall in turn might lead to decreased arterial blood pressure.

Animals↗
PubMed1989

Pathophysiologic role of calcium in the development of vascular smooth muscle tone.

Recent information indicates that the intracellular ionized calcium concentration [Ca2+]i plays a regulatory role not only in determining the magnitude of vascular tone but also in regulating growth of vascular tissue. Studies on living vascular smooth muscle cells using the calcium indicator aequorin have revealed that the relation between [Ca2+]i and contraction of the vascular smooth muscle cell is complex. More than 1 intracellular kinase may be involved, leading to the coexistence of multiple excitation-contraction coupling pathways. However, it appears that all of these pathways may be calcium-dependent. It is not yet known whether the cause of human essential hypertension involves an elevated [Ca2+]i in the vascular smooth muscle cell. However, evidence is presented supporting the concept that a decreased [Ca2+]i in the hypertensive smooth muscle cell will lead to a decrease in vascular tone and total peripheral resistance, and possibly also antagonize the growth response of the vascular smooth muscle cell associated with the secondary effects of hypertension.

Animals↗
PubMed1989

Agonist-specific myosin phosphorylation and intracellular calcium during isometric contractions of arterial smooth muscle.

The relationship between phosphorylation of the 20-kDa myosin light chain, intracellular calcium levels ([Ca2+]i), and isometric force was studied during prolonged activation of arterial smooth muscle. Aequorin, preloaded into ferret aortic strips, was used as a [Ca2+]i indicator. Two dimensional polyacrylamide gel electrophoresis was used to determine the phosphorylation levels of the 20-kDa myosin light chain (LC20). During the 30-min depolarization of arterial smooth muscle by K+ (21 mM), both LC20 phosphorylation and [Ca2+]i increased significantly at all time points examined as did the steady state stress. A transient rise in LC20 phosphorylation and [Ca2+]i occurred within 30 s, followed by suprabasal levels through the 10-min period during a sustained alpha 1-mediated activation by 10(-5) M phenylephrine whereas a higher force was developed at a shorter time compared to K+. An active phorbol ester 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA, 10(-6) M) induced a slow contraction of similar magnitude to that induced by K+ without significantly changing either [Ca2+]i or LC20 phosphorylation over a 90-min period. These results demonstrate that the amount of LC20 phosphorylation correlates with the [Ca2+]i in all three types of activation. The initial levels of [Ca2+]i and LC20 phosphorylation correlate with the onset of force development but not the magnitude of steady state stress, suggesting a role for [Ca2+]i and LC20 phosphorylation in regulating the cross bridge cycling rate during tension development. The lack of a detectable increase in [Ca2+]i and LC20 phosphorylation during DPBA activation suggests that sites other than LC20, phosphorylated by protein kinase C, may be involved in regulating smooth muscle contraction.

Animals↗
PubMed1987

Intracellular calcium levels in phorbol ester-induced contractions of vascular muscle.

Intracellular calcium concentration ([Ca2+]i) was measured with aequorin in ferret and rat aortic strips contracted with phorbol esters. In ferret aorta, 12-deoxyphorbol 13-isobutyrate 20-acetate (DPBA, 1 microM) induced contractions without significantly increasing [Ca2+]i, whereas 21 mM K+ induced smaller contractions with a significant rise in [Ca2+]i. Ca2+-free 2.5 mM ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)-physiological saline solution (PSS) had no effect on DPBA-induced tension, whereas it abolished contractions induced by 66 mM K+. The alpha 1-adrenergic agonist phenylephrine (10(-5) M) induced less than 10% of the tension with no initial [Ca2+]i spike under Ca-free conditions. In rat aorta, both phorbol 12-myristate 13-acetate (PMA, 2 microM) and DPBA (1 microM) induced contractions without increasing [Ca2+]i; Ca2+-free EGTA-PSS or the addition of the calcium channel blocker gallopamil (D600, 1 microM), however, abolished greater than 50% of the tension induced by either phorbol ester with a decrease in [Ca2+]i. These results are consistent with the idea that 1) resting [Ca2+]i is both sufficient and required to support phorbol ester-induced contractions in two vascular smooth muscles, suggesting an increased sensitivity of the contractile apparatus for Ca2+, and 2) there are differences in the mechanisms by which phorbol esters and alpha 1-agonists may activate vascular smooth muscle.

Animals↗
PubMed1983

Properties and function of phosphatases from vascular smooth muscle.

Myosin light chain phosphatase (MLCP) activity was present in extracts from a wide variety of mammalian tissues. A partially purified preparation of bovine aortic MLCP also showed activity against phosphorylase a and p-nitrophenyl phosphate (PNP). Whether these three activities are ascribable to a single multifunctional phosphatase or to three distinct phosphatases is unknown. The three phosphatase activities coelute during gel filtration both before and after treatment with ethanol showing exclusion volumes corresponding to 240,000 and 35,000 daltons, respectively. This indicates that the enzyme is dissociable into a smaller catalytic subunit. The widespread occurrence of MLCP activity and the close parallel among MLCP, phosphorylase a phosphatase, and PNP phosphatase activities suggest that the enzyme (or enzymes) may participate in physiological processes in addition to dephosphorylation of phosphorylated myosin light chains.

Animals↗
PubMed1981

Cytoplasmic and nuclear binding of estradiol in the brain and pituitary of old female rats.

The subcellular distribution of estradiol following the in vivo administration of 3H-estradiol, cytosol and nuclear estrogen receptor after in vitro incubation with 3H-estradiol were studied in young adult female rats (3-6 months old) and old female rats (over 18 months old). After intravenous injection of 3H-estradiol, with or without deithylstilbestrol pretreatment, significantly higher cytosol uptake of estradiol in the pituitary, dorsal hypothalamus and corticomedial amygdala (AMYG) with concomitant significantly lower nuclear uptake in the preoptic area (POA) and basomedial hypothalamus (BMH) and a tendency of lower nuclear uptake in AMYG and pituitary of old noncyclic (NC) rats as compared with young adult female rats were observed. The uterus of NC rats had a low ratio of milligrams of protein per milligram wet weight as compared with young rats and the nuclear binding 1 h after the injection was significantly lower than that of young rats when the value was expressed as disintegrations per minute per milligram weight but not when expressed as disintegrations per minute per milligram protein. Assays of cytosol and nuclear estrogen receptor after in vitro incubation with 3H-estradiol revealed that old prolonged vaginal cornification (PVC) rats did not react estradiol priming with increased nuclear receptors in the pooled brain tissues of AMYG, POA and BMH, anterior pituitary and uterus. These results indicate that there seems to be an impaired translocation of estradiol from cytosol to nucleus in old PVC rats.

Aging↗
PubMed1980

Changes in running-wheel activity, eating and drinking and their day/night distributions throughout the life span of the rat.

Running-wheel activity, eating and drinking of 39 male Long-Evans rats of various ages ranging from four to 33 months old, were recorded twice a day and total activity, and food and water intake per day and percentage nocturnal activity were studied. Running activity and food intake decrease as age advances and water intake first decreases from four months to 17 months of age and later increases proportionally with further increases in age. The percentage of nocturnal activity of the running-wheel decreases from 12 months of age proportionally with an increase of age; food and water intake also have this tendency. However, incidence of disappearance of diurnal difference in running activity, eating and drinking is quite low (7%, 32% and 25% respectively). Seven long-term ovariectomized Sprague-Dawley rats over 24 months of age show similar results to those with male Long-Evans rats. Reversal of the lighting schedule does not induce a phase shift of running activity, eating and drinking in two among 15 rats over 18 months of age, seven among 21 rats and four among 21 rats, respectively. Other rats over 18 months of age can recover their light/dark rhythm in a period not significantly different from rats below 13 months of age. The neuron number of the suprachiasmatic nucleus does not have significant correlations with percentage nocturnal activity in running activity, eating and drinking.

Aging↗