Biomedical subjects

A Xu

Publications and source records attributed to A Xu.

At least 55 records · Page 3Linked to original sources

PubMed2001

[Relation between transcatheter arterial chemoembolization and time of death in patients with hepatic carcinoma].

OBJECTIVE: To evaluate the relationship between transcatheter arterial chemoembolization (TACE) and time of death in hepatic carcinoma patients. METHODS: One hundred and ninety-five patients (male 188, female 7) with liver cancer underwent TACE with patients dying at different intervals after treatment. Dose of antitumor drugs, amount of iodized oil, liver function, size, recurrence of surgical resection, pattern of tumor, metastasis, presence of portal vein thrombosis, post TACE relapse, post TACE liver function and AFP, etc, altogether 15 variables were subjected to statistical analysis with the Cox's hazard proportional model. RESULTS: According to univariate and forward stepwise regression analysis, factors associated with significantly worse survival were diffused type, multi-nodular growth of tumor, and tumor embolus in the portal vein (P < 0.0001). Before death, most patients had symptoms of chest oppression, shortness of breath, ascites, abdominal distension, jaundice, hepatic failure and hematemesis. Causes of death were hepatic coma (27.2%, n = 53), hepatic failure (23.1%, n = 45), hemorrhage from the digestive tract (36.9%, n = 72), multiple organ failure (5.1%, n = 10) and others (7.2%, n = 14). CONCLUSION: Several factors of TACE may lead to failure in liver function and death. TACE may hasten death in patients with severe liver functional embarrassment. Before instituting TACE, careful weighing the pros and cons of the general condition and liver function is important for patients with portal vein embolism, multiple tumors or diffused lesions. Tolerance of liver to the drug, time, dose and method of treatment should be meticulously and carefully planned. Post TACE protection of liver is also very important.

Adolescent↗

PubMed2000

Immobilization of the early secretory pathway by a virus glycoprotein that binds to microtubules.

Membrane trafficking from the endoplasmic reticulum (ER) to the Golgi complex is mediated by pleiomorphic carrier vesicles that are driven along microtubule tracks by the action of motor proteins. Here we describe how NSP4, a rotavirus membrane glycoprotein, binds to microtubules and blocks ER-to-Golgi trafficking in vivo. NSP4 accumulates in a post-ER, microtubule-associated membrane compartment and prevents targeting of vesicular stomatitis virus glycoprotein (VSV-G) at a pre-Golgi step. NSP4 also redistributes beta-COP and ERGIC53, markers of a vesicular compartment that dynamically cycles between the ER and Golgi, to structures aligned along linear tracks radiating throughout the cytoplasm. This block in membrane trafficking is released when microtubules are depolymerized with nocodazole, indicating that vesicles containing NSP4 are tethered to the microtubule cytoskeleton. Disruption of microtubule-mediated membrane transport by a viral glycoprotein may represent a novel pathogenic mechanism and provides a new experimental tool for the dissection of early steps in exocytic transport.

Animals↗

PubMed2000

Hypusine is required for a sequence-specific interaction of eukaryotic initiation factor 5A with postsystematic evolution of ligands by exponential enrichment RNA.

Hypusine is formed through a spermidine-dependent posttranslational modification of eukaryotic initiation factor 5A (eIF-5A) at a specific lysine residue. The reaction is catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase. eIF-5A is the only protein in eukaryotes and archaebacteria known to contain hypusine. Although both eIF-5A and deoxyhypusine synthase are essential genes for cell survival and proliferation, the precise biological function of eIF-5A is unclear. We have previously proposed that eIF-5A may function as a bimodular protein, capable of interacting with protein and nucleic acid (Liu, Y. P., Nemeroff, M., Yan, Y. P., and Chen, K. Y. (1997) Biol. Signals 6, 166-174). Here we used the method of systematic evolution of ligands by exponential enrichment (SELEX) to identify the sequence specificity of the potential eIF-5A RNA targets. The post-SELEX RNA obtained after 16 rounds of selection exhibited a significant increase in binding affinity for eIF-5A with an apparent dissociation constant of 1 x 10(-7) m. The hypusine residue was found to be critical for this sequence-specific binding. The post-SELEX RNAs shared a high sequence homology characterized by two conserved motifs, UAACCA and AAUGUCACAC. The consensus sequence was determined as AAAUGUCACAC by sequence alignment and binding studies. BLAST analysis indicated that this sequence was present in > 400 human expressed sequence tag sequences. The C terminus of eIF-5A contains a cold shock domain-like structure, similar to that present in cold shock protein A (CspA). However, unlike CspA, the binding of eIF-5A to either the post-SELEX RNA or the 5'-untranslated region of CspA mRNA did not affect the sensitivity of these RNAs to ribonucleases. These data suggest that the physiological significance of eIF-5A-RNA interaction depends on hypusine and the core motif of the target RNA.

Base Sequence↗

PubMed2000

Differential regulation of Ca2+/calmodulin-dependent enzymes by plant calmodulin isoforms and free Ca2+ concentration.

Multiple calmodulin (CaM) isoforms are expressed in plants, but their biochemical characteristics are not well resolved. Here we show the differential regulation exhibited by two soya bean CaM isoforms (SCaM-1 and SCaM-4) for the activation of five CaM-dependent enzymes, and the Ca(2+) dependence of their target enzyme activation. SCaM-1 activated myosin light-chain kinase as effectively as brain CaM (K(act) 1.8 and 1.7 nM respectively), but SCaM-4 produced no activation of this enzyme. Both CaM isoforms supported near maximal activation of CaM-dependent protein kinase II (CaM KII), but SCaM-4 exhibited approx.12-fold higher K(act) than SCaM-1 for CaM KII phosphorylation of caldesmon. The SCaM isoforms showed differential activation of plant and animal Ca(2+)-ATPases. The plant Ca(2+)-ATPase was activated maximally by both isoforms, while the erythrocyte Ca(2+)-ATPase was activated only by SCaM-1. Plant glutamate decarboxylase was activated fully by SCaM-1, but SCaM-4 exhibited an approx. 4-fold increase in K(act) and an approx. 25% reduction in V(max). Importantly, SCaM isoforms showed a distinct Ca(2+) concentration requirement for target enzyme activation. SCaM-4 required 4-fold higher [Ca(2+)] for half-maximal activation of CaM KII, and 1.5-fold higher [Ca(2+)] for activation of cyclic nucleotide phosphodiesterase than SCaM-1. Thus these plant CaM isoforms provide a mechanism by which a different subset of target enzymes could be activated or inhibited by the differential expression of these CaM isoforms or by differences in Ca(2+) transients.

Amino Acid Sequence↗

PubMed2000

Reversible inhibition of the calcium-pumping ATPase in native cardiac sarcoplasmic reticulum by a calmodulin-binding peptide. Evidence for calmodulin-dependent regulation of the V(max) of calcium transport.

Calmodulin (CaM) and Ca(2+)/CaM-dependent protein kinase II (CaM kinase) are tightly associated with cardiac sarcoplasmic reticulum (SR) and are implicated in the regulation of transmembrane Ca(2+) cycling. In order to assess the importance of membrane-associated CaM in modulating the Ca(2+) pump (Ca(2+)-ATPase) function of SR, the present study investigated the effects of a synthetic, high affinity CaM-binding peptide (CaM BP; amino acid sequence, LKWKKLLKLLKKLLKLG) on the ATP-energized Ca(2+) uptake, Ca(2+)-stimulated ATP hydrolysis, and CaM kinase-mediated protein phosphorylation in rabbit cardiac SR vesicles. The results revealed a strong concentration-dependent inhibitory action of CaM BP on Ca(2+) uptake and Ca(2+)-ATPase activities of SR (50% inhibition at approximately 2-3 microM CaM BP). The inhibition, which followed the association of CaM BP with its SR target(s), was of rapid onset (manifested within 30 s) and was accompanied by a decrease in V(max) of Ca(2+) uptake, unaltered K(0.5) for Ca(2+) activation of Ca(2+) transport, and a 10-fold decrease in the apparent affinity of the Ca(2+)-ATPase for its substrate, ATP. Thus, the mechanism of inhibition involved alterations at the catalytic site but not the Ca(2+)-binding sites of the Ca(2+)-ATPase. Endogenous CaM kinase-mediated phosphorylation of Ca(2+)-ATPase, phospholamban, and ryanodine receptor-Ca(2+) release channel was also strongly inhibited by CaM BP. The inhibitory action of CaM BP on SR Ca(2+) pump function and protein phosphorylation was fully reversed by exogenous CaM (1-3 microM). A peptide inhibitor of CaM kinase markedly attenuated the ability of CaM to reverse CaM BP-mediated inhibition of Ca(2+) transport. These findings suggest a critical role for membrane-bound CaM in controlling the velocity of Ca(2+) pumping in native cardiac SR. Consistent with its ability to inhibit SR Ca(2+) pump function, CaM BP (1-2.5 microM) caused marked depression of contractility and diastolic dysfunction in isolated perfused, spontaneously beating rabbit heart preparations. Full or partial recovery of contractile function occurred gradually following withdrawal of CaM BP from the perfusate, presumably due to slow dissociation of CaM BP from its target sites promoted by endogenous cytosolic CaM.

Adenosine Triphosphate↗

PubMed2000

Thyroid hormone-induced overexpression of functional ryanodine receptors in the rabbit heart.

Modifications in the Ca(2+)-uptake and -release functions of the sarcoplasmic reticulum (SR) may be a major component of the mechanisms underlying thyroid state-dependent alterations in heart rate, myocardial contractility, and metabolism. We investigated the influence of hyperthyroid state on the expression and functional properties of the ryanodine receptor (RyR), a major protein in the junctional SR (JSR), which mediates Ca(2+) release to trigger muscle contraction. Experiments were performed using homogenates and JSR vesicles derived from ventricular myocardium of euthyroid and hyperthyroid rabbits. Hyperthyroidism, with attendant cardiac hypertrophy, was induced by the injection of L-thyroxine (200 microg/kg body wt) daily for 7 days. Western blotting analysis using cardiac RyR-specific antibody revealed a significant increase (>50%) in the relative amount of RyR in the hyperthyroid compared with euthyroid rabbits. Ca(2+)-dependent, high-affinity [(3)H]ryanodine binding was also significantly greater ( approximately 40%) in JSR from hyperthyroid rabbits. The Ca(2+ )sensitivity of [(3)H]ryanodine binding and the dissociation constant for [(3)H]ryanodine did not differ significantly between euthyroid and hyperthyroid hearts. Measurement of Ca(2+)-release rates from passively Ca(2+)-preloaded JSR vesicles and assessment of the effect of RyR-Ca(2+)-release channel (CRC) blockade on active Ca(2+)-uptake rates revealed significantly enhanced (>2-fold) CRC activity in the hyperthyroid, compared with euthyroid, JSR. These results demonstrate overexpression of functional RyR in thyroid hormone-induced cardiac hypertrophy. Relative abundance of RyR may be responsible, in part, for the changes in SR Ca(2+) release, cytosolic Ca(2+) transient, and cardiac systolic function associated with thyroid hormone-induced cardiac hypertrophy.

Animals↗

PubMed2000

Ascorbic acid and glutathione modulate the biological activity of S-nitrosoglutathione.

Ascorbic acid and glutathione (GSH) are important determinants of the intracellular redox state, and both are known to accelerate the decomposition of S-nitrosoglutathione (GSNO), an endogenous adduct of nitric oxide (NO). The implications of these observations for GSNO bioactivity are not yet clear. We investigated the effect of ascorbic acid and GSH on GSNO bioactivity by using a bioassay with isolated segments of guinea pig aorta suspended in organ chambers. Arterial segments demonstrated relaxation to GSNO (0.1 micromol/L) that was significantly enhanced by 300 micromol/L ascorbic acid (71+/-6% versus 53+/-6%, P<0.05) but not GSH. Both ascorbic acid and GSH significantly shortened the duration of arterial relaxation in response to 0.1 micromol/L GSNO (from >120 minutes to 22.5+/-3.5 and 36.3+/-4.3 minutes, respectively; P<0.05), consistent with accelerated decomposition of GSNO that was confirmed spectrophotometrically. The effect of ascorbic acid was abrogated by either DTPA or the copper(I)-specific agent bathocuproine but not deferoxamine, indicating a dependence on the availability of redox-active copper. Consistent with this notion, the action of ascorbic acid on GSNO bioactivity was also supported by copper-zinc superoxide dismutase, a physiologically relevant source of copper. In contrast, the effect of GSH on GSNO degradation and GSNO-mediated arterial relaxation was independent of transition metal ions, because DTPA had no effect. These data indicate that both ascorbic acid and GSH modulate GSNO bioactivity and suggest a distinction between the mechanism of GSNO degradation by ascorbic acid or GSH. Whereas both ascorbic acid and GSH accelerate the degradation of GSNO, only ascorbic acid is dependent on the presence of transition metal ions.

Animals↗

PubMed2000

[Clinical study on super-selective camptothecin microsphere's embolization of internal iliac artery for bladder carcinoma].

OBJECTIVE: To evaluate the efficacy of camptothecin microsphere's embolization of the internal iliac artery for bladder carcinoma. METHODS: Eighteen patients with inoperable and advanced bladder carcinoma were treated with camptothecin microsphere's super-selective embolization of the internal iliac artery. RESULTS: Tumor size was reduced significantly, and tumor cells were damaged to various degrees in 17 patients. Adverse effects were not found. CONCLUSION: Camptothecin microsphere's embolization of the internal iliac artery is a safe and effective therapy for inoperable and advanced bladder carcinoma.

Adult↗

PubMed2000

[An observation of the immuno-persistence after inoculating with the domestic BRD II strain rubella vaccine among infants and young children].

OBJECTIVE: To study the immuno-persistence after inoculating with the domestic BRD II strain rubella vaccine among infants and young children. METHODS: Hemagglutination inhibition (HI) antibody detection method was used to test on children at age 6 to 18 months without rubella infection or rubella vaccine immunization in Yantai city of Shandong province and were selected for the observation of the immuno-persistence against the domestic BRD II strain rubella vaccine from 1995 to 1998. RESULTS: Positive rates of HI antibody among children of 6, 7, 8, 9, 12 and 18 month-old of inoculation were 94.44%, 97.22%, 96.67%, 100.00%, 100.00%, 100.00% and 93.10%, 93.55%, 96.77%, 96.97%, 100.00%, 100.00% (P > 0.05), in one or two years respectively. The geometric mean reverse titers (GMRTs) were about 50% lower than that after 1 month after 1 year. Similar results were found that after 2 years GMRTs was about 50% lower than that after 1 year of inoculation. There was highly significant difference on GMRTs of HI antibody between infants with 6 to 7 month-olds (29.89) and young children with 8 to 18 month-olds (53.00) after 2 years of inoculation (t = 3.58, P < 0.001). CONCLUSION: The immunization schedule at the first dose for BRD II strain rubella vaccine should be started when the child is 8 month old. For the second dose, the immunization schedule used in other developed countries should be referred adopted in China.

Antibodies, Viral↗

PubMed1999

Phosphorylation of P20 is associated with the actions of insulin in rat skeletal and smooth muscle.

Although a large number of protein kinases/phosphatases involved in insulin's actions have been characterized recently, relatively few of the downstream phosphoproteins have been identified. We have employed two-dimensional gel electrophoresis-based proteome analysis to investigate the insulin-evoked phosphorylation cascade in rat soleus muscle. Insulin reproducibly increased phosphorylation of a 20-kDa protein with a pI value of 6.0, which was identified subsequently as a phospho-isoform of P20, a small heat-shock-related protein. The adenylyl cyclase activator, forskolin, decreased phosphorylation of this P20 isoform and increased phosphorylation of another two P20 isoforms, with pI values of 5.9 and 5.6. Two-dimensional peptide mapping revealed that the phospho-peptides of these three P20 isoforms are different. In contrast to its action in soleus muscle, insulin decreased phosphorylation of the P20 isoform with pI 6.0 and increased phosphorylation of the two isoforms with pI values of 5.9 and 5.6 in vascular smooth muscle. This effect is similar to that induced by vasodilatory stimuli, suggesting that insulin could exert its vasodilatory action by affecting phosphorylation of P20. In summary, these results demonstrate that insulin differently modulates phosphorylation of P20 in skeletal and smooth muscle, and suggest that P20 could be a potential modulator of insulin's functions in these tissues.

Animals↗

PubMed1999

Role of oxyradicals in mutagenicity and DNA damage induced by crocidolite asbestos in mammalian cells.

Crocidolite, one of the most carcinogenic forms of asbestos, is mutagenic in cultured mammalian cells when assayed using a system that can detect multilocus deletions. In the present study, we examined the effect of buthionine sulfoximine (BSO) on mutation frequency and the formation of 8-hydroxydeoxyguanosine (8-OHdG) in human-hamster hybrid (A(L)) cells induced by crocidolite fibers in an attempt to determine the role of oxyradicals in mediating fiber mutagenesis. BSO, a competitive inhibitor of the enzyme gamma-glutamyl cysteine synthetase, depleted nonprotein sulfhydryls to <5% of control within 24 h at a nonmutagenic dose of 25 microM. In cells pretreated with BSO for 24 h, the mutation yield at the CD59 locus induced by a 4 microg/cm2 dose of crocidolite fibers was increased by more than 3-fold (P < 0.05). Using immunoperoxidase staining with a monoclonal antibody specific for 8-OHdG, we demonstrated that crocidolite fibers induced a dose-dependent increase in oxidative DNA damage in A(L) cells. Furthermore, addition of DMSO, a well-established hydroxyl radical (OH*) scavenger, dramatically suppressed 8-OHdG induction (P < 0.005). Our results definitely demonstrate that reactive oxygen species mediate fiber-induced DNA damage mutagenesis in A(L) cells in a concentration-dependent manner.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1999

Insulin and insulin antagonists evoke phosphorylation of P20 at serine 157 and serine 16 respectively in rat skeletal muscle.

We show here that insulin and insulin antagonists differentially modify phosphorylation of three phospho-isoforms of P20 (termed S1, S2 and S3) in rat skeletal muscle. Precise phosphorylation sites of S1 and S2 were mapped to serine 157 and serine 16 respectively. Insulin evoked phosphorylation of P20 at serine 157 through the phosphatidylinositol (PI) 3-kinase pathway. Epinephrine and calcitonin gene-related peptide decreased phosphorylation at serine 157 and increased phosphorylation at serine 16 and other unidentified sites. These results demonstrate that the PI-3-kinase pathway of anabolic insulin and the cAMP pathway of catabolic hormones converge on P20 and suggest a potential role of this protein in regulating energy metabolism of skeletal muscle.

Amino Acid Sequence↗

PubMed1999

Enhanced nitric oxide-mediated vascular relaxation in radial artery compared with internal mammary artery or saphenous vein.

BACKGROUND: The superior long-term patency of internal mammary artery coronary bypass grafts compared with venous grafts has been attributed in part to increased endothelium-derived nitric oxide (. NO) production. Interest in the radial artery as an alternative bypass conduit has recently been revived; however, its biological characteristics remain incompletely defined. The purpose of this study was to compare the.NO-mediated vasomotor properties of the radial artery to those of the internal mammary artery and saphenous vein. METHODS AND RESULTS: Matched segments of radial artery, internal mammary artery, and saphenous vein (n=24 patients) were examined by use of organ-chamber methodology. Endothelium-dependent and -independent vasomotor responses were assessed by dose-response curves to acetylcholine, N(G)-nitro-L-arginine methyl ester (L-NAME), 8-bromo-cyclic 3',5'-guanosine monophosphate (8-bromo-cGMP), and nitroglycerin. Maximum.NO-mediated radial artery relaxation in response to acetylcholine (86+/-10%) was significantly greater than internal mammary artery (56+/-9%) or saphenous vein (11+/-5%, both P<0.0001). Similarly, acetylcholine-stimulated cGMP accumulation in radial artery (9.1+/-1.7 pmol/mg protein) was also greater than internal mammary artery (6.2+/-0.3 pmol/mg protein) or saphenous vein (1.4+/-0.2 pmol/mg protein, both P<0.05). Estimated basal endothelial.NO production, assayed as the percent maximum contraction in response to L-NAME, was greater in radial artery (39+/-5%) than internal mammary artery (23+/-6%) or saphenous vein (5+/-2%, both P<0.05). Maximum relaxation of all vessels to nitroglycerin was similar, although the sensitivity of radial artery to nitroglycerin was greater (EC(50)=33+/-7 nmol/L) than the internal mammary artery (203+/-32 nmol/L) or saphenous vein (97+/-12 nmol/L, both P<0.05). Vascular cGMP in response to 0.1 micromol/L nitroglycerin was significantly higher in the radial artery (8.3+/-1. 4 pmol/mg protein) compared with the internal mammary artery (3. 5+/-1.3 pmol/mg protein) or saphenous vein (1.4+/-0.3 pmol/mg protein, both P<0.0001). Relaxation to 8-bromo-cGMP was identical for all 3 conduits. CONCLUSIONS: These data indicate that. NO-dependent relaxation of radial artery is greater than that of internal mammary artery or saphenous vein. This difference is related to endothelial production of.NO and/or vessel sensitivity to. NO. Such favorable physiological characteristics of radial artery could conceivably contribute to improved long-term patency of this conduit compared with saphenous vein.

Adult↗

PubMed1999

Highly specific recognition of primer RNA structures for 2'-OH priming reaction by bacterial reverse transcriptases.

A minor population of Escherichia coli contains retro-elements called retrons, which encode reverse transcriptases (RT) to synthesize peculiar satellite DNAs called multicopy single-stranded DNA (msDNA). These RTs recognize specific RNA structures in their individual primer-template RNAs to initiate cDNA synthesis from the 2'-OH group of a specific internal G residue (branching G residue). The resulting products (msDNA) consist of RNA and single-stranded DNA, sharing hardly any sequence homology. Here, we investigated how RT-Ec86 recognizes the specific RNA structure in its primer-template RNA. On the basis of structural comparison with HIV-1 RT, domain exchanges were carried out between two E. coli RTs, RT-Ec86 and RT-Ec73. RT-Ec86 (320 residues) and RT-Ec73 (316 residues) share only 71 identical residues (22%). From the analysis of 10 such constructs, the C-terminal 91-residue sequence of RT-Ec86 was found to be essential for the recognition of the unique stem-loop structure and the branching G residue in the primer-template RNA for retron-Ec86. Using the SELEX (systematic evolution of ligands by exponential enrichment) method with RT-Ec86 and primer RNAs containing random sequences, the identical stem-loop structure (including the 3-U loop) to that found in the retron-Ec86 primer-template RNA was enriched. In addition, the highly conserved 4-base sequence (UAGC), including the branching G residue, was also enriched. These results indicate that the highly diverse C-terminal region recognizes specific stem-loop structures and the branching G residue located upstream of the stem-loop structure. The present results with seemingly primitive RNA-dependent DNA polymerases provide insight into the mechanisms for specific protein RNA recognition.

Amino Acid Sequence↗

PubMed1999

Serine phosphorylation of the sarcoplasmic reticulum Ca(2+)-ATPase in the intact beating rabbit heart.

Recent studies have demonstrated that Ca(2+)/calmodulin-dependent protein kinase phosphorylates the Ca(2+)-pumping ATPase of cardiac sarcoplasmic reticulum (SR) in vitro. Also, evidence from in vitro studies suggested that this phosphorylation, occurring at Ser(38), results in stimulation of Ca(2+) transport. In the present study, we investigated whether serine phosphorylation of the SR Ca(2+)-ATPase occurs in the intact functioning heart. Hearts removed from anesthetized rabbits were subjected to retrograde aortic perfusion of the coronary arteries with oxygenated mammalian Ringer solution containing (32)P(i) and contractions were monitored by recording systolic left ventricular pressure development. Following 45-50 min of (32)P perfusion, the hearts were freeze-clamped, SR isolated, and analyzed for protein phosphorylation. SDS-polyacrylamide gel electrophoresis and autoradiography showed phosphorylation of several peptides including the Ca(2+)-ATPase and Ca(2+) release channel (ryanodine receptor). The identity of Ca(2+)-ATPase as a phosphorylated substrate was confirmed by Western immunoblotting as well as immunoprecipitation using a cardiac SR Ca(2+)-ATPase-specific monoclonal antibody. The Ca(2+)-ATPase showed immunoreactivity with a phosphoserine monoclonal antibody indicating that the in situ phosphorylation occurred at the serine residue. Quantification of Ca(2+)-ATPase phosphorylation in situ yielded a value of 208 +/- 12 pmol (32)P/mg SR protein which corresponded to the phosphorylation of approximately 20% of the Ca(2+) pump units in the SR membrane. Since this phosphorylation occurred under basal conditions (i.e., in the absence of any inotropic intervention), a considerable steady-state pool of serine-phosphorylated Ca(2+)-ATPase likely exists in the normally beating heart. These findings demonstrate that serine phosphorylation of the Ca(2+)-ATPase is a physiological event which may be important in the regulation of SR function.

Animals↗

PubMed1999

Expression of translationally controlled tumour protein is regulated by calcium at both the transcriptional and post-transcriptional level.

We have investigated how the programme of protein synthesis is altered in response to a loss of calcium homoeostasis in Cos-7 cells using a differential proteome mapping approach. Exposure of the cells to the calcium ionophore A23187 or thapsigargin, or alternatively, expression of a viral glycoprotein reported to deplete intracellular calcium stores, resulted in the up-regulated expression of a characteristic set of proteins. One of these is the translationally controlled tumour protein (TCTP), a cytoplasmic protein whose expression has not previously been linked to calcium perturbation. Quantitative Northern blot assay demonstrated that steady-state mRNA abundance of TCTP was also increased under these conditions. Clamping the cytosolic calcium concentration by the introduction of bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA) into cells did not affect the increase in steady-state levels of TCTP mRNA observed in response to ionophore. Therefore depletion of endoplasmic reticulum (ER) calcium, but not elevation of the cytosolic calcium concentration, was responsible for increased transcription of the TCTP gene. However, the presence of BAPTA significantly attenuated the ionophore-mediated increase in levels of the protein. Moreover, the level of TCTP in ionophore-treated cells increased in advance of a detectable increase in the corresponding mRNA abundance. These results indicate that expression of TCTP is regulated at two distinct levels in response to the concentration of calcium in different cellular compartments. Whereas depletion of the ER store causes an increase in TCTP mRNA abundance, increased cytosolic calcium concentrations regulate gene expression at the post-transcriptional level.

Animals↗

PubMed1999

Amylin evokes phosphorylation of P20 in rat skeletal muscle.

To investigate the signal transduction events underlying amylin's actions, the amylin-evoked protein phosphorylation cascade was analysed using two-dimensional gel electrophoresis. We found that phosphorylation of three isoelectric variants of P20 (termed ARPP1, ARPP2 and ARPP3) was associated with amylin's actions in rat skeletal muscle. Amylin decreased phosphorylation of ARPP1 and increased phosphorylation of ARPP2 and ARPP3 in a dose-dependent manner. Insulin inhibited amylin-evoked phosphorylation of ARPP2 and ARPP3. The amylin-selective antagonist rat amylin-(8-37) completely reversed amylin's action on ARPP3 and partially decreased phosphorylation of ARPP2. By contrast, the CGRP-selective antagonist, human CGRP-(8-37) blocked phosphorylation of ARPP2 but had little effect on ARPP3. These results suggest that amylin modifies phosphorylation of P20 via two independent mechanisms, and that P20 might be a molecule mediating amylin's biological functions.

Amyloid↗

PubMed1999

Ca2+/calmodulin-dependent phosphorylation of the Ca2+-ATPase, uncoupled from phospholamban, stimulates Ca2+-pumping in native cardiac sarcoplasmic reticulum.

Recent studies have demonstrated phosphorylation of the cardiac and slow-twitch muscle isoform (SERCA2a) of the sarcoplasmic reticulum (SR) Ca2+-ATPase (at Ser38) by a membrane-associated Ca2+/calmodulin-dependent protein kinase (CaM kinase). Analysis of the functional consequence of Ca2+-ATPase phosphorylation in the native SR membranes, however, is complicated by the concurrent phosphorylation of the SR proteins phospholamban (PLN) which stimulates Ca2+ sequestration by the Ca2+-ATPase, and the ryanodine receptor-Ca2+ release channel (RYR-CRC) which likely augments Ca2+ release from the SR. In the present study, we achieved selective phosphorylation of the Ca2+-ATPase by endogenous CaM kinase in isolated rabbit cardiac SR vesicles utilizing a PLN monoclonal antibody (PLN AB) which inhibits PLN phosphorylation, and the RYR-CRC blocking drug, ruthenium red, which inhibits phosphorylation of RYR-CRC. Analysis of the Ca2+ concentration-dependence of ATP-energized Ca2+ uptake by SR showed that endogenous CaM kinase mediated phosphorylation of the Ca2+-ATPase, in the absence of PLN and/or RYR-CRC phosphorylation, results in a significant increase (approximately 50-70%) in the Vmax of Ca2+ sequestration without any change in the k0.5 for Ca2+ activation of the Ca2+ transport rate. On the other hand, treatment of SR with PLN AB (which mimics the effect of PLN phosphorylation by uncoupling Ca2+-ATPase from PLN) resulted in approximately 2-fold decrease in k0.5 for Ca2+ without any change in Vmax of Ca2+ sequestration. These findings suggest that, besides PLN phosphorylation, direct phosphorylation of the Ca2+-ATPase by SR-associated CaM kinase serves to enhance the speed of cardiac muscle relaxation.

Animals↗