OBJECTIVES: To construct a cell line that can stably express human phospholamban(PLN) and initially explore its application in the study of myocardial toxicity mechanism. METHODS: FastCloning method was used to insert the open reading frame sequence of target gene PLN into eukaryotic expression vector pcDNA5/FRT/TO(hereinafter referred to as pDFT) to construct the pDFT-PLN-Flag plasmid. The Flp-In^TM T-REx^TM 293 cells were generated by cotransfection of the constructed plasmid and pOG44 plasmid to express the target gene. Successfully recombined monoclonal cell lines were screened by hygromycin B resistance. Western blot and indirect immunofluorescence (IFA) were used to examine the expression of the target protein in recombinant cells. After the cell line was exposed to aconitine, it was verified by Western blot to detect changes in PLN protein phosphorylation. RESULTS: After PCR amplification of the recombinant plasmid and DNA electrophoresis, the length of the amplified product is the same as the known PLN gene fragment, which is consistent with the open reading frame (ORF) sequence of the human PLN gene after sequencing. IFA and Western blot showed that the constructed proliferation cell line can stably express high levels of human PLN under induction and regulation. Preliminary results showed that the phosphorylation level of Thr17-PLN decreased after two hours of exposure to 1 μmol/L aconitine. CONCLUSIONS This human cell line can stably express PLN and can be used to study the mechanism of action of aconitine on the cell at molecular level.
Calcium-Binding Proteins/metabolism* ; Cell Line ; Humans ; Myocardium/metabolism* ; Phosphorylation

Transglutaminase enzymes (TGases) catalyze the calcium dependent formation of an isopeptide bond between protein-bound glutamine and lysine substrates. Previously we have shown that activated TGase 3 acquires two additional calcium ions at site two and three. The calcium ion at site three results in the opening of a channel. At this site, the channel opening and closing could modulate, depending on which metal is bound. Here we propose that the front of the channel could be used by the two substrates for enzyme reaction. We propose that the glutamine substrate is directed from Trp236 into the enzyme, shown by molecular docking. Then a lysine substrate approaches the opened active site to engage Trp327, leading to formation of the isopeptide bond. Further, direct comparisons of the structures of TGase 3 with other TGases have allowed us to identify several residues that might potentially be involved in generic and specific recognition of the glutamine and lysine substrates.
Animals ; Binding Sites ; Calcium/metabolism ; Calcium-Binding Proteins/*metabolism ; Glutamine/metabolism ; Human ; Lysine/metabolism ; Models, Chemical ; Models, Molecular ; Protein Binding ; Protein Structure, Tertiary ; Transglutaminases/*metabolism

The activation of Ca2+ entry through store-operated channels by agonists that deplete Ca2+ from the endoplasmic reticulum (ER) is a ubiquitous signaling mechanism, the molecular basis of which has remained elusive for the past two decades. Store-operated Ca2+-release-activated Ca2+ (CRAC) channels constitute the sole pathway for Ca2+ entry following antigen-receptor engagement. In a set of breakthrough studies over the past two years, stromal interaction molecule 1 (STIM1, the ER Ca2+ sensor) and Orai1 (a pore-forming subunit of the CRAC channel) have been identified. Here we review these recent studies and the insights they provide into the mechanism of store-operated Ca2+ channels (SOCCs).
Animals ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Calcium Signaling ; drug effects ; Humans ; Membrane Proteins ; antagonists & inhibitors ; metabolism ; Protein Binding

OBJECTIVETo investigate the abnormal abundances of calcium regulatory proteins in rabbit myocytes with failing hearts.

METHODSSixteen rabbits were divided into two groups: 8 rabbits with heart failure induced by volume plus pressure overload and 8 sham-operated animals. The hemodynamic parameters and cardiac structure and function were detected via catheterization and echocardiography respectively. L-type calcium channel (LTCC), Ryanodine receptor 2 (RyR2), Sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), and Na(+)-Ca(2+) exchanger (NCX) protein abundances were determined by Western blot analysis.

RESULTSThe ratio of left ventricular mass to body weight, heart rate and left ventricular end diastolic pressure in heart failure rabbits were significantly increased compared with sham-operated rabbits (P < 0.01), but their left ventricular shorten fraction [(21.3 +/- 4.00)% vs. (36.5 +/- 1.36)%] and ejection fraction (0.45 +/- 0.07 vs. 0.70 +/- 0.02) were decreased (P < 0.01). In heart failure rabbits, the abundances of LTCC and RyR2 were significantly decreased (R(LTCC/actin): 0.287 +/- 0.029 vs. 0.624 +/- 0.009; R(RyR2/actin): 0.106 +/- 0.001 vs. 0.203 +/- 0.011; P < 0.01), whereas the expressions of SERCA2a and NCX were markedly increased (R(NCX/actin): 0.497 +/- 0.015 vs. 0.221 +/- 0.014; R(SERCA2a/actin): 0.611 +/- 0.036 vs. 0.433 +/- 0.008; P < 0.01).

CONCLUSIONSReductions of LTCC and RyR2 might contribute to risk factors of systolic dysfunction in failing hearts. In early stage of heart failure, upregulated SERCA2a and NCX protein levels may be helpful for maintaining cardiac performance.

Animals ; Calcium ; metabolism ; Calcium-Binding Proteins ; biosynthesis ; Female ; Heart Failure ; metabolism ; Male ; Rabbits ; Ryanodine Receptor Calcium Release Channel ; metabolism ; Sarcoplasmic Reticulum ; chemistry ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

To understand the neurochemical properties of the gastric myenteric plexus of ruminants, the expression patterns of calbindin D-28k (CB), calretinin (CR), substance P (SP) and calcitonin gene-related peptide (CGRP) were explored in the Korean native goat. In gastric myenteric plexus, CB and SP immunoreactivity were observed in round- or ovalshaped neurons. CR and CGRP immunoreactivity were detected only in the nerve fibers. This immunohistochemical localization of CB, CR, CGRP and SP in the myenteric plexus of the goat stomach exhibited species-specific patterns. These findings suggest that these substances may be directly or indirectly related to the gastric functions of the goat stomach.
Animals ; Calcitonin Gene-Related Peptide ; Calcium-Binding Protein, Vitamin D-Dependent/metabolism ; Calcium-Binding Proteins/*metabolism ; Goats/*metabolism ; Immunohistochemistry/veterinary ; Myenteric Plexus/*metabolism ; Stomach/*innervation ; Substance P/metabolism

The aim of the present study was to investigate the abnormal calcium re-uptake function of myocardium sarcoplasmic reticulum (SR) in rabbits with heart failure, as well as potential mechanisms. Heart failure model was established in rabbits through aortic insufficiency and constriction of abdominal aorta. The SR Ca(2+) re-uptake function was measured with a calcium imaging device. The activity of myocardium SR calcium adenodine triphosphatase 2a (SERCA2a) was measured by inorganic phosphate. The protein expressions of SERCA2a, CaMKII, PKA, PP1α, phospholamban (PLB), PLB-Ser(16) and PLB-Thr(17) were evaluated by Western blot. The activities of PKA and CaMKII were detected by γ-(32)P substrate incorporation. The results showed that, compared with the sham operation group, the heart failure group exhibited reduced Ca(2+) re-uptake amount (P < 0.01) and the expression and activity of SERCA2a (P < 0.05 or P < 0.01), decreased expression of PLB and its phosphorylation status in sites of Ser(16) and Thr(17) (P < 0.05), increased expressions and activities of PKA and CaMKII (P < 0.05 or P < 0.01), and increased expression of PP1α (P < 0.05). These results suggest that the abnormal Ca(2+) re-uptake function in heart failure is related with reduced expression and activities of SERCA2a, as well as reduced expression of PLB and its phosphorylation status. Both PLB-Ser(16) and -Thr(17) may be involved in the regulation of myocardium SR calcium pump activity in heart failure.
Animals ; Calcium ; metabolism ; Calcium-Binding Proteins ; metabolism ; Heart Failure ; physiopathology ; Myocardium ; metabolism ; Phosphorylation ; Rabbits ; Sarcoplasmic Reticulum ; pathology

OBJECTIVE: To study the relationship between postmortem interval and the myofibril fragmentation index of skeletal muscle. METHODS: Rabbit skeletal muscle were left in the room temperature for different postmortem intervals, and the protein concentration of each sample was detected by using biuret method. Furthermore, the myofibril fragmentation index of each sample was measured under the protein concentration level of 0.5 mg/mL. RESULTS: The myofibril fragmentation index increased obviously according to the postmortem interval prolongation. CONCLUSION The myofibril fragmentation index may be used on the estimation of early postmortem interval.
Animals ; Calcium-Binding Proteins/metabolism* ; Female ; Male ; Muscle, Skeletal/metabolism* ; Myofibrils/metabolism* ; Postmortem Changes ; Proteins/metabolism* ; Rabbits ; Spectrophotometry ; Time Factors

P-selectin, one of the membrane proteins, expresses on platelet and endothelia and interacts with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocyte membrane. This interaction mediates leukocytes rolling on endothelial membrane and then induces leukocyte recruitment to the site of infection or tissue injury. In the present study, we constructed the recombinant wild type human P-selectin, its calcium-binding sites mutants and recombinant PSGL-1-globulin (PSGL-1-Rg). They expressed in Sf9 cells by using the baculovirus expression system and were purified by TalonTM metal or Protein A affinity chromatography. The results showed that the recombinant PSGL-1-Rg interacted with recombinant wild type P-selectin and two P-selectin mutants with 2 calcium-binding sites mutation respectively, but could not bind to the P-selectin mutant with all 4 calcium-binding sites mutation. Therefore, we verified the importance of P-selectin calcium-binding sites for its interaction with PSGL-1.
Binding Sites ; Calcium ; metabolism ; Humans ; Leukocytes ; metabolism ; Membrane Glycoproteins ; metabolism ; Mutation ; P-Selectin ; metabolism ; Recombinant Proteins ; metabolism

OBJECTIVE: To investigate the expression of Calponin-1 and Transgenlin in the uterine smooth muscles during normal labor on-sets, and to evaluate their effect on initiating the normal labor. METHODS: A total of 14 uterine bodies and lower segments of human pregnancy were divided to a non-labor group (NIL) and a labor group(IL). Immunohistochemical technology and Western blot were used to determine the expression of Calponin-1 and Transgelin in the 2 groups. RESULTS: Immunohistochemical detection and Western blot showed that Calponin-1 protein in the uterine smooth muscle tissue of the body and the lower uterine segment of smooth muscle tissues had significant difference (P<0.05). The expression of Transgelin in the uterine body smooth muscle tissue in the IL was higher than that in the NIL(P<0.05). In the lower uterine segments of the smooth muscle, the expression of Transgelin was not significantly different in the 2 groups (P>0.05). CONCLUSION Calponin-1 of the uterine smooth muscle and Transgelin of the uterine body smooth muscle may involve in the regulation of uterine smooth muscle contractility, which is closely related to child birth launch.
Adult ; Calcium-Binding Proteins ; genetics ; metabolism ; Female ; Humans ; Labor Onset ; metabolism ; Microfilament Proteins ; genetics ; metabolism ; Muscle Proteins ; genetics ; metabolism ; Myometrium ; metabolism ; Pregnancy ; Uterine Contraction ; metabolism

The aim of the present study was to investigate the expressions of calpain and calpastatin in the myocardium of simulated weightlessness rats, and to elucidate the underlying mechanism of cardiac troponin I (cTnI) degradations. Tail-suspended (SUS) rats were used as a simulated weightlessness model on the ground. The myocardium of rats was homogenized, and the expressions of calpain-1, calpain-2, calpastatin and cTnI were analyzed by Western blotting technique. Calpastatin expression was significantly decreased in 2- and 4-week SUS groups compared with that in the synchronous controls (P<0.05). Calpain-2 expression was slightly decreased, whereas calpain-1 expression was unaltered in SUS groups. However, calpain-1/calpastatin and calpain-2/calpastatin ratios were increased after tail-suspension, being significantly higher in 2- and 4-week SUS groups than those in the synchronous controls (P<0.05, P<0.01). Cardiac TnI degradation was significantly increased after tail-suspension (P<0.01), but cTnI degradation in both SUS and control groups was significantly inhibited by a non-specific inhibitor of calpain, PD150606 (P<0.01). These results suggest that an increase in calpain activity may enhance cTnI degradation in the myocardium of tail-suspended rats.
Animals ; Calcium-Binding Proteins ; metabolism ; Calpain ; metabolism ; Hindlimb Suspension ; Myocardium ; metabolism ; Proteolysis ; Rats ; Troponin I ; metabolism ; Weightlessness Simulation