IP(3)R-mediated Ca(2+) release is modulated by anandamide in isolated cardiac nuclei

Cannabinoids (CBs) are known to alter coronary vascular tone and cardiac performance. They also exhibit cardioprotective properties, particularly in their ability to limit the damage produced by ischaemia reperfusion injury. The mechanisms underlying these effects are unknown. Here we investigate the intracellular localisation of CB receptors in the heart and examine whether they may modulate localised nuclear Ca²⁺ release. In isolated cardiac nuclear preparations, expression of both the inositol 1,4,5-trisphosphate receptor type 2 (IP₃R) and CB receptors (CB₁R and CB₂R) was demonstrated by immunoblotting. Both receptors localised to the nucleus and purity of the nuclear preparations was confirmed by co-expression of the nuclear marker protein nucleolin but absence of cytoplasmic actin. To measure effects of IP₃R and CBR agonists on nuclear Ca²⁺ release, isolated nuclei were loaded with Fluo5N-AM. This dye accumulates in the nuclear envelope. Isolated nuclei responded to IP₃ with rapid and transient Ca²⁺ release from the nuclear envelope. Anandamide inhibited this IP₃-mediated release. Preincubation of nuclear preparations with either the CB₁R antagonist (AM251) or the CB₂R antagonist (AM630) reversed anandamide-mediated inhibition to 80% and 60% of control values respectively. When nuclei were pre-treated with both CBR antagonists, anandamide-mediated inhibition of IP₃-induced Ca²⁺ release was completely reversed. These results are the first to demonstrate the existence of cardiac nuclear CB receptors. They are also the first to show that anandamide can negatively modulate IP₃-mediated nuclear Ca²⁺ release. As such, this provides evidence for a novel key mechanism underlying the action of CBs and CBRs in the heart.     

Anti-ageing and rejuvenating effects of quercetin

Homeostasis is a key feature of the cellular lifespan. Its maintenance influences the rate of ageing and it is determined by several factors, including efficient proteolysis. The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Alterations of proteasome function have been recorded in various biological phenomena including ageing and replicative senescence. Proteasome activities and function are decreased upon replicative senescence, whereas proteasome activation confers enhanced survival against oxidative stress, lifespan extension and maintenance of the young morphology longer in human primary fibroblasts. Several natural compounds possess anti-ageing/anti-oxidant properties. In this study, we have identified quercetin (QUER) and its derivative, namely quercetin caprylate (QU-CAP) as a proteasome activator with anti-oxidant properties that consequently influence cellular lifespan, survival and viability of HFL-1 primary human fibroblasts. Moreover, when these compounds are supplemented to already senescent fibroblasts, a rejuvenating effect is observed. Finally, we show that these compounds promote physiological alterations when applied to cells (i.e. whitening effect). In summary, these data demonstrate the existence of naturally occurring anti-ageing products that can be effectively used through topical application.     

蜣螂有效部位的初步表征

目的:建立蜣螂有效部位的质量控制方法。方法:采用薄层色谱法对蜣螂有效部位含有的氨基酸进行定性鉴别,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法作分子量的检测,氨基酸自动分析仪对蜣螂有效部位中游离氨基酸与结合氨基酸进行种类和含量检测。结果:蜣螂有效部位的薄层色谱中,在与对照品色谱相应的位置上,显相同颜色的斑点;分子量检测表明蜣螂有效部位主要为多肽的混合物,相对分子质量分布在6×103～14×103;游离氨基酸与构成多肽的氨基酸检测表明有效部位中除色氨酸被破坏无法测出外,共检出17种氨基酸,其中游离型氨基酸占13.41%,结合型氨基酸即多肽占9.27%。结论:表征方法灵敏准确、重复性好、专属性强,可用于蜣螂有效部位的质量控制。     

Polyethylene glycosylation prolongs the stability of recombinant human paraoxonase-1

Paraoxonase-1 (PON1) is a native enzyme that is synthesized in the liver and is capable of hydrolyzing organophosphates (OPs). It is regarded as part of a promising approach for the pretreatment and therapy of OP poisoning. Previous experiments with purified rabbit serum PON1 have established that it can protect rats against many OP exposures. In the current paper, we described a preparation of active recombinant human PON1 (rHuPON1) by engineering an Escherichia coli expression system. Recombinant HuPON1 was purified by Ni-NTA affinity chromatography followed by DEAE sepharose fast-flow chromatography. After purification, rHuPON1 was chemically modified with polyethyleneglycol (PEG)-20K. Recombinant HuPON1 exhibited a mean residence time (MRT) of 8.9h, which was threefold shorter than that of native HuPON1 in rats. However, rHuPON1 chemically modified with PEG-20K displayed an MRT of 19.5h, suggesting that PEG modification can prolong the circulatory stability of rHuPON1. PEG-rHuPON1 had a catalytic efficiency sufficient in protecting rats against OP poisoning, as measured by acetylcholinesterase activity in tissues and signs after poisoning.     

六种中药材电泳鉴别

目的:对山药、当归、百合、天花粉、紫菀、熟地进行电泳分析,以探索其鉴别方法。方法:应用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)技术,对山药、当归、熟地等中药材进行蛋白质电泳。结果:不同中药材的电泳图谱存在明显差异。结论:该电泳方法可作为熟地等几种中药材的鉴别依据之一。     

Root Extract of Polygonum Hydropiper Alters the Expression of Rat Uterine Protein Profile in Presence and Absence of Ovary in-situ during Periimplantation Period: An Evidence on SDS-PAGE

Objective To study its effects on reproductive performance in albino rats.
Methods The chromatographic fraction （CF） of crude methanolic extract of the herb has been subcutaneously administered to female albino rats. Experiments were carried out in adult cyclic females and oavriectomised （OVX） females during early gestation period. Uterine horns were collected following the respective treatment regimen to stud）, the protein profile in 15% gel SDS-PAGE.
Results The CF induced changes in the expression of protein in rat uterus. New proteins have been expressed in uterus of adult cyclic females having ovary in-situ. The OVX females treated with CF showed altered uterine protein profile compared with that of OVX control and OVX estradiol-17β （E2） treated rats. The CF exerted its effect on expression of uterine protein during early gestation period in rats. While uterine proteins of CF treated females were similar to that of controls during preimplantation period; many of the proteins on day 6 of gestation have been found either missing or expressed in lesser intensity.
Conclusion The root of Polygonum hydropiper contains potential compound（s） which can alter the reproductive performance of female rats modulating uterine protein expression.     

Fibrinogen Nový Jicín and Praha II: cases of hereditary Aalpha 16 Arg-->Cys and Aalpha 16 Arg-->His dysfibrinogenemia

INTRODUCTION: Various dysfibrinogenemias have been described worldwide. This paper describes two new cases of dysfibrinogenemia identified in the Czech Republic. MATERIALS AND METHODS: The proposita of fibrinogen Novy Jicín, a 12-year-old girl, presented with hemorrhagic complications, low Clauss fibrinogen level (0.3 g/l) and prolonged both thrombin (70.8 s) and reptilase (>180 s) time. Her mother and sister both presented with normal coagulation tests, normal fibrinogen level and reported no history of bleeding. The carriers of the fibrinogen Praha II were a 31-year-old man and his 11-year-old daughter. They both presented with low fibrinogen Clauss level (0.88 g/l) and prolonged thrombin and reptilase time. To identify the genetic mutation responsible for these dysfibrinogens, genomic DNA extracted from the blood was analyzed. The presence of the mutant chains in the circulation was determined by MALDI-TOF mass spectroscopy. Scanning electron micrographs of the patients' fibrin clots were obtained. RESULTS: The kinetics of fibrinopeptide release and fibrin polymerization were impaired for both fibrinogen Novy Jicín and Praha II. DNA sequencing showed heterogeneous fibrinogen Aalpha R16C mutation in the fibrinogen Novy Jicín case and heterogeneous fibrinogen Aalpha R16H in the fibrinogen Praha II case. The mutant chains were found to be expressed to the circulation by MALDI-TOF mass spectroscopy. Scanning electron micrographs of the patient's fibrin clot were found to be abnormal. CONCLUSIONS: The case of dysfibrinogenemia Aalpha R16C-fibrinogen Novy Jicín and the case of dysfibrinogenemia Aalpha R16H were found by routine coagulation testing and were genetically identified.     

Interaction of calcium/calmodulin-dependent protein kinase IIdeltaC with sorcin indirectly modulates ryanodine receptor function in cardiac myocytes

Calcium/calmodulin dependent protein kinase II delta C (CaMKIIdelta(C)) and the EF-hand Ca(2+)-binding protein, sorcin have both been shown to regulate the excitation-contraction coupling process. This study explores the possibility that these two proteins interact directly and, as a result of this interaction, modulate cardiac calcium handling. Two independent methods (surface plasmon resonance (SPR) and overlay assays) were used to determine whether CaMKIIdelta(C) and sorcin interacted in a direct manner. The nature of this interaction was explored by (i) examining the effects of sorcin on CaMKIIdelta(C) activity using a selective kinase assay and (ii) studying whether sorcin was a substrate for CaMKIIdelta(C) using autoradiography. Ryanodine binding assays on mouse ventricular cardiomyocytes were used to determine specific functional effects of this interaction. SPR studies suggested that sorcin interacts with CaMKIIdelta(C) in a concentration-dependent manner. This interaction occurs in the presence of Ca(2+) and in the presence or absence of calmodulin (CaM). Overlay assays confirmed the existence of this interaction. Further experiments suggested that this interaction is reciprocal. Firstly, sorcin significantly inhibited both recombinant and native CaMKIIdelta(C) activity to similar extents. Secondly, sorcin was phosphorylated by CaMKIIdelta(C). Thirdly, sorcin inhibition of CaMKII activity occurred under conditions where sorcin remained dephosphorylated. Functionally, CaMKIIdelta(C)-mediated phosphorylation of sorcin served to abolish the inhibitory effect of sorcin on ryanodine receptor (RyR(2)) open probability (Po). Since both proteins are capable of directly modulating RyR(2) activity, this interaction may serve as an additional or alternative indirect route by which both proteins can regulate RyR(2) opening status in cardiac myocytes.     

Comparative study on the stability of soybean (Glycine max) β-conglycinin in vivo

The immunoreactivity and structural variation of β-conglycinin in digesta from digestive tracts of pigs were measured by inhibition ELISA and sodium dodecyl sulphate polyacrylamide gel electrophoresis, respectively. Results showed that the immunoreactivity disappearance proportion of β-conglycinin significantly increased from stomach to the caecum in all groups (P<0.05). In the stomach, upper-jejunum and middle-jejunum, the immunoreactivity disappearance proportion of β-conglycinin significantly increased among these three groups (P<0.05), while it has no significant difference in ileum and caecum (P>0.05). The α′, α subunits of β-conglycinin were easier to digest than the β subunit. It indicated that immunoreactivity disappearance proportion of β-conglycinin tended to increase with the growth of age and the descending down of digestive tract, while the β subunits of β-conglycinin are more stable to digestion than α′, α subunits.