Expression of Na(+)/K(+)-ATPase alpha subunit isoforms in rat salivary glands: occurrence of sense and antisense RNAs of the alpha3 isoform in the sublingual gland

We examined the expression of Na(+)/K(+)-ATPase alpha-subunit isoforms in rat salivary glands by RT-PCR. Isoform alpha1 was expressed strongly in all three major salivary glands. The alpha2 isoform was expressed in both submandibular gland (SMG) and sublingual gland (SLG) but faintly in the parotid gland (PG). The alpha3 was detected only in the SLG, and the alpha3 mRNA in the SLG was 1/8 of its level in the brain. Na(+)/K(+)-ATPase alpha3 isoform in the SLG, was localized predominantly on the basolateral plasma membranes in serous cells by immunohistochemical method. We also found the presence of natural antisense RNA of the alpha3 isoform in rat SLG: the 1st-strand cDNA prepared with gene-specific forward primers targeted to the CDS region and to the promoter region of the alpha3 gene in place of oligo(dT) or gene-specific reverse primers produced reasonable PCR products corresponding to the alpha3 cDNA sequence by the subsequent PCR reaction. Synthesis of the 1st-strand cDNA with the gene-specific forward primers was prevented by RNase digestion of the total RNA preparation, indicating that the PCR products in the RT-PCR system were not due to the contaminated genomic DNA, if any. The alpha3 protein level in rat SLG increased with aging, and levels of both alpha3 mRNA (sense RNA) and alpha3 antisense RNA were higher in SLGs of aged rats than in those of young rats, respectively.     

变形链球菌耐氟菌株与亲代菌株质子移位膜ATP酶活性的比较

目的:通过测定并比较变形链球菌耐氟菌株及其亲代菌株体内质子移位膜ATP酶的活性,以阐明耐氟菌株耐酸能力提高的原因。方法:将变链菌株Ingbritt及其耐氟突变株Ingbritt-FR通过甲苯处理,2个循环的液氮冷冻和37℃解冻,制成透性细胞。将透性细胞悬液加到含有10mmol/LMgSO4的50mmol/LTris-maleate测试缓冲液中(pH6.0),加温至37℃,再加入5mmol/LATP(pH6.0)起动反应。分别于10、20、60min取样,测定样品中水解ATP所释出的无机磷量。采用磷钼酸比色法在紫外分光光度计上进行比色分析(660nm),所得数据采用双因素方差分析。结果:在10、20和60min时,耐氟菌株H+-ATP酶活性分别为308.48、136.67和82.80μmolPi/g细胞干重/min,显著高于亲代菌株的相应酶活性:104.77,、64.69和30.70(P<0.01)。随时间推移,两类菌株的H+-ATP酶活性均逐渐降低,在酶的作用时间差别上有统计学意义(P<0.01)。结论:耐氟菌株ATP酶活性增高为其耐酸性增高的原因;H+-ATP酶活性及耐酸性的增高将会增加变链菌耐氟菌株的致龋潜能。     

电针预处理对心肌缺血再灌注模型大鼠心肌组织FXR基因及心肌细胞线粒体功能的影响

目的探讨电针预处理改善心肌缺血再灌注损伤的可能作用机制。方法40只Wistar大鼠随机分为假手术组、缺血再灌注组、FXR抑制剂组及电针预处理组,每组10只。各组大鼠均予捆绑固定7天后,除假手组外其余各组均进行心肌缺血再灌注造模处理。FXR抑制剂组第8天尾静脉注射法尼酯X受体(FXR)抑制剂z-Guggulsterone 0.4μl/g后再造模,电针预处理组从第1天开始取"内关""足三里""关元"电针7天,第8天再造模。造模后测定大鼠血清白细胞介素8(IL-8)浓度及心肌细胞线粒体呼吸链酶活性及Na^+-K^+-ATP酶、Ca^2+-Mg^2+-ATP酶活性,检测心肌组织FXR基因表达水平。结果与假手术组相比,缺血再灌注组IL-8浓度升高,呼吸链酶Ⅰ、Ⅱ、Ⅲ、Ⅳ及Na^+-K^+-ATP酶、Ca^2+-Mg^2+-ATP酶活性均降低,FXR基因表达水平上调(P<0.01)。与缺血再灌注组相比,FXR抑制剂组与电针预处理组IL-8浓度均降低,呼吸链酶Ⅰ、Ⅱ、Ⅲ、Ⅳ及Na^+-K^+-ATP酶、Ca^2+-Mg^2+-ATP酶活性升高,FXR基因表达水平降低(P<0.01)。与FXR抑制剂组比较,电针预处理组IL-8浓度、FXR基因表达水平升高,Na^+-K^+-ATP酶、Ca^2+-Mg^2+-ATP酶活性及呼吸链酶Ⅲ、Ⅳ均降低(P<0.05)。结论电针预处理可能通过降低血清IL-8浓度,提高心肌细胞线粒体呼吸链复合酶、ATP酶活性,下调FXR基因表达,从而改善心肌缺血再灌注损伤。     

Unitary step of gliding machinery in Mycoplasma mobile

Among the bacteria that glide on substrate surfaces, Mycoplasma mobile is one of the fastest, exhibiting smooth movement with a speed of 2.0–4.5 μm⋅s⁻¹ with a cycle of attachment to and detachment from sialylated oligosaccharides. To study the gliding mechanism at the molecular level, we applied an assay with a fluorescently labeled and membrane-permeabilized ghost model, and investigated the motility by high precision colocalization microscopy. Under conditions designed to reduce the number of motor interactions on a randomly oriented substrate, ghosts took unitary 70-nm steps in the direction of gliding. Although it remains possible that the stepping behavior is produced by multiple interactions, our data suggest that these steps are produced by a unitary gliding machine that need not move between sites arranged on a cytoskeletal lattice.The fastest of the Mycoplasma species is Mycoplasma mobile (M. mobile); they glide with a speed of 2.0–4.5 μm⋅s⁻¹ (1, 2). Under an optimal-growth condition, cultivated single M. mobile cells are flask-shaped (Fig. 1A) and glide smoothly across a substrate covered with surface-immobilized sialylated oligosaccharides (3) in the direction of protrusion at a constant speed (Movie S1). Genomic sequencing and analysis have revealed that the mechanism must differ from other forms of motor protein systems and bacterial motility, because M. mobile lacks genes encoding conventional motor proteins in eukaryotes, such as myosin, kinesin, and dynein, in addition to lacking other motility structures in bacteria, such as flagella and pili (4). So far, three proteins have been identified as a part of the gliding machinery (Fig. 1B, Bottom): Gli123 (5), Gli521 (6), and Gli349 (7). The machinery units localize around the cell neck, and their number has been estimated to be ∼450 (2, 5, 8). Gli349 extends out from the cell membrane and shows a rod structure, ∼100 nm in total, with two flexible hinges when isolated (9). Notably, the machinery is driven by hydrolysis of ATP to ADP and inorganic phosphate, caused by an unknown ATPase (10). Because of the large size and characteristic structure of Gli349, and a series of studies with mutants and inhibitory antibodies (2, 11), it has been hypothesized that Gli349 works as a “leg” by binding to and releasing from a substrate covered with randomly arranged sialylated oligosaccharides (2) consuming the chemical energy of ATP. In addition, the pivoting movement of an elongated cell suggests that there are units working not simultaneously but rather independently to propel the cell forward (12). To test this hypothesis and identify conformational changes of a key part of the gliding machinery, we here designed an assay to detect the movement of M. mobile by high precision colocalization microscopy. In the presence of an excess number of binding targets in the solution, which decreased the number of active legs, stepwise displacement was shown for the first time, to our knowledge, to occur in gliding bacteria.Open in a separate windowFig. 1.Nanometer-scale tracking of Mycoplasma gliding. (A) A dark-field image of M. mobile. The image was captured with center-stop optics to maintain the high numerical aperture of the objective, which enabled a high spatial resolution (35). (Scale bar: 1 μm.) (B, Upper) Illustration of the fluorescent ghost. The gliding machinery was distributed around the neck portion, but only the active machinery bound to the glass is shown for simplicity. (Bottom) A construction model of the gliding machinery comprising three proteins: Gli123, Gli521, and Gli349. See the review by Miyata (2) for more detail. (C) A fluorescent image of the labeled ghost was acquired with a time resolution of 2 ms. (Scale bar: 1 μm; pixel size: 240 nm.) (D) The intensity profile of C. The XY area is 5 × 5 μm. (E) Gaussian fitting to D. Nanometer-scale tracking is achieved by positioning the peak of the 2D Gaussian function fitting to the intensity profile of the ghost. (F, Left) The speed of gliding ghosts at different [ATP]s in the solution (n = 129). The cyan curve shows a fit with Michaelis–Menten kinetics; Vmaxspeed and K_m are 2.6 µm⋅s⁻¹ and 61 µM, respectively. The dotted cyan curve shows a fit with the kinetics including the Hill coefficient; Vmaxspeed, [ATP₅₀] and n are 2.2 µm⋅s⁻¹, 43 µM, and 2.4, respectively. (Right) The speed of living cells with no ATP in the solution (2.1 ± 0.1 µm⋅s⁻¹; n = 22). (G) Effect of SL on the gliding velocity of the ghost at saturated [ATP]s, 0.3–1.0 mM (n = 50).     

Diabetes mellitus and subjects' ageing: a study on the ATP content and ATP-related enzyme activities in human erythrocytes

Na⁺/K⁺- and Ca²⁺-ATPase are the major ATP-dependent membrane-bound enzymes that regulate the cation transmembrane gradient which is altered both in red blood cell (RBC) senescence and in RBCs of diabetic patients. In an attempt to clarify the possible connection between diabetes mellitus and ageing, we investigated the relationship between RBC ATP content, Na⁺/K⁺-ATPase, Ca²⁺-ATPase activities and ageing in healthy, insulin-dependent (IDDM) and non-insulin-dependent (NIDDM) subjects. A significant correlation was found (r = ?0.82; P < 0.001) between RBC ATP content and subject's age only in the control group. A significant reduction in Na⁺/K⁺-ATPase activity was observed in the older group (C₂) of control subjects, in comparison with the younger (C₁) one. In both IDDM and NIDDM subjects, the enzymatic activity was significantly decreased when compared with healthy subjects of similar age (P < 0.001). A significant negative correlation was found between age and enzymatic activity in healthy subjects (r = ?0.60; P < 0.001). No difference was observed in the RBC membrane Ca²⁺-ATPase activity between younger (C₁) and older (C₂) healthy subjects. Ca²⁺-ATPase activity was significantly increased both in IDDM patients compared with C₁ (P < 0.001) and in NIDDM patients compared with C₂ (P < 0.001). The present data indicate that ageing causes a reduction in the erythrocyte ATP content in both healthy and diabetic subjects. In diabetic patients Na⁺/K⁺-ATPase activity decreases independently of age.     

前胡丙素对大鼠缺血-再灌注心肌肌浆网钙泵功能及其蛋白水平的影响

目的研究前胡丙素对大鼠缺血-再灌注(I-R)心肌肌浆网钙泵(SERCA)的影响及机制。方法 24只雄性SD大鼠随机均分为正常对照组、I-R组、前胡丙素+I-R组和聚乙二醇400+I-R组。检测再灌注15min时冠状动脉流出量(CF)和冠状动脉流出液中乳酸脱氢酶(LDH)漏出量,采用改良对硝基苯酚棕榈酸酯(p-NPP)法和Millipore过滤技术测定SERCA的活性和三磷酸腺苷依赖的SERCA45 Ca2+摄取率,Western blot法检测心肌细胞SERCA蛋白表达水平。结果与I-R组相比,前胡丙素+I-R组CF、SERCA蛋白表达量、SERCA的活性和45 Ca2+摄取率增高(P<0.01),LDH漏出量降低(P<0.01)。结论前胡丙素对离体大鼠心脏I-R损伤有保护作用,可能与其减轻I-R损伤引起的心肌SERCA功能和蛋白表达抑制有关。     

Status and prospect of current inotropic agents

Critical reassessment of established inotropic drugs such as the phosphodiesterase inhibitors and the digitalis glycosides has reaffirmed the need for novel cardiotonic agents that will not only beneficially affect the haemodynamic and functional impairment of patients with overt congestive heart failure, but also prevent its clinical manifestation and reduce the high mortality. None of the drugs examined in these directions - calcium sensitisers, β-receptor blockers, sodium channel modulators, digitalis derivatives - have been shown to achieve these goals. The research on endogenous digitalis did not, as was hoped, reveal a general strategy for improving the therapeutic index of cardiac glycosides. The proof that Na+/K+-transporting ATPase of cardiac muscle is the molecular point of attack (receptor) for the inotropic and toxic effects of digitalis-like acting C/D-cis and C/D-trans steroids revealed the cyclopentano-perhydrophenanthrene nucleus as their common pharmacophoric lead structure. This has opened a wide field for lead development in the direction of derivatives that favourably discriminate between the inotropy-linked α1-isoform and the toxicity-linked α3-isoform of Na+/K+-ATPase as the basis for the design of inotropic agents with high therapeutic margin.     

A Non-Ouabain Na/K Atpase Inhibitor Isolated from Bovine Hypothalamus. Its Relation to Hypothalamic Ouabain

We have isolated from bovine hypothalamic and pituitary tissue the sodium pump inhibitor HHIF that is structurally different from ouabain. By mass spectrometric analysis this purified factor revealed a single unique molecular ion with an accurate mass of 412.277 and a mass spectra different from ouabain. It has been previously shown that HHIF inhibits the Ca²⁺-ATPase of the plasma membrane of synaptosomes. HHIF increases free calcium levels in cultured rat mesangial cells as well as mesangial cell contraction and proliferation. With the same purification procedure we have isolated in parallel HHIF and Ouabain from central nervous tissue. Ouabain elutes prior to HHIF in the final purification HPLC systems. This endogenous Ouabain has, in all the systems tested, the same chromatographic behavior that synthetic cold or [³H] Ouabain     

Rabeprazole: the role of proton pump inhibitors in Helicobacter pylori eradication

Proton pump inhibitors have become one of the cornerstones in the treatment of Helicobacter pylori infection. Rabeprazole (Pariet^®) is a substituted benzimidazole proton pump inhibitor with potent gastric acid suppression properties. Its high acid–base dissociation constant allows activation over a broader pH range, resulting in quick, irreversible binding to the H⁺/K⁺-ATPase pump, and a more rapid onset of action compared with omeprazole, lansoprazole and pantoprazole. Unlike other proton pump inhibitors, the metabolism of rabeprazole is primarily via a nonenzymatic reduction to the thioether derivative, and the cytochrome P450 isoenzyme 2C19 is only partly involved in its metabolism. The effect of genetic polymorphism in cytochrome P450 isoenzyme 2C19 on the pharmacokinetics and pharmacodynamics of rabeprazole is therefore limited. In humans, once-daily dosing of 5–40 mg of rabeprazole inhibits gastric acid secretion in a dose-dependent manner. In vitro studies have shown that rabeprazole possesses more potent antibacterial properties against the growth of H. pylori than other proton pump inhibitors. Furthermore, its thioether derivative has more potent inhibitory in vitro activity against the growth and motility of clarithromycin-resistant H. pylori than other proton pump inhibitors or commonly used antimicrobials. Despite these inherent favorable characteristics of rabeprazole, randomized controlled trials have largely shown equivalence amongst proton pump inhibitors when used with two antibiotics in the eradication of H. pylori, with cure rates of 75–89% on an intent-to-treat basis. However, rabeprazole appears to consistently achieve such comparable eradication rates even when used at reduced doses (10 mg twice daily) as part of clarithromycin-based triple therapy.