血管钠肽抑制异丙肾上腺素增强的大鼠心肌细胞钙瞬变

采用光谱荧光法研究血管钠肽(vasonatrin peptide,VNP)对心肌细胞内钙瞬变的作用及其机制,观察钠尿肽鸟苷酸环化酶(guanylate cyclase,GC)受体的特异性阻断剂(HS-142-1)、8-溴-环磷酸鸟苷(8-Br-cGMP)和镁蓝(methylene blue,MB)对心肌细胞内钙瞬变的影响。结果显示,异丙肾上腺素(isoproterenol,Iso)(10~(-10)～10~(-6)mol/L)可剂量依赖性地引起心肌细胞内钙瞬变增强,相对于对照组分别增强(13±8)%(P>0.05)、(26±13)%(P<0.05)、(66±10)%(P<0.01)、(150±10)%(P<0.01)和(300±25)%(P<0.01)。此效应可被β肾上腺素受体阻断剂普萘洛尔(10~(-6)mol/L)所阻断。VNP(10~(-10)～10~(-6)mol/L)可剂量依赖性地抑制Iso(10~(-8)mol/L)引起的心肌细胞内钙瞬变幅值的升高,相对于Iso(10~(-8)mol/L)分别减弱(99±3)%(P>0.05)、(96±2)%(P<0.05)、(84±6)%(P<0.01)、(66±3)%(P<0.01)和(62±3)%(P<0.01)。8-Br-cGMP(10~(-7)～10~(-3)mol/L)也可剂量依赖性地抑制Iso(10~(-8)mol/L)引起心肌细胞内钙瞬变的增强。HS-142-1(2×10~(-5)mol/L)使VNP的作用几乎完全消失。MB是GC的抑制剂,10~(-5)mol/L MB不但使VNP的作用完全消失,而且增强Iso对心肌细胞内钙瞬变的效应。VNP和HS-142-1本身对心肌细胞内钙瞬变无显著影响。而MB使心     

Mitochondrial Respiratory Chain Complex Patterns from Acanthamoeba castellanii and Lycopersicon esculentum: Comparative Analysis by BN-PAGE and Evidence of Protein–Protein Interaction Between Alternative Oxidase and Complex III

We have previously shown that a kinetic interplay exists between the cytochrome pathway and the alternative oxidase in mitochondria from amoeba Acanthamoeba castellanii . Native interaction analyses using blue native gel electrophoresis coupled to denaturating electrophoresis and immunodetection have indicated associations between alternative oxidase and oxidative phosphorylation complexes in both amoeba and tomato mitochondria. These associations are dependent on the expression level of alternative oxidase according to the physiological state in both organisms. Alternative oxidase associates broadly with large complexes of the respiratory chain when it is expressed in large amount, i.e., in ripe tomato and exponentially growing amoeba. On the contrary, alternative oxidase interacts specifically with complex III even if expression of the oxidase is low, i.e., in green tomato and stationary phase amoeba. This specific interaction represents a higher level of regulation driven by protein-protein interactions leading to a direct kinetic interplay between the cytochrome pathway and alternative oxidase in both plant and amoeba mitochondria.     

Influence of 50 Hz frequency sinusoidal magnetic field on the blood-brain barrier permeability of diabetic rats

The combined effects of diabetes and a 50 Hz, 5 mT RMS flux density sinusoidal magnetic field for 8 h a day, for 21 consecutive days on the permeation of Evans-blue dye through the blood-brain barrier were studied in male Wistar albino rats. Our results suggest that magnetic field has no effect on the blood-brain barrier permeability in normoglycemic animals, but that diabetic rats are vulnerable to magnetic fields.     

Mate recognition in a freshwater fish: geographical distance, genetic differentiation, and variation in female preference for local over foreign males

It often is assumed that more distant allopatry should reflect reduced rates of contemporary gene flow and/or greater divergence in mate recognition systems. This assumption, however, is rarely tested and may not always be appropriate. Here we investigated female preference for local and foreign males in a morphologically variable Australian freshwater fish, the Pacific blue-eye Pseudomugil signifer. Using a multidisciplinary approach that combined molecular phylogeography with conventional mate choice experiments, we found female blue-eyes spent more time in association with local males only when the alternative was a foreigner from a geographically and genetically more distant population. When offered the choice between two foreign males, females associated more with males from the population that was more closely adjacent to their own. Our results suggest that female preference for local over foreign males in blue-eyes may depend on how genetically and geographically separated populations are from one another.     

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (₁O₂) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of ₁O₂ with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (•OH) adduct of DMPO (DMPO-OH) resulting from ₁O₂-dependent generation of •OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its •OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of •OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of •OH from ₁O₂, and that spin trap-mediated •OH generation hardly occurs with DEPMPO.     

细胞固定化载体比表面的测定

为了衡量细胞固定化载体的性能。基于单分子层吸附理论,利用溶液中亚甲蓝染料在固形物表面的吸附倾向;建立了用于测定细胞固定化载体比表面的“动态染料吸附法”,方法学考察时以PVA-海藻酸钠的混合载体为例,结果表明四批次测量同一载体比表面的结果变异系数为5.5%,测量的载体比表面能精确反映载体内PVA,或是海藻酸钠浓度的变化,说明方法重复性好,灵敏度高,同时讨论了文献中“染料吸附法”测定比表面的不足。     

Supramolecular organization in streptococcal pericellular capsules is based on hyaluronan tertiary structures

Supramolecular organization involving a polyanionic glycan in a bacterial capsule (hyaluronan, HA, in streptococcal capsules) is revealed, for the first time, by electron-histochemical methodology previously used to elucidate ultrastructure in extracellular matrix. Capsular HA filaments thereby revealed closely resemble aligned linear structures demonstrated by similar technology in HA solution. These parallel arrays, spontaneously formed, are based on HA tertiary structures (beta sheet-like) which are stabilized by hydrophobic and hydrogen bonds. HA tertiary structures in aqueous solutions resist shear stress as shown by rheo-NMR. Thus, supramolecular HA wrapping covering many cells probably stabilizes chains of bacteria. Streptococci possibly templated the ordered structures since eukaryotic B6 cell HA did not produce similarly organized envelopes. Supramolecular organization in streptococcal and pneumococcal capsules are compared. Their glycans are very similar but the potential for HA-like tertiary structures is not present in the pneumococcal type 3 polysaccharide and chains of cells are not formed to the same extent by pneumococci. We suggest that the streptococcal capsule exemplifies a simple extracellular matrix analogous to those in animal connective tissues, which contain glycans (chondroitin, keratan, and dermochondan sulfates) of the HA family, capable of undergoing aggregation to similar tertiary structures.     

Microsatellite identification of extrapair sires in a socially monogamous warbler

Few studies of avian mating systems have identified the siresof extrapair young, and hence it has been difficult to determinethe scale at which reproductive interactions occur. For instance,females may be free to copulate with any male in the population(a "global" scale of interactions), or females may be restrictedto copulating only with males on neighboring territories (a"local" scale). The scale of such interactions has importantconsequences for an understanding of the evolutionary causesand consequences of extrapair fertilizations. We used five hypervariable microsatellite loci and multilocus DNA fingerprintingto examine parentage of more than 400 nestling black-throatedblue warblers (Dendroica caerulescens). Extrapair fertilizationswere common, and the microsatellite markers allowed us to identifythe sires for 89% of the young analyzed. Most identified extrapairsires were males on neighboring or nearby territories, andmost nestlings for whom we could not identify a sire came fromterritories at the edge of the study plot. Thus, reproductive interactions appear to be more local than global in this population.Extrapair fertilizations contributed significantly to totalvariation in male reproductive success. However, the standardizedvariance in male reproductive success (0.68-0.74) was not substantiallygreater than that for females (0.53-0.60), and the contributionof extrapair fertilizations (9-14%) was much lower than thecontribution of within-pair fertilizations (75-77%). This suggeststhat the local scale of reproductive interactions may limitvariation in male reproductive success and hence the opportunityfor selection.     

Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NAD(+). When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions.     

Selective recovery of lactate dehydrogenase using affinity foam

Selective isolation of lactate dehydrogenase (LDH) from porcine muscle extract was studied using foam generated from the vigorous stirring of a non-ionic surfactant, Triton X-114 derivatized with Cibacron blue. The cloud point of the surfactant-dye conjugate was higher than that of the native Triton X-114, and also the foam prepared from the affinity surfactant was more rigid taking a longer time to collapse. The equilibrium dissociation constant between pure LDH and surfactant-dye conjugate was 5.0 microM as compared to the value of 2.2 microM for the enzyme and free dye as measured by differential spectroscopy. The isolation procedure involved mixing of the porcine muscle extract with the affinity foam, separating and collapsing the foam, and warming the solution formed to 37 degrees C to yield the surfactant-dye phase and an aqueous phase containing the enzyme. The effect of surfactant concentration and protein load on enzyme recovery and purification was investigated. Under optimal conditions, LDH was quantitatively recovered with high purification factor in a very short time. Both recovery and purification were higher when foam prepared from an equivalent mixture of surfactant-dye conjugate and unmodified surfactant was used. The selectivity of interaction between LDH and detergent-dye conjugate was confirmed by lowered recovery when NADH was included during the binding step.