Recovery of force during postcontractile depression in single Xenopus muscle fibers

This study examined the relationship between force and cytosolic free calcium concentration ([Ca2+]c) in different fiber types from Xenopus before, during, and after cells underwent postcontractile depression (PCD). During a standardized fatigue run, force in the two fast fatiguing (FF) fiber types (types 1 and 2, n = 10) fell more quickly (5.8 vs. 8.1 min) and to a greater degree [0.36 vs. 0.51 of initial (P(o))] than in the slow fatiguing (SF) fiber type (type 3, n = 11). After the initial fatigue run, both FF and SF experienced a drop in force to <15% P(o) (PCD) at a similar time (20.6 vs. 21.4 min). A second stimulation period, undertaken during PCD, produced significant recovery of force in both groups, but significantly more so in SF than FF (64 +/- 7 vs. 29 +/- 2% P(o)). This force recovery during PCD was accompanied by a significant increase in peak [Ca2+]c, particularly in SF. However, despite the significant recovery of force during stimulation while in PCD, the amount of force produced for a given peak [Ca2+]c was significantly lower in both groups during PCD than at any other point in the experiment. A final stimulation period, initiated when all fibers had recovered from PCD, demonstrated a recovery of both force and peak [Ca2+]c in both groups, but this recovery was significantly greater in SF vs. FF. These data demonstrate that with continuous electrical stimulation, it is possible to produce a significant recovery of force production during the normally quiescent period of PCD, but that it occurs with a decreased muscle force production for a given peak [Ca2+]c. This suggests that factors other than structural alterations of the sarcoplasmic reticulum are likely the cause of PCD in these fibers.     

Regulation of contraction and relaxation by membrane potential in cardiac ventricular myocytes

Control of contraction and relaxation by membrane potential was investigated in voltage-clamped guinea pig ventricular myocytes at 37 degrees C. Depolarization initiated phasic contractions, followed by sustained contractions that relaxed with repolarization. Corresponding Ca(2+) transients were observed with fura 2. Sustained responses were ryanodine sensitive and exhibited sigmoidal activation and deactivation relations, with half-maximal voltages near -46 mV, which is characteristic of the voltage-sensitive release mechanism (VSRM) for sarcoplasmic reticulum Ca(2+). Inactivation was not detected. Sustained responses were insensitive to inactivation or block of L-type Ca(2+) current (I(Ca-L)). The voltage dependence of sustained responses was not affected by changes in intracellular or extracellular Na(+) concentration. Furthermore, sustained responses were not inhibited by 2 mM Ni(2+). Thus it is improbable that I(Ca-L) or Na(+)/Ca(2+) exchange generated these sustained responses. However, rapid application of 200 microM tetracaine, which blocks the VSRM, strongly inhibited sustained contractions. Our study indicates that the VSRM includes both a phasic inactivating and a sustained noninactivating component. The sustained component contributes both to initiation and relaxation of contraction.     

Simultaneous measurement of the frequencies of intrachromosomal recombination and chromosome gain using the yeast DEL assay

The yeast DEL assay measures the frequency of intrachromosomal recombination between two partially-deleted his3 alleles on chromosome XV. The his3Delta alleles share approximately 400bp of overlapping homology, and are separated by an intervening LEU2 sequence. Homologous recombination between the his3Delta alleles results in deletion of the intervening LEU2 sequence (DEL), and reversion to histidine prototrophy. In this study we have attempted to further extend the use of the yeast DEL assay to measure the frequency of chromosome XV gain events. Reversion to His(+)Leu(+) in the haploid yeast DEL tester strain RSY6 occurs upon non-disjunction of chromosome XV sister chromatids, coupled with a subsequent DEL event. Here we have tested the ability of the yeast DEL assay to accurately predict the aneugenic potential of the diversely-acting, known or suspected aneugens actinomycin D, benomyl, chloral hydrate, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and methotrexate. Actinomycin D and benomyl strongly induced aneuploidy. EMS and methotrexate modestly induced aneuploidy, while chloral hydrate and MMS failed to illicit any significant induction. In addition, by FACS-analysis of DNA content it was shown that the majority of both spontaneous- and chemically-induced His(+)Leu(+) revertants were heterodiploid. Thus, our results indicate endoreduplication of almost entire chromosome sets as a major mechanism of aneuploidy induction in haploid Saccharomyces cerevisiae.     

Genetic monogamy in blue-headed vireos and a comparison with a sympatric vireo with extrapair paternity

Based on the breeding synchrony hypothesis, we predicted, intwo congeners that nest in simiilar habitat but differ in nestingsynchrony, that blue-headed vireos (Vireo solitarius) wouldhave fewer extrapair fertilizations (EPFs) thaii red-eyed vireos(V. olivaceus EPFs were rare in blue-headed vireos (1/37 nestlings),but common in red-eyed vireos (11/19 nestlings). We studiedthe behavior of blue-headed vireos to determine what factorscould promote genetic monogamy. We found no evidence that malesmate guarded to prevent extrapair copulations from occurring.Males did not follow fertile mates closely when mates left thenest (14–25% of female departures) and, during the egg-layingperiod, males were often alone on the nest (22.3 mm/h). Femaleblue-headed vireos, but not red-eyed vireos, obtain direct benefitsfrom social mates such as nest building and incubation (49.1%of the total), and they assess male quality long before becomingfertile. Female blue-headed vireos spent more time incubatingwhen their mates had low incubation effort. Furthermore, maleincubation effort was positively correlated with nest survivalduring incubation. We discuss the evolution of genetic monogamyand sex role convergence in blue-headed vireos in relation toasynchronous breeding.     

细胞因子基因转导对人乳腺癌细胞MHC抗原及细胞膜糖蛋白表达的影响

为探讨细胞因子基因(人IL-2、IL-6)转导对于肿瘤细胞膜MHC抗原及细胞膜糖蛋白表达调控的影响,本文利用脂质体介导的方法,将含人IL-6、IL-2基因的逆转录病毒载体分别导入人乳腺癌细胞系MCF-7细胞中,采用间接免疫荧光染色流式细胞仪测定法,对基因转导的瘤细胞细胞膜糖蛋白及MHC抗原表达进行测定。结果表明经两种基因修饰的MCF-7细胞MHCⅠ型抗原表达均获得增强,此外,基因转导细胞可程度不同地表现出细胞膜多种糖蛋白表达的变化。提示肿瘤细胞膜抗原及糖蛋白表达的改变可能是细胞因子基因转导影响肿瘤细胞免疫原性的重要结构基础。     

Song ranging by incubating male Blue-headed Vireos: the importance of song representation in repertoires and implications for song delivery patterns and local/foreign dialect discrimination

ABSTRACT.   Mechanisms used by birds to range their distance from singing conspecifics are being debated. In particular, the idea that an incoming song must be in a bird's repertoire for it to be ranged accurately is controversial, but important to our appreciation of the role ranging plays in song evolution. We tested the relation between ranging accuracy and songs in repertoires in playback experiments to male Blue-headed Vireos ( Vireo solitarius ) whose precise locations were known because they were incubating eggs. Males ranged songs heard while incubating and, when their mates relieved them at the nest, flew directly to the silent playback sites, suggesting that they remembered the locations of simulated intruders. Male vireos approached playback sites of local songs, likely in their own repertoires, more precisely than foreign songs recorded 95–645 km from our study site. Songs included in local and foreign playback tapes differed primarily in frequency modulation, but were similar in other measurements. These results support ranging theory as described by Morton (1986) . If the songs within an individual's repertoire are ranged with greater accuracy, we discuss how the stability of neighborhoods becomes a factor as to whether or not selection will favor repertoire sharing in song evolution. As well, singing style is affected by ranging. Because Blue-headed Vireos present their songs in a stereotyped order, a listener can compare ordered sequential changes in signal degradation. Comparing degradation in a sequence of songs adds a temporal element that should result in more accurate ranging of the singer's location.     

Molecular dynamics simulations of a fibrillogenic peptide derived from apolipoprotein C-II

The pathway to amyloid fibril formation in proteins involves specific structural changes leading to the combination of misfolded intermediates into oligomeric assemblies. Recent NMR studies showed the presence of “turns” in amyloid peptides, indicating that turn formation may play an important role in the nucleation of the intramolecular folding and possible assembly of amyloid. Fully solvated all-atom molecular dynamics simulations were used to study the structure and dynamics of the apolipoprotein C-II peptide 56 to 76, associated with the formation of amyloid fibrils. The peptide populated an ensemble of turn structures, stabilized by hydrogen bonds and hydrophobic interactions enabling the formation of a strong hydrophobic core which may provide the conditions required to initiate aggregation. Two competing mechanisms discussed in the literature were observed. This has implications in understanding the mechanism of amyloid formation in not only apoC-II and its fragments, but also in other amyloidogenic peptides.     

Oxidized cholesterol metabolites found in human atherosclerotic lesions promote apolipoprotein C-II amyloid fibril formation

Apolipoprotein amyloid deposits and lipid oxidation products are colocalized in human atherosclerotic tissue. In this study we show that the primary ozonolysis product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (KA), rapidly promotes human apolipoprotein (apo) C-II amyloid fibril formation in vitro. Previous studies show that hydrophobic aldehydes, including KA, modify proteins by the formation of a Schiff base with the lysine epsilon-amino group or N-terminal amino group. High-performance liquid chromatography, mass spectrometry, and proteolysis of KA-modified apoC-II revealed that KA randomly modified six different lysine residues, with primarily one KA attached per apoC-II molecule. Competition experiments showed that an aldehyde scavenging compound partially inhibited the ability of KA to hasten apoC-II fibril formation. Conversely, the acid derivative of KA, lacking the ability to form a Schiff base, accelerated apoC-II fibril formation, albeit to a lesser extent, suggesting that amyloidogenesis triggered by KA involves both covalent and noncovalent mechanisms. The viability of a noncovalent mechanism mediated by KA has been observed previously with alpha-synuclein aggregation, implicated in Parkinson's disease. Electron microscopy demonstrated that fibrils formed in the presence of KA had a similar morphology to native fibrils; however, the isolated KA-apoC-II covalent adducts in the absence of unmodified apoC-II formed fibrillar structures with altered ropelike morphologies. KA-mediated fibril formation by apoC-II was inhibited by the addition of the amine-containing compound hydralazine and the lipid-binding protein apoA-I. These in vitro studies suggest that the oxidized small molecule pool could trigger or hasten the aggregation of apoC-II to form amyloid deposits.