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941.
Petronel Tuluc Bettina Schlick Bernhard E. Flucher Karin Jurkat-Rott 《Biophysical journal》2009,96(1):35-44
The Ca2+ channel α1S subunit (CaV1.1) is the voltage sensor in skeletal muscle excitation-contraction (EC) coupling. Upon membrane depolarization, this sensor rapidly triggers Ca2+ release from internal stores and conducts a slowly activating Ca2+ current. However, this Ca2+ current is not essential for skeletal muscle EC coupling. Here, we identified a CaV1.1 splice variant with greatly distinct current properties. The variant of the CACNA1S gene lacking exon 29 was expressed at low levels in differentiated human and mouse muscle, and up to 80% in myotubes. To test its biophysical properties, we deleted exon 29 in a green fluorescent protein (GFP)-tagged α1S subunit and expressed it in dysgenic (α1S-null) myotubes. GFP-α1SΔ29 was correctly targeted into triads and supported skeletal muscle EC coupling. However, the Ca2+ currents through GFP-α1SΔ29 showed a 30-mV left-shifted voltage dependence of activation and a substantially increased open probability, giving rise to an eightfold increased current density. This robust Ca2+ influx contributed substantially to the depolarization-induced Ca2+ transient that triggers contraction. Moreover, deletion of exon 29 accelerated current kinetics independent of the auxiliary α2δ-1 subunit. Thus, characterizing the CaV1.1Δ29 splice variant revealed the structural bases underlying the specific gating properties of skeletal muscle Ca2+ channels, and it suggests the existence of a distinct mode of EC coupling in developing muscle. 相似文献
942.
Sander Verbrugge Bettina Lechner Günther Woehlke Erwin J.G. Peterman 《Biophysical journal》2009,97(1):173-182
Kinesin-1 motor proteins move along microtubules in repetitive steps of 8 nm at the expense of ATP. To determine nucleotide dwell times during these processive runs, we used a Förster resonance energy transfer method at the single-molecule level that detects nucleotide binding to kinesin motor heads. We show that the fluorescent ATP analog used produces processive motility with kinetic parameters altered <2.5-fold compared with normal ATP. Using our confocal fluorescence kinesin motility assay, we obtained fluorescence intensity time traces that we then analyzed using autocorrelation techniques, yielding a time resolution of ∼1 ms for the intensity fluctuations due to fluorescent nucleotide binding and release. To compare these experimental autocorrelation curves with kinetic models, we used Monte-Carlo simulations. We find that the experimental data can only be described satisfactorily on the basis of models assuming an alternating-site mechanism, thus supporting the view that kinesin's two motor domains hydrolyze ATP and step in a sequential way. 相似文献
943.
Monica Busuioc Katarzyna Mackiewicz Bettina A. Buttaro Patrick J. Piggot 《Journal of bacteriology》2009,191(23):7315-7322
Intracellular polysaccharide (IPS) is accumulated by Streptococcus mutans when the bacteria are grown in excess sugar and can contribute toward the cariogenicity of S. mutans. Here we show that inactivation of the glgA gene (SMU1536), encoding a putative glycogen synthase, prevented accumulation of IPS. IPS is important for the persistence of S. mutans grown in batch culture with excess glucose and then starved of glucose. The IPS was largely used up within 1 day of glucose starvation, and yet survival of the parental strain was extended by at least 15 days beyond that of a glgA mutant; potentially, some feature of IPS metabolism distinct from providing nutrients is important for persistence. IPS was not needed for persistence when sucrose was the carbon source or when mucin was present.Streptococcus mutans is a facultative colonizer of the human dental plaque, the microbial pellicle that covers the surface of the teeth. It is the major etiological agent of dental caries (17). Sugar metabolism is central to the behavior of S. mutans (4, 7). It can use a variety of sugars. The sugars are fermented by glycolysis with production of organic acids, particularly lactic acid (4, 7). In addition to providing energy, sucrose is used to produce extracellular polysaccharides to form the biofilm matrix that aids in the association of S. mutans with the dental plaque. Once the S. mutans biofilm becomes part of the dental plaque, the acidic by-products of sugar fermentation dissolve tooth enamel, eventually resulting in dental caries (17). The presence of sugars in the dental plaque is periodic and reflects the intake of dietary sugars. If there is excess sugar available, in addition to producing organic acids and matrix, intracellular (iodophilic) polysaccharide (IPS; glycogen) is formed.The IPS of S. mutans is a polymer of the glycogen-amylopectin type, with α-(1, 4)- and α-(1, 6)-linked glucose, and is stored as intracellular granules (10). Intracellular glycogen storage reserves in various bacterial species are synthesized from glucose-1-P via ADP-glucose (1). The synthesis involves at least three enzymes: glycogen synthase, glucose-1-phosphate pyrophosphorylase, and branching enzyme. The genes encoding these enzymes are commonly found in a glg operon, although the order of genes differs between species. In two gram-positive species, Bacillus subtilis and Bacillus stearothermophilus, the gene order is glgB-glgC-glgD-glgA-glgP (15, 29): glgA encodes glycogen synthase, glgB encodes glucan branching enzyme, and glgC and glgD encode subunits of glucose-1-phosphate pyrophosphorylase. The glgP gene encodes glycogen phosphorylase, which is unlikely to be involved in glycogen synthesis (29). Genes putatively encoding similar enzymes are present in the same order in the genome of S. mutans (29); they are thought also to form an operon.The IPS can be used as a source of carbohydrate for fermentation upon nutrient depletion (11, 13). In planktonic cultures, IPS reserves are largely consumed within 12 h of the imposition of sugar starvation (11, 13, 32). In S. mutans, IPS utilization may prolong acid production and hence the period of lowered pH of the resting (between meals) plaque, a factor that contributes to the incidence of caries. Indeed, IPS is implicated in dental caries: a mutant that synthesized elevated levels of IPS was hypercariogenic in germfree rats (27). Strains isolated from human carious lesions were nearly all stable IPS producers, whereas most strains from caries-inactive persons were variable IPS producers (13, 33).Since S. mutans deep in the dental plaque may not have access to nutrients because of competition with the bacteria at the surface of the plaque, the bacteria may need to survive longer periods of nutrient starvation. Previous studies in our laboratory showed that S. mutans can survive under sugar starvation conditions, provided that the pH remains above ∼5.5 (22). The presence of spent medium and mucin significantly prolonged survival of sugar-starved biofilms and batch cultures (22; also unpublished observations). Here we examine the role of IPS.The role of IPS (glycogen) in bacterial survival has been tested for several other bacterial species. It was found to extend survival of Aerobacter aerogenes (8) and Escherichia coli (28). Intracellular glycogen was also shown to support the survival of Streptococcus mitis during stationary-phase starvation (32). In contrast, glycogen-rich Sarcina lutea died at a higher rate during starvation than did bacteria without glycogen (2).In order to test the role of IPS in S. mutans survival, we constructed an IPS-deficient mutant by inactivating glgA (GenBank SMU.1536) (http://www.oralgen.lanl.gov/), putatively encoding the glycogen synthase. We also constructed a mutant potentially altered in IPS metabolism by inactivating the putative pullulanase structural gene, pul (SMU.1541). Pullulanases are responsible for hydrolyzing α-(1,6) linkages (and in some cases 1,4 linkages) in pullulan and in other polysaccharides (35) and may be important in determining the branching in IPS and/or affecting the catabolism of IPS. We studied the persistence of bacteria under conditions of sugar limitation and of sugar excess in both batch cultures and biofilms. We found that IPS can play a role in supporting S. mutans persistence in batch cultures but found no role for IPS in survival in biofilms. 相似文献
944.
Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres
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Bettina A Moser Lakxmi Subramanian Ya‐Ting Chang Chiaki Noguchi Eishi Noguchi Toru M Nakamura 《The EMBO journal》2009,28(7):810-820
To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle‐dependent recruitment of telomere‐specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S‐phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase ε (Polε) arrived at telomeres earlier than the lagging strand DNA polymerases α (Polα) and δ (Polδ). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polε, whereas S‐phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polα. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited. 相似文献
945.
Haberland A Zaitsev S Waldöfner N Erdmann B Böttger M Henke W 《Molecular biology reports》2009,36(5):1083-1093
Efficient non-viral vectors for the in vivo siRNA transfer are still being searched for. Comparing the differences of the
structural appearance of siRNA and pDNA one would assume differences in the assembling behaviour between these polyanions
when using polycationic vectors such as nuclear proteins. The spontaneous assembly of nuclear proteins such as histone H1
(H1) with pDNA as polyanion which has intensively been investigated over the last decade, showed a particulate structure of
the resulting complexes. For an efficient in vivo use small almost monomolecular structures are searched for. Using siRNA
as the polyanion might enforce this structural prerequisite lacking unwanted aggregation processes, exploiting the molecular
size of siRNA. We therefore investigated the structure of H1/siRNA complexes. Five commonly used methods characterizing the
resulting assemblies such as retardation gels, static and dynamic light scattering, reduction of ethidium bromide fluorescence,
analytical ultracentrifugation, and electron microscopy were used. From analytical ultracentrifugation we learned that under
physiological salt conditions the siRNA-H1 binding was not cooperative, even though the gel analysis showed disproportionation
which would be an indication for a cooperative binding mode. H1 formed very small and stable complexes with siRNA at a molar
ratio of 1:1 and 1:2. In order to find out if the observed structural appearance of the H1/siRNA complexes is due to unspecific
charge effects only or to special features of H1, polylysine was included in the study. Low molecular weight polylysine (K16) showed also non-cooperative binding with siRNA. 相似文献
946.
947.
948.
Pekka Santtila Anna-Lotta Högbacka Patrick Jern Ada Johansson Markus Varjonen Katarina Witting Bettina von der Pahlen N. Kenneth Sandnabba 《Evolution and human behavior》2009,30(1):58-65
The genetic background of male homosexuality presents an evolutionary paradox, since homosexuality could be considered a reproductive disadvantage. We tested E.M. Miller's (2000) balanced polymorphism explanation, which states that alleles partially preventing androgenization in male fetuses during pregnancy would be associated with a homosexual orientation. Having all the alleles produces homosexuality, while heterosexual carriers with only a few of these alleles instead have a reproductive advantage; that is, they have more traits, which, by controlling for excessive aggressiveness and psychopathy, make them more attractive mates. Pairs of brothers were used to test these assumptions. If homosexuality is due to having all the androgenization-preventing alleles, then heterosexual men with homosexual brothers are more likely to also have some of the these alleles compared to heterosexual men with heterosexual brothers. These two categories were compared on variables related both directly and indirectly to reproductive success, with heterosexual men with a homosexual brother hypothesized to have an advantage on the variables. However, no statistically significant findings in support of the theory were detected. The results were discussed together with alternative explanations. 相似文献
949.
950.
Aims: To evaluate the potential role of fungal community structure in predisposing Kenyan maize to severe aflatoxin contamination by contrasting aflatoxin‐producing fungi resident in the region with repeated outbreaks of lethal aflatoxicosis to those in regions without a history of aflatoxicosis. Methods and Results: Fungi belonging to Aspergillus section Flavi were isolated from maize samples from three Kenyan provinces between 2004 and 2006. Frequencies of identified strains and aflatoxin‐producing abilities were assessed, and the data were analysed by statistical means. Most aflatoxin‐producing fungi belonged to Aspergillus flavus. The two major morphotypes of A. flavus varied greatly between provinces, with the S strain dominant in both soil and maize within aflatoxicosis outbreak regions and the L strain dominant in nonoutbreak regions. Conclusions: Aspergillus community structure is an important factor in the development of aflatoxins in maize in Kenya and, as such, is a major contributor to the development of aflatoxicosis in the Eastern Province. Significance and Impact of the Study: Since 1982, deaths caused by aflatoxin‐contaminated maize have repeatedly occurred in the Eastern Province of Kenya. The current study characterized an unusual fungal community structure associated with the lethal contamination events. The results will be helpful in developing aflatoxin management practices to prevent future outbreaks in Kenya. 相似文献