Properties of immunoaffinity purified 106-kDa Ca2+ release channels from the skeletal sarcoplasmic reticulum.

The sulfhydryl-gated 106-kDa Ca(2+)-release channel (SG-106) was purified by biotin-avidin chromatography from skeletal sarcoplasmic reticulum (SR) vesicles and used as an antigen to raise polyclonal antibodies. Western blots showed that the antisera crossreacted with the antigenic SG-106 and not with SR Ca2+, Mg(2+)-ATPase or with junctional foot proteins (JFPs) (Zaidi et al., 1989, J. Biol. Chem. 264(36), 21, 725-21, 736; 21, 737-21, 747). Polyclonal antibody-affinity columns were used to selectively purify SG-106-kDa proteins which, upon incorporation in planar bilayers, revealed the presence of a cationic channels with properties similar to "native" Ca(2+)-release channels obtained through the fusion of SR vesicles with planar bilayers. In agreement with measurements of Ca2+ release from SR vesicles, sulfhydryl oxidizing and reducing agents (i.e., 2,2'-dithiodipyridine and dithiothreitol) respectively increased and decreased the open-time probability of 106-kDa Ca(2+)-release channels. In contrast with reports on JFPs, ryanodine at 0.5-1 nM increased the open-time probability and at 2-10 nM locked 106-kDa Ca(2+)-release channels in a closed state rather than an open subconductance state. The SG-106 was activated by millimolar ATP, inhibited by millimolar Mg2+, and blocked by micromolar ruthenium red. Adriamycin (2-10 microM) caused a transient activation of SG-106 Ca(2+)-release channels, followed by closure in about 5 min, and intermittent activation to a subconductance state. Polyclonal antibodies used to purify the SG-106 also activated the channel when added to the cis side but not the trans side of the bilayer. Thus, SG-106 channels possess features that are similar to "native" SR Ca(2+)-release channels, are immunologically distinct from JFPs, and interact in seconds with nanomolar ryanodine in planar bilayers.     

Disulfide linkage of biotin identifies a 106-kDa Ca2+ release channel in sarcoplasmic reticulum

Reactive disulfide reagents (RDSs) with a biotin moiety have been synthesized and found to cause Ca2+ release from sarcoplasmic reticulum (SR) vesicles. The RDSs oxidize SH sites on SR proteins via a thiol-disulfide exchange, with the formation of mixed disulfide bonds between SR proteins and biotin. Biotinylated RDSs identified a 106-kDa protein which was purified by biotin-avidin chromatography. Disulfide reducing agents, like dithiothreitol, reverse the effect of RDSs and thus promoted active re-uptake of Ca2+ and dissociated biotin from the labeled protein indicating that biotin was covalently linked to the 106-kDa protein via a disulfide bond. Several lines of evidence indicate that this protein is not Ca2+, Mg2+-ATPase and is not a proteolytic fragment or a subunit of the 400-kDa Ca2+-ryanodine receptor complex (RRC). Monoclonal antibodies against the ATPase did not cross-react with the 106-kDa protein, and polyclonal antibodies against the 106-kDa did not cross-react with either the ATPase or the 400-kDa RRC. RDSs did not label the 400-kDa RRC with biotin. Linear sucrose gradients used to purify the RRC show that the 106-kDa protein migrated throughout 5-20% linear sucrose gradients, including the high sucrose density protein fractions containing 400-kDa RRC. Protease inhibitors diisopropylfluorophosphate used to prevent proteolysis of 400-kDa proteins did not alter the migration of 106-kDa in sucrose gradients nor the patterns of biotin labeling of the 106-kDa protein. Incorporation of highly purified 106-kDa protein (free of RRC) in planar bilayers revealed cationic channels with large Na+ (gNa+ = 375 +/- 15 pS) and Ca2+ (gCa2+ = 107.7 +/- 12 pS) conductances which were activated by micromolar [Ca2+]free or millimolar [ATP] and blocked by micromolar ruthenium red or millimolar [Mg2+]. Thus, the SR contains a sulfhydryl-activated 106-kDa Ca2+ channel with apparently similar characteristics to the 400-kDa "feet" proteins.     

Reactive disulfides trigger Ca2+ release from sarcoplasmic reticulum via an oxidation reaction

Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to the S-S bond such as 2,2'-dithiodipyridine (2,2'-DTDP), 4,4'-dithiodipyridine, and N-succinimidyl 3(2-pyridyldithio)propionate (SPDP) trigger Ca2+ release from sarcoplasmic reticulum (SR) vesicles. They are known to specifically oxidize free SH sites via a thiol-disulfide exchange reaction with the stoichiometric production of thiopyridone. Thus, the formation of a mixed S-S bond between an accessible SH site on an SR protein and a RDS causes large increases in SR Ca2+ permeability. Reducing agents, glutathione (GSH) or dithiothreitol reverse the effect of RDSs and permit rapid re-uptake of Ca2+ by the Ca2+, Mg2+-ATPase. The RDSs, 2,2'-DTDP, 4,4'-dithiodipyridine and SPDP displaced [3H]ryanodine binding to the Ca2+-receptor complex at IC50 values of 7.5 +/- 0.2, 1.5 +/- 0.1, and 15.4 +/- 0.1 microM, respectively. RDSs did not alter the rapid initial phase of Ca2+ uptake by the pump, stimulated ATPase activity, and induced release from passively loaded vesicles with nonactivated pumps; thus they act at a Ca2+ release channel and not at the Ca2+, Mg2+-ATPase. Efflux rates increased in 0.25-1.0 mM [Mg2+]free then decreased in 2-5 mM [Mg2+]free. Adenine nucleotides inhibited the oxidation of SHs on SR protein by RDSs and thus reduced Ca2+ efflux rates. However, once RDSs oxidized these SH sites and opened the Ca2+ release pathway, subsequent additions of nucleotides stimulated Ca2+ efflux. In skinned fibers, 2,2'-dithiodipyridine elicited rapid twitches which were blocked by ruthenium red. These results indicate that RDSs trigger Ca2+ release from SR by oxidizing a critical SH group, and thus provide a method to covalently label the protein(s) involved in causing these changes in Ca2+ permeability.     

Proteome of the phytopathogen <Emphasis Type="Italic">Xanthomonas citri</Emphasis> subsp. <Emphasis Type="Italic">citri</Emphasis>: a global expression profile

Background  

Citrus canker is a disease caused by Xantomonas citri subsp.citri (Xac), and has emerged as one of the major threats to the worldwide citrus crop because it affects all commercial citrus varieties, decreases the production and quality of the fruits and can spread rapidly in citrus growing areas. In this work, the first proteome of Xac was analyzed using two methodologies, two-dimensional liquid chromatography (2D LC) and tandem mass spectrometry (MS/MS).     

Visual acuity in larval zebrafish: behavior and histology

Background

Mating plugs that males place onto the female genital tract are generally assumed to prevent remating with other males. Mating plugs are usually explained as a consequence of male-male competition in multiply mating species. Here, we investigated whether mating plugs also have collateral effects on female fitness. These effects are negative when plugging reduces female mating rate below an optimum. However, plugging may also be positive when plugging prevents excessive forced mating and keeps mating rate closer to a females' optimum. Here, we studied these consequences in the gonochoristic nematode Caenorhabditis remanei. We employed a new CO₂-sedation technique to interrupt matings before or after the production of a plug. We then measured mating rate, attractiveness and offspring number.

Results

The presence of a mating plug did not affect mating rate or attractiveness to roving males. Instead, females with mating plugs produced more offspring than females without copulatory plugs.

Conclusions

Our experiment suggests that plugging might have evolved under male-male competition but represents a poor protection against competing males in our experiment. Even if plugging does not reduce mating rate, our results indicate that females may benefit from being plugged in a different sense than remating prevention.     

Microsatellite variation in Drosophila melanogaster and Drosophila simulans: a reciprocal test of the ascertainment bias hypothesis

Interspecific comparisons of microsatellite loci have repeatedly shown that the loci are longer and more variable in the species from which they are derived (the focal species) than are homologous loci in other (nonfocal) species. There is debate as to whether this is due to directional evolution or to an ascertainment bias during the cloning and locus selection processes. This study tests these hypotheses by performing a reciprocal study. Eighteen perfect dinucleotide microsatellite loci identified from a Drosophila simulans library screen and 18 previously identified in an identical Drosophila melanogaster library screen were used to survey natural populations of each species. No difference between focal and nonfocal species was observed for mean PCR fragment length. However, heterozygosity and number of alleles were significantly higher in the focal species than in the nonfocal species. The most common allele in the Zimbabwe population of both species was sequenced for 31 of the 36 loci. The length of the longest stretch of perfect repeat units is, on average, longer in the focal species than in the non-focal species. There is a positive correlation between the length of the longest stretch of perfect repeats and heterozygosity. The difference in heterozygosity can thus be explained by a reduction in the length of the longest stretch of perfect repeats in the nonfocal species. Furthermore, flanking-sequence length difference was noted between the two species at 58% of the loci sequenced. These data do not support the predictions of the directional-evolution hypothesis; however, consistent with the ascertainment bias hypothesis, the lower variability in nonfocal species is an artifact of the microsatellite cloning and isolation process. Our results also suggest that the magnitude of ascertainment bias for repeat unit length is a function of the microsatellite size distribution in the genomes of different species.      

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.      

Cell adhesion molecules in the early developing mouse retina: Retinal neurons show preferential outgrowth in vitro on L1 but not N-CAM

Both L1 and N-CAM are present on optic axons early in the developing mouse retina and optic nerve. In in vitro assays on substrates of purified cell adhesion molecules cells derived from E13 mouse retinae showed vigorous neurite extension on L1 but not on N-CAM. Although retinal neurons on N-CAM showed only limited attachment to the substrate, they were able to form lamellipodia immediately around the cell perimeter. In contrast, similarly derived cortical cells showed extensive neurite outgrowth on both substrates. Under these culture conditions, nearly all of the L1 and N-CAM present in the cell membrane appeared to be sequestered on the lower surface of the growth cones and neurites, indicating that most of these cell adhesion molecules were involved in homophilic interactions. Our results suggest differential roles for L1 and N-CAM in intitiation and establishment of the optic pathway. © 1994 John Wiley & Sons, Inc.