The voltage-dependent calcium channel beta subunit contains two stable interacting domains

Voltage-dependent calcium channels selectively enable Ca2+ ion movement through cellular membranes. These multiprotein complexes are involved in a wide spectrum of biological processes such as signal transduction and cellular homeostasis. alpha1 is the membrane pore-forming subunit, whereas beta is an intracellular subunit that binds to alpha1, facilitating and modulating channel function. We have expressed, purified, and characterized recombinant beta3 and beta2a using both biochemical and biophysical methods, including electrophysiology, to better understand the beta family's protein structural and functional correlates. Our results indicate that the beta protein is composed of two distinct domains that associate with one another in a stable manner. The data also suggest that the polypeptide regions outside these domains are not structured when beta is not in complex with the channel. In addition, the beta structural core, comprised of just these two domains without other sequences, binds tightly to the alpha interaction domain (AID) motif, a sequence derived from the alpha1 subunit and the principal anchor site of beta. Domain II is responsible for this binding, but domain I enhances it.     

Histochemical localization of the PBAN receptor in the pheromone gland of Heliothis peltigera

The presence of the pyrokinin (PK)/ Pheromone biosynthesis activating neuropeptide (PBAN) receptor in pheromone gland cells of Heliothis peltigera females was demonstrated, and its spatial distribution in the ovipositor was visualized with two photo-affinity biotinilated ligands: BpaPBAN1-33NH(2) and BpaArg(27)-PBAN28-33NH(2). Light microscopy histological studies revealed that the gland is contained within the inter-segmental membrane (ISM) between the 8th and 9th abdominal segments. The gland was found to be composed of a single layer of columnar epithelial cells positioned under the inter-segmental cuticle. Similar epithelial cells were also found in the dorsal and ventral regions of the 9th abdominal segment. All regions containing the glandular cells bound both ligands, indicating presence of the PK/PBAN receptor. The patterns obtained with both ligands were similar, hinting at the possibility that either both ligands bind to the same receptor, or, that if there are two distinct receptors, their spatial distribution throughout the gland is very similar.     

Large-scale identification of leaf senescence-associated genes

Leaf senescence is a form of programmed cell death, and is believed to involve preferential expression of a specific set of "senescence-associated genes" (SAGs). To decipher the molecular mechanisms and the predicted complex network of regulatory pathways involved in the senescence program, we have carried out a large-scale gene identification study in a reference plant, Arabidopsis thaliana. Using suppression subtractive hybridization, we isolated approximately 800 cDNA clones representing SAGs expressed in senescing leaves. Differential expression was confirmed by Northern blot analysis for 130 non-redundant genes. Over 70 of the identified genes have not previously been shown to participate in the senescence process. SAG-encoded proteins are likely to participate in macromolecule degradation, detoxification of oxidative metabolites, induction of defense mechanisms, and signaling and regulatory events. Temporal expression profiles of selected genes displayed several distinct patterns, from expression at a very early stage, to the terminal phase of the senescence syndrome. Expression of some of the novel SAGs, in response to age, leaf detachment, darkness, and ethylene and cytokinin treatment was compared. The large repertoire of SAGs identified here provides global insights about regulatory, biochemical and cellular events occurring during leaf senescence.     

Comparison of normalization methods for CodeLink Bioarray data

Background  

The quality of microarray data can seriously affect the accuracy of downstream analyses. In order to reduce variability and enhance signal reproducibility in these data, many normalization methods have been proposed and evaluated, most of which are for data obtained from cDNA microarrays and Affymetrix GeneChips. CodeLink Bioarrays are a newly emerged, single-color oligonucleotide microarray platform. To date, there are no reported studies that evaluate normalization methods for CodeLink Bioarrays.     

Technical delivery of myogenic cells through an endocardial injection catheter for myocardial cell implantation

BACKGROUND: The next clinical frontier in the therapeutics of ischemic heart disease may involve the development and delivery of specific molecules and cells into the myocardium. The aim of the present study was to evaluate the efficiency and safety of the MyoStar injection catheter (Biosense-Webster Inc.) that has recently been developed to deliver molecules and cells to the myocardium. The 8 Fr (110 cm length) catheter comprises a navigation sensor with a 27 gauge needle at the distal tip. METHODS: Mouse myogenic cells (C2) were delivered to a tissue culture dish through different modalities: a standard laboratory pipette, a syringe needle (27 gauge) and the injection catheter. The cells were counted and monitored for growth and differentiation in the tissue culture immediately after delivery and two, three and six days later. Cells that were injected through a regular syringe needle or through the injection catheter demonstrated the same capacity to proliferate in tissue culture up to six days. RESULTS: The behavior of the cells in culture (fusion) was identical for the cells delivered to the tissue culture by a pipette or by the injection catheter. CONCLUSION: The results of the present study indicate that delivery of cells through the MyoStar injection catheter is a method with no significant loss or adverse effects to the cells along the path of the catheter. The catheter, which possesses both injection and navigation capabilities, can be used to deliver cell therapy to patients with ischemic heart disease.     

Mutations in genes of Saccharomyces cerevisiae encoding pre-mRNA splicing factors cause cell cycle arrest through activation of the spindle checkpoint

Previous work has identified a group of genes whose products play important roles in two seemingly unrelated processes: cell cycle progression and splicing. The products of these genes show a network of physical and genetic interactions suggestive of the existence of a protein complex, the cell cycle and splicing complex (CSC). Here we analyze the genetic interactions between ISY1, SYF2 and NTC20, three non-essential components of the CSC. We show that mutations in ISY1 cause lethality in the absence of Ntc20p, and that the double mutant isy1Δ syf2Δ shows a temperature-dependent cell cycle arrest. This arrest is due to lower levels of α-tubulin, a protein encoded by TUB1 and TUB3, two intron-containing genes. We show that the low levels of α-tubulin in isy1Δ syf2Δ trigger activation of the spindle checkpoint, causing cell cycle arrest. Thus, our results have uncovered an unexpected role for pre-mRNA splicing in the maintenance of the fidelity of chromosome transmission during cell division.     

Calcium Dependence of Muscarinic Receptor-Mediated Catecholamine Secretion from the Perfused Rat Adrenal Medulla

It had previously been thought that muscarinic cholinergic receptors utilize an influx of extracellular calcium for activation of adrenomedullary catecholamine secretion. However, it has recently been demonstrated that muscarinic receptors on isolated adrenal chromaffin cells can elevate cytosolic free calcium levels in a manner independent of extracellular calcium, presumably by mobilizing intracellular calcium stores. We now demonstrate that muscarinic receptor-mediated catecholamine secretion from perfused rat adrenal glands can occur under conditions of extracellular calcium deprivation that are sufficient to block both nicotine- and electrically stimulated release. Three independent conditions of extracellular calcium deprivation were used: nominally calcium-free perfusion solution (no calcium added), EGTA-containing calcium-free perfusion solution, and perfusion solution containing the calcium channel blocker verapamil. Secretion was evoked from the perfused glands by either transmural electrical stimulation or injection of nicotine or muscarine into the perfusion stream. Each condition of calcium deprivation was able to block nicotine- and electrically stimulated catecholamine release in an interval that left muscarine-evoked release largely unaffected. The above results demonstrate that muscarine-evoked catecholamine secretion from perfused rat adrenal glands can occur in the absence of extracellular calcium, presumably by mobilization of intracellular calcium. The latter may be due to muscarinic receptor-mediated generation of inositol trisphosphate.