Nonselective currents and channels in plasma membranes of protoplasts from coats of developing seeds of bean

In developing bean (Phaseolus vulgaris) seeds, phloem-imported nutrients move in the symplast from sieve elements to the ground parenchyma cells where they are transported across the plasma membrane into the seed apoplast. To study the mechanisms underlying this transport, channel currents in ground parenchyma protoplasts were characterized using patch clamp. A fast-activating outward current was found in all protoplasts, whereas a slowly activating outward current was observed in approximately 25% of protoplasts. The two currents had low selectivity for univalent cations, but the slow current was more selective for K(+) over Cl(-) (P(K):P(Cl) = 3.6-4.2) than the fast current (P(K):P(Cl) = 1.8-2.5) and also displayed Ca(2+) selectivity. The slow current was blocked by Ba(2+), whereas both currents were blocked by Gd(3+) and La(3+). Efflux of K(+) from seed coat halves was inhibited 25% by Gd(3+) and La(3+) but was stimulated by Ba(2+) and Cs(+), suggesting that only the fast current may be a component in the pathway for K(+) release. An "instantaneous" inward current observed in all protoplasts exhibited similar pharmacology and permeability for univalent cations to the fast outward current. In outside-out patches, two classes of depolarization-activated cation-selective channels were observed: one slowly activating of low conductance (determined from nonstationary noise to be 2.4 pS) and another with conductances 10-fold higher. Both channels occurred at high density. The higher conductance channel in 10 mM KCl had P(K):P(Cl) = 2.8. Such nonselective channels in the seed coat ground parenchyma cell could function to allow some of the efflux of phloem-imported univalent ions into the seed apoplast.     

Radiotherapy: effects on expanded skin

Although the combination of radiation and tissue expansion has been associated with a significant rate of complications, the specific pathophysiology has yet to be clearly elucidated. The objective of this study was to develop a model to identify and examine specific histologic changes associated with tissue expansion and irradiation. Rectangular 50-cc silicone tissue expanders were placed subcutaneously over the midline dorsum of 18 adult New Zealand white rabbits. Preoperative radiographic dosimetry demonstrated that the radiation portal was away from vital intraabdominal structures. The expanders were inflated with 10 cc of saline every other day for a total of 80 cc. Expanders were left in place for 2 to 3 weeks to allow fibrovascular capsule formation. The rabbits were then divided into three groups (six rabbits per group), each receiving one of three nonfractionated doses of radiation (20, 25, or 35 Gy). Half of the expanded skin was irradiated using a single dose, and the other half served as a nonirradiated control. Capsules and skin were harvested 6 weeks after the delivery of radiation, allowing the beginning of chronic radiation changes to occur. Using hematoxylin and eosin staining, histomorphometric analysis was performed. The data were analyzed using Student's test. Although irradiation did not affect dermal thickness, it did cause a statistically significant increase in epidermal thickness. At 20, 25, and 35 Gy the increase in epidermal thickness was 43, 90, and 130 percent, respectively. Although significant epidermal changes could be identified, capsular and dermal alterations were not evident. Further studies evaluating the long-term effects of alterations in capsular formation caused by radiation may be required.     

Signaling microdomains define the specificity of receptor-mediated InsP(3) pathways in neurons

M(1) muscarinic (M(1)AChRs) and B(2) bradykinin (B(2)Rs) receptors are two PLCbeta-coupled receptors that mobilize Ca(2+) in nonexcitable cells. In many neurons, however, B(2)Rs but not M(1)AChRs mobilize intracellular Ca(2+). We have studied the membrane organization and dynamics underlying this coupling specificity by using Trp channels as biosensors for real-time detection of PLCbeta products. We found that, in sympathetic neurons, although both receptors rapidly produced DAG and InsP(3) as messengers, only InsP(3) formed by B(2)Rs has the ability to activate IP(3)Rs. This exclusive coupling results from spatially restricted complexes linking B(2)Rs to IP(3)Rs, a missing partnership for M(1)AChRs. These complexes allow fast and localized rises of InsP(3), necessary to activate the low-affinity neuronal IP(3)R. Thus, these signaling microdomains are of critical importance for the induction of selective responses, discriminating proinflammatory information associated with B(2)Rs from cholinergic neurotransmission.     

Junctional signaling microdomains: bridging the gap between the neuronal cell surface and Ca2+ stores

Growing evidence suggests that plasma membranes are locally differentiated into microdomains that are important interaction sites for organization of signaling molecules. These signaling microdomains create local conditions that enhance molecular interactions, excluding others, thereby ensuring speed, spatial localization, and specificity of signal transduction. With the special emphasis on InsP(3) and Ca(2+) signaling pathways, we will discuss here the evolving concept of signaling microdomains that provide a key framework for understanding the differential regulation of many cellular target proteins.     

VPAC(1) receptors have different agonist efficacy profiles on membrane and intact cells

The vasoactive intestinal peptide receptor VPAC(1) is preferentially coupled to G(alpha s) protein but also increases [Ca(2+)](i) through interaction with G(alpha i)/G(alpha q) protein. We evaluated a panel of full, partial and null agonists for their capability to stimulate adenylate cyclase activity in both intact cells and membrane and [Ca(2+)](i) in intact cells transfected with the reporter gene aequorin. In intact cells, the agonists efficacy for cAMP and calcium increase were well, but not linearly correlated: VPAC(1) receptors activated G(alpha s) protein more efficiently but with the same pharmacological profile as the other G proteins. In contrast, there was a difference between cAMP increase in intact and broken cell membranes: EC(50) values were generally lower in intact cells whereas the efficacy was higher. There was, however, no correlation between the shift in the EC(50) value and the intrinsic activity. Of interest, the (4-28) fragment, a reported antagonist on cell membrane, was a full agonist in intact cells. We concluded that the active states of the VPAC(1) receptor resulting from the coupling to different effector are undistinguishable by the VIP analogs tested but that receptor properties are different when evaluated in intact cells or cell membranes.     

Characterization of Burkholderia pseudomallei infection and identification of novel virulence factors using a Caenorhabditis elegans host system

The environmental saphrophyte Burkholderia pseudomallei is the causative agent of melioidosis, a systemic, potentially life-threatening condition endemic to many parts of south-east Asia and northern Australia. We have used the soil nematode Caenorhabditis elegans as a model host to characterize the mechanisms by which this bacterium mounts a successful infection. We find that C. elegans is susceptible to a broad range of Burkholderia species, and that the virulence mechanisms used by this pathogen to kill nematodes may be similar to those used to infect mammals. We also find that the specific dynamics of the C. elegans-B. pseudomallei host-pathogen interaction can be highly influenced by environmental factors, and that nematode killing results at least in part from the presence of a diffusible toxin. Finally, by screening for bacterial mutants attenuated in their ability to kill C. elegans, we genetically identify several new potential virulence factors in B. pseudomallei. The use of C. elegans as a model host should greatly facilitate future investigations into how B. pseudomallei can interact with host organisms.     

Identification,cloning, purification,and enzymatic characterization of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate synthase

The enzyme encoded by Rv2682c in Mycobacterium tuberculosis is a functional 1-deoxy-D-xylulose 5-phosphate synthase (DXS), suggesting that the pathogen utilizes the mevalonate-independent pathway for isopentenyl diphosphate and subsequent polyprenyl phosphate synthesis. These key precursors are vital in the biosynthesis of many essential aspects of the mycobacterial cell wall. Rv2682c encodes the conserved DRAG sequence that has been proposed as a signature motif for DXSs and also all 13 conserved amino acid residues thought to be important to the function of transketolase enzymes. Recombinant Rv2682c is capable of utilizing glyceraldehyde 3-phosphate and erythrose 4-phosphate as well as D- and L-glyceraldehyde as aldose substrates. The enzyme has K(m) values of 40 microM, 6.1 microM, 5.6 mM, and 4.5 mM for pyruvate, D-glyceraldehyde 3-phosphate, D-glyceraldehyde, and L-glyceradehyde, respectively. Rv2682c has an absolute requirement for divalent cation and thiamin diphosphate as cofactors. The K(d) (thiamin diphosphate )for the native M. tuberculosis DXS activity partially purified from M. tuberculosis cytosol is 1 microM in the presence of Mg(2+).     

Synthesis,radiolabeling and preliminary biological evaluation of radiolabeled 5-methyl-6-nitroquipazine,a potential radioligand for the serotonin transporter

5-Methyl-6-nitroquipazine, a novel analogue of the potent and selective serotonin transporter inhibitor 6-nitroquipazine was synthesized and radiolabeled with tritium and the positron emitter carbon-11. [³H]5-methyl-6-nitroquipazine was found to have a K_d=51±7 pM. The high affinity and the facile labeling of [¹¹C]5-methyl-6-nitroquipazine makes it a promising radioligand for visualization of the serotonin transporter with positron emission tomography.