Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.     

BTK-2, a new inhibitor of the Kv1.1 potassium channel purified from Indian scorpion Buthus tamulus

A novel inhibitor of voltage-gated potassium channel was isolated and purified to homogeneity from the venom of the red scorpion Buthus tamulus. The primary sequence of this toxin, named BTK-2, as determined by peptide sequencing shows that it has 32 amino acid residues with six conserved cysteines. The molecular weight of the toxin was found to be 3452 Da. It was found to block the human potassium channel hKv1.1 (IC(50)=4.6 microM). BTK-2 shows 40-70% sequence similarity to the family of the short-chain toxins that specifically block potassium channels. Multiple sequence alignment helps to categorize the toxin in the ninth subfamily of the K+ channel blockers. The modeled structure of BTK-2 shows an alpha/beta scaffold similar to those of the other short scorpion toxins. Comparative analysis of the structure with those of the other toxins helps to identify the possible structure-function relationship that leads to the difference in the specificity of BTK-2 from that of the other scorpion toxins. The toxin can also be used to study the assembly of the hKv1.1 channel.     

Comparison of three rat models of cerebral vasospasm

A substantial number of rat models have been used to research subarachnoid hemorrhage-induced cerebral vasospasm; however, controversy exists regarding which method of selection is appropriate for this species. This study was designed to provide extensive information about the three most popular subarachnoid hemorrhage rat models: the endovascular puncture model, the single-hemorrhage model, and the double-hemorrhage model. In this study, the basilar artery and posterior communicating artery were chosen for histopathological examination and morphometric analysis. Both the endovascular puncture model and single-hemorrhage model developed significant degrees of vasospasm, which were less severe when compared with the double-hemorrhage model. The endovascular puncture model and double-hemorrhage model both developed more vasospasms in the posterior communicating artery than in the basilar artery. The endovascular puncture model has a markedly high mortality rate and high variability in bleeding volume. Overall, the present study showed that the double-hemorrhage model in rats is a more suitable tool with which to investigate mechanism and therapeutic approaches because it accurately correlates with the time courses for vasospasm in humans.     

Asp1080 upstream of the calmodulin-binding domain is critical for autoinhibition of hPMCA4b

The role of the plasma membrane Ca(2+) pump (PMCA) is to remove excess Ca(2+) from the cytosol to maintain low intracellular Ca(2+) levels. Asp(1080) lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, A. (2002) J. Biol. Chem. 277, 6822-6829). Asp(1080) had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp(1080) to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp(1080) mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp(1077) had no effect on basal activity or calmodulin activation. We propose that the conserved Asp(1080), even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.     

GD3 recruits reactive oxygen species to induce cell proliferation and apoptosis in human aortic smooth muscle cells

Sialic acid containing glycosphingolipids (gangliosides) are expressed on the surface of all mammalian cells and have been implicated in regulating various biological phenomena; however, the detailed signaling mechanisms involved in this process are not known. We report here a novel aspect of disialoganglioside, GD3-mediated regulation of cell proliferation and cell death via the recruitment of reactive oxygen species (ROS). A low concentration (2.5-10 microm) of GD3, incubated with human aortic smooth muscle cells for a short period of time (10-30 min), stimulates superoxide generation via the activation of both NADPH oxidase and NADH oxidase activity. This leads to downstream signaling leading to cell proliferation and apoptosis. However, [(3)H]GD3 incubated with the cells under such conditions was found in a trypsin-sensitive fraction that was separable from endogenous GD3. The exact mechanism causing ROS generation and downstream signaling remains to be elucidated. The uptake of GD3 was accompanied by a 2.5-fold stimulation in the activity of mitogen-activated protein (MAP) kinase and 5-fold stimulation in cell proliferation. Preincubation of cells with membrane-permeable antioxidants, pyrrolidine dithiocarbamate, and N-acetylcysteine abrogated the superoxide generation and cell proliferation. In contrast, at higher concentrations (50-200 microm) GD3 inhibited the generation of superoxides but markedly stimulated the generation of nitric oxide (NO) (10-fold compared with control). This in turn stimulated mitochondrial cytochrome c release and intrachromosomal DNA fragmentation, which lead to apoptosis. In sum, at a low concentration, GD3 recruits superoxides to activate p44 MAPK and stimulates cell proliferation. In contrast, at high concentrations GD3 recruits nitric oxide to scavenge superoxide radicals that triggered signaling events that led to apoptosis. These observations might have relevance in regard to the potential role of GD3 in aortic smooth muscle cell proliferation and apoptosis that may contribute to plaque rupture in atherosclerosis.     

Effect of low level exposure of lead and cadmium on hepatic estradiol metabolism in female rats

Toxic effect of metal cations on female reproduction and gonadal functions was studied. Adult synchronized female rats were treated intraperitoneally with lead acetate and cadmium acetate separately and in combination (0.025, 0.05 and 0.1 mg/kg body wt) for 15 days. The metabolizing enzymes (17beta-hydroxy steroid oxidoreductase and UDP glucoronyl transferase) activities decreased with increasing dose showing significant change compared to control. Also, significant decrease in cytochrome P450 (CYP450) content was found after the treatment. Displacement of zinc bound to metallothionein was more in cadmium treated rats compared to other groups. In all these parameters, treatment in combination of lead and cadmium showed intermediate results indicating some kind of competition between the two metals. But the histological studies showed that combined treatment caused more cytotoxic effect than cadmium and lead alone. These results indicated that metal cations tested did have a direct inhibitory effect on metabolizing enzyme activities.     

The Th1-specific costimulatory molecule,m150, is a posttranslational isoform of lysosome-associated membrane protein-1

In an earlier report, we had shown a 150-kDa protein termed as M150, isolated from the surface of activated macrophages, to possess costimulatory activity for CD4(+) T cells. Significantly, this protein was found to specifically elicit Th1 responses. In this study, we characterize M150, which belongs to a unique subset of the lysosome-associated membrane protein-1 glycoprotein. Interestingly, the costimulatory activity of M150 depends on its posttranslational modification, which has a distinct glycosylation pattern restricted to macrophages. Furthermore, it has been demonstrated that in addition to stimulating Th1-specific responses, M150 is also capable of driving differentiation of naive CD4(+) T cells into the Th1 subset. This altered posttranslational modification of housekeeping protein appears to represent a novel pathway by which APCs can additionally regulate T cell responses.     

Differential action of iodine on mitochondria from human tumoral- and extra-tumoral tissue in inducing the release of apoptogenic proteins

Iodide is actively concentrated in the thyroid gland for thyroid hormone biosynthesis. Excess iodine has been observed to induce apoptosis in thyrocytes and mammary cells. The mechanism of iodine induced apoptosis is poorly understood. Among various cell organelles, mitochondria is known to provide conducive environment for the organification of iodine, i.e. iodination of different proteins. Mitochondria also play a central role in execution of apoptosis. To study the role of mitochondria in iodine induced apoptosis, we investigated the direct interaction of iodine and human breast mitochondria vis-a-vis its role in the initiation of apoptosis in vitro. We observed that mitochondria isolated from the tumor (TT) and extra-tumoral tissue (ET) of human breast display significant uptake of iodine. Mitochondrial proteins were observed to be predominantly iodinated in ET but not in TT mitochondria. Treatment with iodine showed an increase in mitochondrial permeability transition of TT and decrease in ET. Iodine induced released factor(s) other than cytochrome c from tumor mitochondria initiate(s) apoptosis in vitro, while those from ET mitochondria were non-apoptogenic in nature. To our knowledge, this is first report demonstrating that iodine acts differentially on mitochondria of tumor and extratumoral origin to release apoptogenic proteins from TT and has a protective effect on ET.     

Defining a pathway of communication from the C-terminal peptide binding domain to the N-terminal ATPase domain in a AAA protein

AAA proteins remodel other proteins to affect a multitude of biological processes. Their power to remodel substrates must lie in their capacity to couple substrate binding to conformational changes via cycles of nucleotide binding and hydrolysis, but these relationships have not yet been deciphered for any member. We report that when one AAA protein, Hsp104, engages polypeptide at the C-terminal peptide-binding region, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2) drives a conformational change in the middle region. This, in turn, drives ATP hydrolysis in the N-terminal ATPase domain (NBD1). This interdomain communication pathway can be blocked by mutation in the middle region or bypassed by antibodies that bind there, demonstrating the crucial role this region plays in transducing signals from one end of the molecule to the other.