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.     

Apoptosis in multiple myeloma: Therapeutic implications

Apoptosis is the primary means by which most radio- and chemotherapy modalities kill cancer cells, and abnormalities in the apoptotic pathways may contribute to disease pathogenesis of cancer. Multiple Myeloma (MM) is a hematological malignancy which will affect 14,000 new individuals in the United States in 2001 and remains irreversibly fatal despite all available therapies. The current review focuses on the studies of apoptotic and survival signaling pathways in MM cells, which have both identified novel apoptotic and anti-apoptotic proteins and provided targets for novel therapeutics.     

The major subacrosomal occupant of bull spermatozoa is a novel histone H2B variant associated with the forming acrosome during spermiogenesis

Recent studies on the structural composition of mammalian sperm heads have shown a congregate of unidentified proteins occupying the periphery of the mammalian sperm nucleus, forming a layer of condensed cytosol. These proteins are the perinuclear theca (PT) and can be categorized into SDS-soluble and SDS-insoluble components. The present study focused on identifying the major SDS-insoluble PT protein, which we localized to the subacrosomal layer of bovine spermatozoa and cloned by immunoscreening a bull testicular cDNA library. The isolated clones encode a protein of 122 amino acids that bears 67% similarity with histone H2B and contains a predicted histone fold motif. The novel amino terminus of the protein contains a potential bipartite nuclear targeting sequence. Hence, we identified this prominent subacrosomal component as a novel H2B variant, SubH2Bv. Northern blot analyses of SubH2Bv mRNA expression showed that it is testis-specific and is also present in murid testes. Immunocytochemical analysis showed SubH2Bv intimately associates, temporally and spatially, with acrosome formation. While the molecular features of SubH2Bv are common to nuclear proteins, it is never seen developmentally within the nucleus of the spermatid. Considering its developmental and molecular characteristics, we have postulated roles of SubH2Bv in acrosome assembly and acrosome-nuclear docking. Copyright 2001 Academic Press.     

The role of IL-18 in blood-stage immunity against murine malaria Plasmodium yoelii 265 and Plasmodium berghei ANKA

A possible protective role of IL-18 in host defense against blood-stage murine malarial infection was studied in BALB/c mice using a nonlethal strain, Plasmodium yoelii 265, and a lethal strain, Plasmodium berghei ANKA. Infection induced an increase in mRNA expression of IL-18, IL-12p40, IFN-gamma, and TNF-alpha in the case of P. yoelii 265 and an increase of IL-18, IL-12p40, and IFN-gamma in the case of P. berghei ANKA. The timing of mRNA expression of IL-18 in both cases was consistent with a role in the induction of IFN-gamma protein expression. Histological examination of spleen and liver tissues from infected controls treated with PBS showed poor cellular inflammatory reaction, massive necrosis, a large number of infected parasitized RBCs, and severe deposition of hemozoin pigment. In contrast, IL-18-treated infected mice showed massive infiltration of inflammatory cells consisting of mononuclear cells and Kupffer cells, decreased necrosis, and decreased deposition of the pigment hemozoin. Treatment with rIL-18 increased serum IFN-gamma levels in mice infected with both parasites, delayed onset of parasitemia, conferred a protective effect, and thus increased survival rate of infected mice. Administration of neutralizing anti-IL-18 Ab exacerbated infection, impaired host resistance and shortened the mean survival of mice infected with P. berghei ANKA. Furthermore, IL-18 knockout mice were more susceptible to P. berghei ANKA than were wild-type C57BL/6 mice. These data suggest that IL-18 plays a protective role in host defense by enhancing IFN-gamma production during blood-stage infection by murine malaria.     

Immunogenicity and protective efficacy of three DNA vaccines encoding pre-erythrocytic- and erythrocytic-stage antigens of Plasmodium cynomolgi in rhesus monkeys

Although several malaria vaccine candidate antigens have been identified, the most suitable methods for their delivery are still being investigated. In this regard, direct immunization with DNA encoding these vaccine target antigens is an attractive alternative. Here, we have investigated the immune responses to DNA immunization with three major vaccine target antigens: the apical membrane antigen-1 and the 19-kDa C-terminal fragment of merozoite surface protein-1 from the erythrocytic stage, and the thrombospondin-related adhesive protein from the pre-erythrocytic stage of Plasmodium cynomolgi in rhesus monkeys. Antigen-specific antibodies were developed in all the immunized monkeys and peripheral blood mononuclear cells from all immunized monkeys proliferated to different extents upon in vitro stimulation with the corresponding recombinant proteins. The immunized monkeys were challenged with P. cynomolgi sporozoites. All of the immunized animals developed infection but although there was no significant difference between the control and vaccinated animals in terms of pre-patent period, total duration of patency and primary peak parasitemia, the vaccinated animals had significantly lower secondary peak parasitemia than the control animals.     

Mitotic regulation: the fine tuning of separase activity

Mitotic progression requires the dissolution of cohesion between sister chromatids. Cohesion is dissolved by an essential protease known as separase. Separase is highly conserved throughout evolution and is subjected to multiple levels of regulation. Here we discuss recent studies that unravel several key mechanisms for regulating separase activity.     

Genetic and physical mapping of a rice blast resistance locus,Pi-CO39(t), that corresponds to the avirulence gene AVR1-CO39 of Magnaporthe grisea

We have identified, genetically mapped and physically delineated the chromosomal location of a new rice blast resistance locus, designated Pi-CO39(t). This locus confers resistance to Magnaporthe grisea isolates carrying the AVR1-CO39 avirulence locus. The AVR1-CO39 locus is conserved in non-rice (cereals and grasses)-infecting isolates of M. grisea, making Pi-CO39(t) useful for engineering M. grisea resistance in rice and other cereals. The resistance in the rice line CO39 was inherited as a single dominant locus in segregating populations derived from F(2) and F(3) crosses between disease-resistant (CO39) and susceptible (51583) rice genotypes. Microsatellite, RFLP and resistance gene analog (RGA) markers were used to map the Pi-CO39(t) locus to a 1.2-cM interval between the probenazole-responsive ( RPR1) gene (0.2 cM) and RFLP marker S2712 (1.0 cM) on the short arm of rice chromosome 11. RFLP markers G320 and F5003, and resistance gene analogs RGA8, RGA38 and RGACO39 were tightly linked to the Pi-CO39(t) locus (no recombination detected in a sample of ~2400 gametes). A large-insert genomic library of CO39 was constructed in the binary plant transformation vector pCLD04541. A library screen using RGA8, RGA38 and probes derived from the ends of CO39 clones, as well as BAC end probes from the corresponding locus in the rice cv. Nipponbare, resulted in the assembly of three CO39 contigs of 180 kb, 110 kb and 145 kb linked to the Pi-CO39(t) locus. A 650-kb contig was also constructed representing the susceptible locus, pi-CO39(t), in the Nipponbare genome. The two genomes are highly divergent with respect to additions, deletions and translocations at the Pi-CO39(t) locus, as revealed by the presence or absence of mapping markers.