SIMILARITIES OF β-BUNGAROTOXIN AND PHOSPHOLIPASE A2 AND THEIR MECHANISM OF ACTION

Abstract— β-Bungarotoxin, a presynaptic neurotoxin isolated from the venom of Bungarus multicinctus , has been shown to initially cause an increase in the frequency of miniature endplate potentials and subsequently block neuromuscular transmission by inhibiting nerve impulse induced release of acetylcholine. In rat brain synaptosomes it causes a Ca²⁺-dependent release of acetylcholine together, with a strong inhibition of the high affinity choline uptake system. In this report we demonstrate that β-bungarotoxin acts as a phospholipase A₂ (phosphatide 2-acyl hydrolase, EC 3.1.1.4), liberating fatty acids from synaptic membrane phospholipids. It also exhibits a striking similarity in a number of neurochemical properties with that of a purified phospholipase A₂ from Naja naja siamensis. In addition, both agents produce a marked depolarization of synaptosomal preparations as measured by a fluorescent dye. We propose that disruption of the membrane phospholipids by phospholipase activity can lead to depolarization of the synaptosomal preparation which promotes both transmitter release and inhibition of the energy-dependent high affinity choline uptake system. With this decreased supply of choline, the acetylcholine content of the cell would be gradually depleted leading to a decrease in transmission.     

Subunit dependence of Na channel slow inactivation and open channel block in cerebellar neurons

Purkinje and cerebellar nuclear neurons both have Na currents with resurgent kinetics. Previous observations, however, suggest that their Na channels differ in their susceptibility to entering long-lived inactivated states. To compare fast inactivation, slow inactivation, and open-channel block, we recorded voltage-clamped, tetrodotoxin-sensitive Na currents in Purkinje and nuclear neurons acutely isolated from mouse cerebellum. In nuclear neurons, recovery from all inactivated states was slower, and open-channel unblock was less voltage-dependent than in Purkinje cells. To test whether specific subunits contributed to this differential stability of inactivation, experiments were repeated in Na(V)1.6-null (med) mice. In med Purkinje cells, recovery times were prolonged and the voltage dependence of open-channel block was reduced relative to control cells, suggesting that availability of Na(V)1.6 is quickly restored at negative potentials. In med nuclear cells, however, currents were unchanged, suggesting that Na(V)1.6 contributes little to wild-type nuclear cells. Extracellular Na(+) prevented slow inactivation more effectively in Purkinje than in nuclear neurons, consistent with a resilience of Na(V)1.6 to slow inactivation. The tendency of nuclear Na channels to inactivate produced a low availability during trains of spike-like depolarization. Hyperpolarizations that approximated synaptic inhibition effectively recovered channels, suggesting that increases in Na channel availability promote rebound firing after inhibition.     

Modification of Delivery System Enhances MHC Nonrestricted Immunogenicity of V3 Loop Region of HIV-1 gp120

A successful peptide vaccine for AIDS is desired to elicit T-helper and cytotoxic T lymphocyte responses besides neutralizing antibodies. The V3 loop peptide of HIV-1 has been shown to contain the principal neutralizing domain, one of the most immunodominant regions, having both B-cell and T-cell determinants. In this study, the tip of the V3 loop region was mutated from GPGR to GPGQ based on the sequence of Indian isolates (CKRKIHIGPGQAFYT). To further enhance the immunogenicity of this epitope, two delivery systems of immune stimulating complexes (ISCOMs) and liposomes were used to incorporate the peptide. Mice of differing haplotypes, H-2^b, H-2^d, H-2^k and H-2^S, showed no MHC restriction when immunized with these formulations. The IgG levels as assessed by ELISA were found to be significantly higher (P<0.05 to P< 0.001) for even five-fold lower doses of the peptide in ISCOMs and liposomes as compared to the conventional alum-based preparation. The major subtype elicited was IgG2a/IgG2b, suggestive of a Th1-like response for all the formulations. Thus, it would appear that the same peptide incorporated in ISCOMs and liposomes selects a Th1 response and may therefore be important not only for neutralization but also for virus clearance.     

Effects function simulation of residential appliance field exposures

This paper demonstrates the application of effects function analysis to residential magnetic field exposure, focusing on appliance sources and mitigation choices. Residential field exposure time series were synthesized by using a sample of background household field measurements, a model of average daily appliance use, and a small sample of EMDEX data of field exposure from 12 household appliances. Four alternative effects functions (average field strength with or without a threshold, field strength window, sudden field changes) were simulated by using the synthesized time series data for different exposure situations, such as high and low levels of appliance use, simple avoidance, and use of a set of hypothetical “low field” appliances (50% lower fields). In particular, field exposure from the use of bedside clocks and electric blankets was examined. Results demonstrate that the choice of effects function is critical for the ranks of field sources and exposure reduction choices. For the effects function of average field strength with or without a threshold, exposure from background fields dominated exposure from all appliances except for bedside clocks and electric blankets. In the case of the field strength window effects function, the dominant field sources changed with the width of the window. For the effects function based on rapid field changes, appliance use was the major source of exposure. Because of the small sample size of our data set and other simplifications, specific results should be viewed as illustrative. Bioelectromagnetics 18:116–124, 1997. © 1997 Wiley-Liss, Inc.     

Molecular phylogenetics and biogeography of the Neocellia Series of Anopheles mosquitoes in the Oriental Region

Molecular studies of population divergence and speciation across the Oriental Region are sparse, despite the region’s high biodiversity and extensive Pliocene and Pleistocene environmental change. A molecular phylogenetic study of the Neocellia Series of Anopheles mosquitoes was undertaken to identify patterns of diversification across the Oriental Region and to infer the role of Pleistocene and Pliocene climatic change. A robust phylogeny was constructed using CO2 and ND5 mitochondrial genes and ITS2 and D3 nuclear ribosomal markers. Bayesian analysis of mitochondrial genes was used to date divergence events. The repeated contraction and expansion of forest habitat resulting from Pleistocene climatic fluctuations appears to have had a substantial impact on intraspecific diversification, but has not driven speciation within this group. Primarily early to mid Pliocene speciation was detected within the Annularis Group, whereas speciation within the Maculatus and Jamesii Groups occurred during the mid and late Pliocene. Both allopatric divergence driven by late Pliocene environmental changes and ecological adaptation, involving altitudinal replacement and seasonality, are likely to have influenced speciation in the Maculatus Group.     

Knotting the NETs: Analyzing histone modifications in neutrophil extracellular traps

Neutrophil extracellular chromatin traps (NETs) are a recently described mechanism of innate immune responses to bacteria and fungi. Evidence indicates that NETs are induced by inflammation, that they contribute to diverse disease pathologies, and that they associate with bactericidal substances. Genomic DNA is released in NETs, leading to a cell death that has been labeled NETosis. Although NETosis clearly differs from apoptosis, the classical form of cell death, recent experiments indicate a connection between NETosis and autophagy. The regulated deployment of NETs may require covalent modification of histones, the basic DNA-binding proteins that organize chromatin in the cell''s nucleus and within NETs. Histone modification by peptidylarginine deiminase 4 (PAD4) is necessary for NET release. The functions of additional histone modifications, however, remain to be tested.Less than a decade since their discovery, neutrophil extracellular traps (NETs) remain in the headlines. Initially, interest focused on the structure of extracellular NET chromatin and its capacity to capture and damage bacteria. Soon, however, researchers began to see the implications of extracellular chromatin for the development of autoimmune diseases. One quintessential autoimmune disease, systemic lupus erythematosus (SLE), is known to arise together with autoantibodies to DNA and chromatin, although the immediate trigger for the production of these autoantibodies is unclear. A connection between NETs and autoimmunity was made by discovering that histones, a set of proteins that act as a structural harness for DNA in chromatin, are modified by peptidylarginine deiminase 4 (PAD4), an enzyme that converts arginines to citrullines. Researchers had long suspected that autoantigen modifications could provide the initial stimuli in autoimmunity because subtle alterations in a protein''s primary sequence can break tolerance. PAD4 is implicated in the development of rheumatoid arthritis (RA) because the most reliable clinical test for RA uses the detection of anti-citrulline antibodies in the sera of patients.In a sophisticated set of experiments reported in the previous issue of Arthritis Research & Therapy, Liu and colleagues [1] accomplished an extensive inventory of post-translational modifications in NET histones. The researchers induced NETs from human neutrophils, as well as two cell lines that assume neutrophil-like characteristics, and used a panel of 40 commercially available antisera to identify histone modifications that arise in parallel with NETs. Stimuli that were used to elicit NET release also induced histone H3 and H4 citrullination in human neutrophils and the EPRO cell line. However, other modifications such as histone H4 lysine 20 methylation and H4 lysine 16 acetylation showed inconsistent results in neutrophils versus the EPRO cells. To survey histone modifications, Liu and colleagues [1] confronted technical difficulties in that histone amino terminal tails contain the highest concentration of histone modifications yet are also highly susceptible to proteases secreted by activated neutrophils [2,3]. The histone tails act as flexible tethers that organize chromatin into higher-order structures. Interestingly, purified NETs failed to induce an immune response in mice, although a subset of SLE sera reacted strongly with citrullinated histone H3 [1]. Therefore, mechanisms that regulate histone modification deserve further attention.Neeli and colleagues [4] were the first to identify citrullinated histone H3 in NETs, a discovery that was confirmed by others [5]. Neeli and colleagues [4] provided a second important insight, namely that PAD4-citrullinated histone H3 is a reliable marker of inflammation. Thus, it became clear that the release of NETs is not an ''accident'' caused by a barrage of proteases and reactive oxygen species unleashed from neutrophils. Instead, production of NETs requires enzymatic activity and input from neutrophil surface receptors and the cytoskeleton [6]. By analyzing PAD4-deficient mice, Li and colleagues [7] demonstrated that PAD4 is essential for the production of NETs in response to bacterial infections. The regulation of PAD4 activity thus moved to the forefront of the research on NETs.It is now clear that NET release takes advantage of NADPH (nicotinamide adenine dinucleotide phosphate) oxidase and the main granule proteases to trigger and construct the extended chromatin network [3,8]. In addition, myeloperoxidase is found in NETs after their release from the cells, and this enzyme and its products are the main components in NETs that kill bacteria [9]. In a notable study from the labs of Banchereau and Pascual [10], it was reported that SLE neutrophils are poised to undergo NETosis upon stimulation with anti-ribonucleo-protein autoantibodies and that NETs released by these neutrophils contain LL37 and HMGB-1, well-known stimulators of immune responses. In subsequent analyses using sera from patients with connective tissue disease, anti-citrullinated histone antibodies were observed in Felty''s syndrome, a rare disorder that shares serologic features with RA and SLE, whereas such autoantibodies were infrequent in SLE and RA [11]. These findings indicate that the process of NETosis is highly relevant to the development of human autoimmune responses, although a direct cause and effect may not connect the release of NETs to the production of autoantibodies.The detailed characterization of NET histone modifications, as accomplished by Liu and colleagues [1], invites speculations about the possible functions of these modifications. Several questions deserve further study: Will NET histone modifications, such as methylation, acetylation, and citrullination, be found to participate in gene regulation that sets the stage for NET release? Will the primary function of histone modifications turn out to be the decondensation of nuclear chromatin that is required for NETs expand to their optimal size and internal structure? Alternatively, NET histone modifications may serve non-traditional purposes. For example, certain modifications may anchor other NET components such as elastase, LL37, or myeloperoxidase to the chromatin meshwork. Unique modifications in NETs may attract phagocytes and stimulate them to ingest the trapped microorganisms. Other histone modifications may activate or dampen the inflammatory response by acting on innate pattern recognition receptors. The answers to these questions will, no doubt, keep research on NETs in leading immunology and microbiology journals for years to come.     

Methimazole complexes of platinum(II): Synthesis, characterization and redox behavior

A variety of platinum(II) complexes of methimazole (2-mercapto-1-methylimidazole; HImS = neutral form and ImS = thiolate form), coordinated in both thione and thiolate forms, have been isolated by reacting methimazole with [PtCl(terpy)]Cl (terpy = 2,2′:6′,2″ terpyridine), [PtCl₂(bipy)] (bipy = bipyridine), [PtCl₂(o-phen)] (o-phen = o-phenanthroline), [PtCl₂(CH₃CN)₂] and [PtCl₂(COD)] (COD = 1,5-cyclooctadiene). These complexes were characterized by electronic absorption, IR and NMR (¹H, ¹³C, ¹⁹⁵Pt) spectroscopies. Molecular structure of [Pt(bipy)(HImS)₂]Cl₂·3H₂O (3a·3H₂O) has been established by single crystal X-ray crystallography. Platinum thiolate complex, [Pt(ImS)₂(HImS)₂] (5), could be obtained by treatment of [Pt(HImS)₄]Cl₂ with sodium methoxide in methanol. The solution of 5 in organic solvents yielded bi- and tri-nuclear platinum complexes. The effect of diimine ligands on oxidation of methimazole moiety in the complexes has been studied by electrochemical oxidation and pulse radiolytic oxidation employing specific one-electron oxidant, radical.     

Cloning and characterization of fxp, the flexible pilin gene of Aeromonas hydrophila

The flexible pilus of Aeromonas hydrophila is a morphologically and biochemically unique organelle which binds eukaryotic cell surfaces and whose expression is induced by specific physiochemical conditions. fxp, the structural gene coding for the flexible pilus subunit, was localized on a 7.6kb plasmid of A. hydrophila strain AH26. A putative Shine-Dalgarno sequence and -10 and -35 regions were identified, a signal peptide sequence delineated, and the coding sequence compared with other bacterial sequences and found to be unique. Plasmid and chromosomal DNA was prepared from 66 other Aeromonas strains and 12 strains from other bacterial genera and examined by Southern blot hybridization using a labelled fxp oligonucleotide and the 7.6kb plasmid as probes. No hybridizing sequences were identified except in the original strain, AH26. It is proposed that fxp codes for a highly evolved organelle, possibly widely distributed in nature, but that it is carried on a genetic element that is rapidly lost from most strains upon in vitro cultivation and storage.