Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.     

State-space analysis of time-varying higher-order spike correlation for multiple neural spike train data

Precise spike coordination between the spiking activities of multiple neurons is suggested as an indication of coordinated network activity in active cell assemblies. Spike correlation analysis aims to identify such cooperative network activity by detecting excess spike synchrony in simultaneously recorded multiple neural spike sequences. Cooperative activity is expected to organize dynamically during behavior and cognition; therefore currently available analysis techniques must be extended to enable the estimation of multiple time-varying spike interactions between neurons simultaneously. In particular, new methods must take advantage of the simultaneous observations of multiple neurons by addressing their higher-order dependencies, which cannot be revealed by pairwise analyses alone. In this paper, we develop a method for estimating time-varying spike interactions by means of a state-space analysis. Discretized parallel spike sequences are modeled as multi-variate binary processes using a log-linear model that provides a well-defined measure of higher-order spike correlation in an information geometry framework. We construct a recursive Bayesian filter/smoother for the extraction of spike interaction parameters. This method can simultaneously estimate the dynamic pairwise spike interactions of multiple single neurons, thereby extending the Ising/spin-glass model analysis of multiple neural spike train data to a nonstationary analysis. Furthermore, the method can estimate dynamic higher-order spike interactions. To validate the inclusion of the higher-order terms in the model, we construct an approximation method to assess the goodness-of-fit to spike data. In addition, we formulate a test method for the presence of higher-order spike correlation even in nonstationary spike data, e.g., data from awake behaving animals. The utility of the proposed methods is tested using simulated spike data with known underlying correlation dynamics. Finally, we apply the methods to neural spike data simultaneously recorded from the motor cortex of an awake monkey and demonstrate that the higher-order spike correlation organizes dynamically in relation to a behavioral demand.     

Novel system evaluating in vivo pathogenicity of desmoglein 3-reactive T cell clones using murine pemphigus vulgaris

Autoreactive T cells are thought to be involved in the pathogenesis of autoimmune diseases, but evidence for their direct pathogenicity is almost lacking. Herein we established a unique system for evaluating the in vivo pathogenicity of desmoglein 3 (Dsg3)-reactive T cells at a clonal level in a mouse model for pemphigus vulgaris (PV), an autoimmune blistering disease induced by anti-Dsg3 autoantibodies. Dsg3-reactive CD4(+) T cell lines generated in vitro were adoptively transferred into Rag-2(-/-) mice with primed B cells derived from Dsg3-immunized Dsg3(-/-) mice. Seven of 20 T cell lines induced IgG anti-Dsg3 Ab production and acantholytic blister, a typical disease phenotype, in recipient mice. Comparison of the characteristics between pathogenic and nonpathogenic Dsg3-reactive T cell lines led to the identification of IL-4 and IL-10 as potential factors associated with pathogenicity. Further in vitro analysis showed that IL-4, but not IL-10, promoted IgG anti-Dsg3 Ab production by primed B cells. Additionally, adenoviral expression of soluble IL-4Ralpha in vivo suppressed IgG anti-Dsg3 Ab production and the PV phenotype, indicating a pathogenic role of IL-4. This strategy is useful for evaluating the effector function of autoreactive T cells involved in the pathogenesis of various autoimmune diseases.     

Short-term preservation of mouse oocytes at 5 degrees C.

The temporary preservation of oocytes without freezing would be useful for some experiments. ICR mouse oocytes were kept in a preservation medium under mineral oil for 1, 2, 3, 4 or 7 days at 5 degrees C, and 1 or 2 days at 37 degrees C. In vitro fertilization was attempted on oocytes rinsed with TYH medium after preservation. More than 70% of morphologically normal oocytes were recovered from each preservation group. Fertilization rates of oocytes preserved for 1, 2, 3, 4 or 7 days at 5 degrees C were 69.9, 66.5, 45.3, 26.7 and 8.8% respectively. Fertilization rates of oocytes preserved for 1 or 2 days at 37 degrees C were 9.6 and 1.6%, respectively. Preservation of oocytes at 5 degrees C has some capability as a method of short-term storage without freezing.     

Production of recombinant granulocyte colony-stimulating factor by knocking into the active immunoglobulin heavy chain gene locus in the hybridoma cell line

The hybridoma cell line KM50 originally produces a monoclonal antibody at a concentration of ∼40 mg ml^-1 in ascites. To investigate the possibility to apply this expression system to the production of useful proteins, the cDNA encoding human granulocyte colony-stimulating factor was inserted by homologous recombination into just downstream of the promoter of the active immunoglobulin heavy chain gene of KM50. Site directed integration of targeting DNAs resulted in the disruption of expression of the immunoglobulin heavy chain proteins with a frequency of 1 in 10 ∼ 100 G418-resistance transfectants. One of the monoclonal antibody-deficient transfectants produced25 ng ml^-1 of granulocyte colony-stimulating factor in the supernatant of its cell culture the number of molecules of which corresponds to that of the monoclonal antibody originally produced by KM50. However, when this transfectant was injected intraperitoneally, it produced only a 9 μg ml^-1 concentration of granulocyte colony-stimulating factor in ascites, which is approximately 3 orders of magnitude less than the monoclonal antibody. This method may be applicable to production of other recombinant proteins, although further optimization in the conditions of production would be needed in order to reach much higher yields. This revised version was published online in August 2006 with corrections to the Cover Date.     

A New H-Phosphonate Approach Using N-Unprotected Monomers and Phosphonium Condensing Reagents

Abstract

Oligodexyribonucleotides were synthesized by using N-unprotected H-phosphonate monomers and a phosphonium-type of new condensing reagent. In the present H-phosphonate approach, N-sulfonyloxaziridine derivatives were successfully employed as new reagents for oxidation of the H-phosphonate linkages in the presence of silylating reagents.     

Fungal Infection of Mantis Shrimp (<Emphasis Type="Italic">Oratosquilla oratoria</Emphasis>) Caused by Two Anamorphic Fungi Found in Japan

Two fungal pathogens of the mantis shrimp (Oratosquilla oratoria) in Yamaguchi and Aichi Prefectures, Japan are described as the new species Plectosporium oratosquillae and Acremonium sp. (a member of the Emericellopsis marine clade). Both fungi infect the gills of the mantis shrimp, which become brown or black due to melanization. The former species is characterized by its slow growth on artificial seawater yeast extract peptone glucose (PYGS) agar, pale yellow to pale orange or grayish yellow colonies, short cylindrical solitary phialides with a wavy tip, and one-celled ellipsoidal conidia. Although lacking the two-celled conidia demonstrated by the type species Plectosporium tabacinum, the taxonomic placement of the new species was confirmed by DNA sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA (ITS1, 5.8S rDNA and ITS2). Acremonium sp., the other causal pathogen, differs from P. oratosquillae by its fast growth on PYGS agar, pale orange to salmon-colored colonies, long, slender conidiophores consisting of solitary phialides with tips lacking an undulate outline, and typically cylindrical conidia. Analysis of ITS and β-tubulin gene sequences placed this fungus within the phylogenetically distinct Emericellopsis (anam. Acremonium) marine clade. Various physiological characteristics of both pathogens were also investigated. This is the first report of a fungal infection found on the mantis shrimp in Japan.     

Heterogeneity in the fluidity of intact erythrocyte membrane and its homogenization upon hemolysis

Intact erythrocytes were spin-labeled with various classes of phospholipid label. The ESR spectrum for phosphatidylcholine spin label was distinctly different from those for phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol and phosphatidic acid spin labels. The overall splitting for the former (52.5 G) was markedly larger than those for the others (approx. 47 G), suggesting a more rigid phosphatidylcholine bilayer phase and more fluid phosphatidylethanolamine and phosphatidylserine phases in the erythrocyte membrane. Evidence for asymmetric distribution of phospholipids in the membrane was obtained. Spin-labeled phosphatidylcholine incorporated into erythrocytes was reduced immediately by cystein and Fe³⁺, while the reduction of spin-labeled phosphatidylserine was very slow. The present results therefore suggest asymmetric fluidity in erythrocyte membrane; a more rigid outer layer and a more fluid inner layer. The heterogeneity in the lipid structure was also manifested in the temperature dependence of the fluidity. The overall splitting for phosphatidylcholine spin label showed two inflection points at 18 and 33 °C, while that for phosphatidylserine spin label had only one transition at 30 °C.When the spin-labeled erythrocytes were hemolyzed, the marked difference in the ESR spectra disappeared, indicating homogenization of the heterogeneous fluidity. Mg²⁺ or $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ prevented the hemolysis-induced spectral changes. Ca²⁺ did not prevent the homogenization and acted antagonistically to Mg²⁺. The heterogeneity preservation by Mg²⁺ was nullified by trypsin, pronase or $\text{N-}\text{ethylmaleimide}$ added inside the cell. Some inner proteins may therefore be involved in maintaining the heterogeneous structure. The protecting action of Mg²⁺ was dependent on hemolysis temperature, starting to decrease at 18 °C and vanishing at 40 °C. The present study suggests that the heterogeneity in the fluidity of intact erythrocyte membranes arises from interactions between lipids and proteins in the membrane and also from interactions between the membrane constituents and the inner proteins. Concentration of cholesterol in the outer layer may also partly contribute to the heterogeneity.     

A novel small protein of Bacillus subtilis involved in spore germination and spore coat assembly

Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T(5) of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine-SDS-PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.     

Adenosine A2a Receptor-Mediated Modulation of Striatal Acetylcholine Release In Vivo

Abstract: To determine the functions of striatal adenosine A_2a receptors in vivo, the effects of a selective agonist, 2-[4-(2-carboxyethyl)phenethylamino]-5'- N -ethylcarboxamidoadenosine hydrochloride (CGS 21680), and an antagonist, ( E )-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837), on acetylcholine release were investigated in the striatum of awake freely moving rats using microdialysis. Intracerebroventricular injection of CGS 21680 (10 µg) increased acetylcholine release in striatum and KF17837 (30 mg/kg p.o.) antagonized the CGS 21680-induced acetylcholine elevation. To investigate the contribution of dopaminergic and GABAergic neurons on A_2a receptor-mediated acetylcholine release, the effects of CGS 21680 were studied by using dopamine-depleted rats in the presence or absence of GABA antagonists. In the dopamine-depleted striatum, the intrastriatal application of CGS 21680 (0.3–30 µ M ) increased extracellular acetylcholine, which was significantly greater than that in normal striatum. The CGS 21680-induced elevation of acetylcholine release was still observed in the presence of GABA antagonists bicuculline (30 µ M ) and 2-hydroxysaclofen (100 µ M ) and was similar in both normal and dopamine-depleted striatum. These results suggest that A_2a agonist stimulates acetylcholine release in vivo, and this effect of A_2a agonist is modulated by dopaminergic and GABAergic neurotransmission.