Monoclonal antibodies against alpha toxin of Clostridium perfringens

Ten distinct monoclonal antibodies (MAbs) against alpha toxin of Clostridium perfringens were produced by the fusion of SP2/O with spleen cells of mice immunized with alpha toxoid, and alpha toxin mixed with or without ethylenediamine-tetraacetate (EDTA). The antibody activity was evaluated by antigen-binding activity in an enzyme linked immunosorbent assay (ELISA), by phospholipase C (PLC)-neutralizing activity using both egg yolk lecithin and p-nitrophenylphosphoryl-choline (PNPPC) hydrolysis reactions and by anti-lethal activity in mice. Since the toxin-neutralizing activities of each MAb were not parallel, it has been suggested that the three biological activities may not be located in the same site in the toxin molecule. This report also describes the development of a simple purification of the toxin by affinity chromatography and a sensitive immunoassay for quantitation of the toxin using the monoclonal antibody.     

Confirmation of the anomeric structure of galacturonic acid in the galacturonosyl-ceramide of Sphingomonas yanoikuyae

The anomeric structure of glycosphingolipids significantly influences their activity to stimulate natural killer T cells. In this study the chemical structure of the galacturonosyl-ceramide in Sphingomonas yanoikuyae, designated GSL-1'sy, was re-examined to prove the anomeric structure of the Dgalacturonic acid (GalA) in the lipid, which was reported as beta-configuration by Naka et al., but was suggested as alpha-configuration in our preliminary study. GSL-1'sy was purified from the bacterial cells with the same procedure as Naka et al. The 1H-NMR analysis of GSL-1'sy revealed that the coupling constant of the anomeric proton of GalA was 3.0 Hz, indicating that GalA in GSL-1'sy is alpha-anomer, the configuration active for the stimulation of natural killer T cells.     

Receptor-like protein kinase 2 (RPK 2) is a novel factor controlling anther development in Arabidopsis thaliana

Receptor-like kinases (RLK) comprise a large gene family within the Arabidopsis genome and play important roles in plant growth and development as well as in hormone and stress responses. Here we report that a leucine-rich repeat receptor-like kinase (LRR-RLK), RECEPTOR-LIKE PROTEIN KINASE2 (RPK2), is a key regulator of anther development in Arabidopsis. Two RPK2 T-DNA insertional mutants (rpk2-1 and rpk2-2) displayed enhanced shoot growth and male sterility due to defects in anther dehiscence and pollen maturation. The rpk2 anthers only developed three cell layers surrounding the male gametophyte: the middle layer was not differentiated from inner secondary parietal cells. Pollen mother cells in rpk2 anthers could undergo meiosis, but subsequent differentiation of microspores was inhibited by tapetum hypertrophy, with most resulting pollen grains exhibiting highly aggregated morphologies. The presence of tetrads and microspores in individual anthers was observed during microspore formation, indicating that the developmental homeostasis of rpk2 anther locules was disrupted. Anther locules were finally crushed without stomium breakage, a phenomenon that was possibly caused by inadequate thickening and lignification of the endothecium. Microarray analyses revealed that many genes encoding metabolic enzymes, including those involved in cell wall metabolism and lignin biosynthesis, were downregulated throughout anther development in rpk2 mutants. RPK2 mRNA was abundant in the tapetum of wild-type anthers during microspore maturation. These results suggest that RPK2 controls tapetal cell fate by triggering subsequent tapetum degradation, and that mutating RPK2 impairs normal pollen maturation and anther dehiscence due to disruption of key metabolic pathways.     

Polyglutamylated Tubulin Binding Protein C1orf96/CSAP Is Involved in Microtubule Stabilization in Mitotic Spindles

The centrosome-associated C1orf96/Centriole, Cilia and Spindle-Associated Protein (CSAP) targets polyglutamylated tubulin in mitotic microtubules (MTs). Loss of CSAP causes critical defects in brain development; however, it is unclear how CSAP association with MTs affects mitosis progression. In this study, we explored the molecular mechanisms of the interaction of CSAP with mitotic spindles. Loss of CSAP caused MT instability in mitotic spindles and resulted in mislocalization of Nuclear protein that associates with the Mitotic Apparatus (NuMA), with defective MT dynamics. Thus, CSAP overload in the spindles caused extensive MT stabilization and recruitment of NuMA. Moreover, MT stabilization by CSAP led to high levels of polyglutamylation on MTs. MT depolymerization by cold or nocodazole treatment was inhibited by CSAP binding. Live-cell imaging analysis suggested that CSAP-dependent MT-stabilization led to centrosome-free MT aster formation immediately upon nuclear envelope breakdown without γ-tubulin. We therefore propose that CSAP associates with MTs around centrosomes to stabilize MTs during mitosis, ensuring proper bipolar spindle formation and maintenance.     

The subunit structure of a major glutathione S-transferase form, MT, in rat testis. Evidence for a heterodimer consisting of subunits with different isoelectric points

A major glutathione S-transferase form (pI 5.7) in rat testis (MT) purified by S-hexyl-glutathione affinity chromatography, followed by chromatofocusing, showed two polypeptide of pI 6.7 (Yn1) and 6.0 (Yn2), having apparently the same molecular mass of 26 kDa on two-dimensional gel electrophoresis. Rechromatofocusing of the MT preparation after 4 M guanidine hydrochloride treatment revealed two additional protein peaks (pI 6.2 and 5.4). These were identified as the two homodimers consisting of the subunits of MT, Yn1Yn1 and Yn2Yn2, respectively. Furthermore, MT could be reconstituted from Yn1Yn1 and Yn2Yn2. These results indicate that MT is a heterodimer, Yn1Yn2, consisting of subunits with very similar molecular masses but different isoelectric points. The Yn1Yn1 form had glutathione S-transferase activities towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene. However, the Yn2Yn2 form had no activity towards any of the substrates examined. N-terminal amino acid sequences of subunits Yn1 and Yn2 revealed differences at two positions in the first 20 residues; the amino acid compositions of these subunits were also similar but not identical, indicating that these two subunits are different in the primary structure. Subunits Yn1 and Yn2 are immunologically related to each other and also to subunits 3 (Yb1) and 4 (Yb2) but they are not identical. These four subunits also showed a high degree of similarity in N-terminal amino acid sequences. Subunits Yn1 and Yn2 seem to belong to the rat GST 3-4 family or class mu. Subunits Yn1 and 4 can make a heterodimer, which is detectable not only in rat testis, but also in the heart, kidney and lung. The Yn1Yn1 form was not detected in the testis, but is present in rat brain [Tsuchida et al. (1987) Eur. J. Biochem. 170, 159-164]. The Yn2Yn2 form seemed to differ from GST 5-5 and may be a new form of rat glutathione S-transferase.     

Structure and organization of the mitochondrial genome of the unicellular red alga Cyanidioschyzon merolae deduced from the complete nucleotide sequence.

The complete nucleotide sequence of the mitochondrial genome of a very primitive unicellular red alga, Cyanidioschyzon merolae , has been determined. The mitochondrial genome of C.merolae contains 34 genes for proteins including unidentified open reading frames (ORFs) (three subunits of cytochrome c oxidase, apocytochrome b protein, three subunits of F1F0-ATPase, seven subunits of NADH ubiquinone oxidoreductase, three subunits of succinate dehydrogenase, four proteins implicated in c-type cytochrome biogenesis, 11 ribosomal subunits and two unidentified open reading frames), three genes for rRNAs and 25 genes for tRNAs. The G+C content of this mitochondrial genome is 27.2%. The genes are encoded on both strands. The genome size is comparatively small for a plant mitochondrial genome (32 211 bp). The mitochondrial genome resembles those of plants in its gene content because it contains several ribosomal protein genes and ORFs shared by other plant mitochondrial genomes. In contrast, it resembles those of animals in the genome organization, because it has very short intergenic regions and no introns. The gene set in this mitochondrial genome is a subset of that of Reclinomonas americana , an amoeboid protozoan. The results suggest that plant mitochondria originate from the same ancestor as other mitochondria and that most genes were lost from the mitochondrial genome at a fairly early stage of the evolution of the plants.     

Predation risk and resource abundance mediate foraging behaviour and intraspecific resource partitioning among consumers in dominance hierarchies

Dominance hierarchies and the resulting unequal resource partitioning among individuals are key mechanisms of population regulation. The strength of dominance hierarchies can be influenced by size‐dependent tradeoffs between foraging and predator avoidance whereby competitively inferior subdominants can access a larger proportion of limiting resources by accepting higher predation risk. Foraging‐predation risk tradeoffs also depend on resource abundance. Yet, few studies have manipulated predation risk and resource abundance simultaneously; consequently, their joint effect on resource partitioning within dominance hierarchies are not well understood. We addressed this gap by measuring behavioural responses of masu salmon Oncorhynchus masou ishikawae to experimental manipulations of predation risk and resource abundance in a natural temperate forest stream. Responses to predation risk depended on body size and social status such that larger fish (often social dominants) exhibited more risk‐averse behaviour (e.g. lower foraging and appearance rates) than smaller subdominants after exposure to a simulated predator. The magnitude of this effect was lower when resources were elevated, indicating that dominant fish accepted a higher predation risk to forage on abundant resources. However, the influence of resource abundance did not extend to the population level, where predation risk altered the distribution of foraging attempts (a proxy for energy intake) from being skewed towards large individuals to being skewed towards small individuals after predator exposure. Our results imply that size‐dependent foraging–predation risk tradeoffs can weaken the strength of dominance hierarchies by allowing competitively inferior subdominants to access resources that would otherwise be monopolized.     

Immunological relationships among subunits of glutathione S-transferases A, AA, B and ligandin and hybrid formation between AA and ligandin by guanidine hydrochloride

Immunological properties of ligandin(Lig) and glutathione S-transferase(GST)-A, -AA and -B were investigated for elucidating their subunit relationships. By using either anti-Lig or -AA antibody, GST-B made a clear common precipitin line with Lig or AA in double immunodiffusion and the activity was inhibited intermediately between Lig and AA, whereas Lig and AA reacted very weakly with antibodies to each other. A hybrid between Lig and AA formed by guanidine hydrochloride treatment was identified immunochemically to be GST-B. GST-A had no immunological relationship with any of other three forms.     

Parthenogenetic development of bovine oocytes treated with ethanol and cytochalasin B after in vitro maturation.

The present study was conducted to investigate the effects of different culture durations (24-36 hr) on bovine oocyte maturation in vitro and the effect of the presence or absence of cumulus cells at the time of treatment to induce parthenogenetic activation (exposure to ethanol and cytochalasin B; CB) (experiment I). The effects of dosage (2.5 or 5.0 micrograms/ml) and incubation time (2.5, 5, or 10 hr) in CB (experiment II) on the subsequent development to the blastocyst stage in vitro was also investigated. In experiment I, cleavage and development to the blastocyst stage were not affected by the presence or absence of cumulus cells at the time of parthenogenetic activation. However, the 24-hr culture duration for in vitro maturation had a significantly lower rate of development to the blastocyst stage than the longer culture durations (27-36 hr). In experiment II, treatment with 5 micrograms/ml CB for 5 hr showed the highest percentage of development to blastocyst in the oocytes matured for both 27 and 30 hr. To determine the viability of the parthenogenetic embryos (morulae and blastocysts), four recipient heifers received two embryos each, and one heifer was found to be pregnant on day 35 following transfer. Although fetal heartbeat was not observed, the subsequent estrus was prolonged in all heifers. The present results demonstrate development of in vitro-matured, parthenogenetically activated bovine embryos up to the preimplantation stage.