Evidence for tryptophan being a signal molecule that inhibits conidial anastomosis tube fusion during colony initiation in Neurospora crassa

Mycelial interconnectedness achieved by hyphal fusion has been hypothesized to facilitate the distribution and sharing of nutrients between different parts of a mycelium, especially when nutrients are heterogeneously distributed in the environment. However, the link between environmental nutrient availability and hyphal fusion is little understood. Here, we report that amino acids and extracellular pH regulate conidial anastomosis tube (CAT) fusion during colony initiation in Neurospora crassa. Quantitative analyses revealed that low extracellular pH and certain amino acids, particularly tryptophan, inhibit CAT fusion. Conidial germination was also inhibited by tryptophan but this inhibition was mitigated by the presence of other amino acids. This provides evidence for tryptophan having a role as a signal molecule that regulates CAT fusion. Tryptophan acts intracellularly because two amino acid permease mutants (Δmtr and Δaap-20) exhibited resistance against tryptophan-mediated inhibition of CAT fusion. Tryptophan and low pH did not significantly affect vegetative hyphal fusion in mature colonies, indicating that the latter is regulated in a different manner to CAT fusion.     

Arabidopsis ABERRANT PEROXISOME MORPHOLOGY9 is a peroxin that recruits the PEX1-PEX6 complex to peroxisomes

Peroxisomes have pivotal roles in several metabolic processes, such as the detoxification of H₂O₂ and β-oxidation of fatty acids, and their functions are tightly regulated by multiple factors involved in peroxisome biogenesis, including protein transport. This study describes the isolation of an embryonic lethal Arabidopsis thaliana mutant, aberrant peroxisome morphology9 (apem9), which is compromised in protein transport into peroxisomes. The APEM9 gene was found to encode an unknown protein. Compared with apem9 having the nucleotide substitution, the knockdown mutants showed severe defects in peroxisomal functions and plant growth. We showed that expression of APEM9 altered PEROXIN6 (PEX6) subcellular localization from the cytosol to peroxisomes. In addition, we showed that PEX1 and PEX6 comprise a heterooligomer and that this complex was recruited to peroxisomal membranes via protein–protein interactions of APEM9 with PEX6. These findings show that APEM9 functions as an anchoring protein, similar to Pex26 in mammals and Pex15p in yeast. Interestingly, however, the identities of amino acids among these anchoring proteins are quite low. These results indicate that although the association of the PEX1-PEX6 complex with peroxisomal membranes is essential for peroxisomal functions, the protein that anchors this complex evolved uniquely in plants.     

Structure-function analysis of NADE: identification of regions that mediate nerve growth factor-induced apoptosis

Nerve growth factor (NGF) can induce apoptosis in neural cells via activation of the low affinity neurotrophin receptor p75NTR. NADE (p75NTR-associated cell death executor) is a p75NTR-associated protein that mediates apoptosis in response to NGF by interacting with the death domain of p75NTR in 293T, PC12, and nnr5 cells (Mukai, J., Hachiya, T., Shoji-Hoshino, S., Kimura, M. T., Nadano, D., Suvanto, P., Hanaoka, T., Li, Y., Irie, S., Greene, L. A., and Sato, T. A. (2000) J. Biol. Chem. 275, 17566-17570). We performed extensive mutational analysis on NADE, to better characterize its structural and functional features. Truncation of a minimal region, including amino acid residues 41-71 of NADE, was found to be sufficient to induce apoptosis. The designated regulatory region includes the C-terminal amino acid residues (72-112) and is essential for NGF-dependent regulation of NADE-induced apoptosis. Furthermore, the mutants with amino acid substitutions in the leucine-rich nuclear export signal (NES) sequence (residues 90-100) abolished the export of NADE from the nucleus to the cytoplasm. Mutation of the NES also abolished self-association of NADE, its interaction with p75NTR, and NGF-dependent apoptosis. Expression of a fragment of NADE (amino acid residues 81-124) blocked NGF-induced apoptosis in oligodendrocytes, suggesting that this region has a dominant negative effect on NGF/p75NTR-induced apoptosis. These studies identify distinct regions of NADE that are involved in regulating specific functions involved in p75NTR signal transduction.     

Hepatitis C virus infection induces apoptosis through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway

We previously reported that cells harboring the hepatitis C virus (HCV) RNA replicon as well as those expressing HCV NS3/4A exhibited increased sensitivity to suboptimal doses of apoptotic stimuli to undergo mitochondrion-mediated apoptosis (Y. Nomura-Takigawa, et al., J. Gen. Virol. 87:1935-1945, 2006). Little is known, however, about whether or not HCV infection induces apoptosis of the virus-infected cells. In this study, by using the chimeric J6/JFH1 strain of HCV genotype 2a, we demonstrated that HCV infection induced cell death in Huh7.5 cells. The cell death was associated with activation of caspase 3, nuclear translocation of activated caspase 3, and cleavage of DNA repair enzyme poly(ADP-ribose) polymerase, which is known to be an important substrate for activated caspase 3. These results suggest that HCV-induced cell death is, in fact, apoptosis. Moreover, HCV infection activated Bax, a proapoptotic member of the Bcl-2 family, as revealed by its conformational change and its increased accumulation on mitochondrial membranes. Concomitantly, HCV infection induced disruption of mitochondrial transmembrane potential, followed by mitochondrial swelling and release of cytochrome c from mitochondria. HCV infection also caused oxidative stress via increased production of mitochondrial superoxide. On the other hand, HCV infection did not mediate increased expression of glucose-regulated protein 78 (GRP78) or GRP94, which are known as endoplasmic reticulum (ER) stress-induced proteins; this result suggests that ER stress is not primarily involved in HCV-induced apoptosis in our experimental system. Taken together, our present results suggest that HCV infection induces apoptosis of the host cell through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway(s).     

Inbreeding avoidance and female mate choice shape reproductive skew in capuchin monkeys (Cebus capucinus imitator)

Reproductive skew in multimale groups may be determined by the need for alpha males to offer reproductive opportunities as staying incentives to subordinate males (concessions), by the relative fighting ability of the alpha male (tug‐of‐war) or by how easily females can be monopolized (priority‐of‐access). These models have rarely been investigated in species with exceptionally long male tenures, such as white‐faced capuchins, where female mate choice for novel unrelated males may be important in shaping reproductive skew. We investigated reproductive skew in white‐faced capuchins at Sector Santa Rosa, Costa Rica, using 20 years of demographic, behavioural and genetic data. Infant survival and alpha male reproductive success were highest in small multimale groups, which suggests that the presence of subordinate males can be beneficial to the alpha male, in line with the concession model's assumptions. None of the skew models predicted the observed degree of reproductive sharing, and the probability of an alpha male producing offspring was not affected by his relatedness to subordinate males, whether he resided with older subordinate males, whether he was prime aged, the number of males or females in the group or the number of infants conceived within the same month. Instead, the alpha male's probability of producing offspring decreased when he was the sire of the mother, was weak and lacked a well‐established position and had a longer tenure. Because our data best supported the inbreeding avoidance hypothesis and female choice for strong novel mates, these hypotheses should be taken into account in future skew models.     

Novel polyvalent live vaccine against varicella–zoster and mumps virus infections

The varicella–zoster virus (VZV) Oka vaccine strain (vOka) is a highly immunogenic and safe live vaccine that has long been used worldwide. Because its genome is large, making it suitable for inserting foreign genes, vOka is considered a candidate vector for novel polyvalent vaccines. Previously, a recombinant vOka, rvOka‐HN, that expresses mumps virus (MuV) hemagglutinin‐neuraminidase (HN) was generated by the present team. rvOka‐HN induces production of neutralizing antibodies against MuV in guinea pigs. MuV also expresses fusion (F) protein, which is important for inducing neutralizing antibodies, in its viral envelope. To induce a more robust immune response against MuV than that obtained with rvOka‐HN, here an rvOka expressing both HN and F (rvOka‐HN‐F) was generated. However, co‐expression of HN and F caused the infected cells to form syncytia, which reduced virus titers. To reduce the amount of cell fusion, an rvOka expressing HN and a mutant F, F(S195Y) were generated. Almost no syncytia formed among the rvOka‐HN‐F(S195Y)‐infected cells and the growth of rvOka‐HN‐F(S195Y) was similar to that of the original vOka clone. Moreover, replacement of serine 195 with tyrosine had no effect on the immunogenicity of F in mice and guinea pigs. Although obvious augmentation of neutralizing antibody production was not observed after adding F protein to vOka‐HN, the anti‐F antibodies did have neutralizing activity. These data suggest that F protein contributes to induction of immune protection against MuV. Therefore this recombinant virus is a promising candidate vaccine for polyvalent protection against both VZV and MuV.     

Studies on the Biosynthesis of Kanamycins

Incorporation of the radioactive degradation products of kanamycin A or related metabolites into kanamycin A by growing cells of Streptomyces kanamyceticus was examined. ³H-Deoxystreptamine was incorporated into deoxystreptamine moiety of kanamycin, but neither ¹⁴C-3-amino-3-deoxy-d-glucose nor ¹⁴C-6-amino-6-deoxy-d-glucose was incorporated. ³H-Kanamycin A added to medium was modified and inactivated.     

Biological Reduction of Ferric Iron by Iron- and Sulfur-oxidizing Bacteria

B-1625 FA_2β, one of the minor acidic actinomycin congeners produced by Streptomyces antibioticus No. B-1625, was produced selectively on the combined use of sarcosine and ferrous sulfate in a complex medium. The FA_2β was further separated into two components, MeOH- insoluble FA_{2β – 1} and MeOH-soluble FA_{2β – 2}, by methanol treatment. After repeating the MeOH treatment, the purified FA_{2β – 1} was examined as to its physicochemical properties, and subjected to amino acid analysis, and ¹³C-, ¹H-NMR and FAB mass spectral measurements. As a result, the structure of FA_{2β – 1} was deduced to be 2-amino-1-carboxyl-4,6-dimethyl-3-phenoxazone-9- carbonyl-threonyl-valyl-sarcosyl-sarcosyl-N-methylvaline. As much as 1 mg/ml of FA_{2β – 1} showed no antibacterial activity against Gram-negative or Gram-positive bacteria, although FA_{2β – 2} shows antibiotic activity.     

Purification and Characterization of Molecular and Immunological Properties of Rice Ferredoxin-Nitrite Reductase

Ferredoxin-nitrite reductase [EC 1.7.7.1] has been purified to apparent homogeneity from rice (Oryza satira cv. Kinmaze) leaves by a procedure used for the spinach enzyme [S. Ida and B. Mikami, Biochim. Biophys. Acta, 681, 167 (1986)]. The rice enzyme consists of a single polypeptide of % molecular weight of 60,000 with 536 amino acid residues. The enzyme showed nearly identical absorption, circular dichroism, and magnetic circular dichroism spectra to those of the spinach enzyme, indicating the presence of the same prosthetic groups and protein conformation in both enzymes. The apparent Km values for nitrite and methyl viologen were 360 μm and 63 μm, respectively. The pH optimum was 7.6. These kinetic parameters are indistinguishable from those reported for spinach nitrite reductase. Monospecific antiserum against purified rice enzyme cross-reacted with nitrite reductases from a variety of higher plants and some phylogenetically divergent plants. Immunological comparisons indicated the rice enzyme is much more closely related to the other monocot enzymes in antigenic structure than to the dicot enzyme proteins. The results lend further support to our previous study [S. Ida, Plant Sci., 49, 111 (1987)] that spinach ferredoxin-nitrite reductase is serologically more related to the dicot enzymes than to the monocot nitrite reductases. Conspicuous differences between the rice and spinach enzymes were found in their molecular sizes and antigenicity. Relatedness of amino acid compositions of the enzyme proteins is discussed in relation to antigenic properties of ferredoxin-nitrite reductase.