Valentin Sergeevich Kirpichnikov: on the 100th anniversary of his birth (1908-1991)

Valentin Sergeevich Kirpichnikov is an outstanding Russian biologist, geneticist, and evolutionist. In his work, the true interest to the theory of evolution was harmoniously combined with long-term successful research into fish genetics and breeding. In 1987, Kirpichnikov published a fundamental treatise entitled "Fish Genetics and Breeding," which was immediately translated into English, German, and Japanese and still remains the most comprehensive handbook in this field. The authority of Kirpichnikov was confirmed by his election to the International Association of Aquaculture and an expert of the leading international organization on food and agriculture, FAO, with the United Nations. During the hard years in the history of Soviet biology, the courage and fidelity to the principles in defending the scientific biology and opposing Lysenko's obscurantism won Valentin Sergeevich the deserved name of a "knight of science". His mode of speech at the seminars and in discussions was unhurried, calm, and fitting. Yet especially important moments supplemented his baritone with hard metal modulations, which revealed the concealed passion and conviction in the defended truth.     

Altered behaviour and reduced survival of juvenile olive flounder, Paralichthys olivaceus, infected by an invasive monogenean, Neoheterobothrium hirame

Neoheterobothriumhirame is a blood feeding monogenean of olive flounder Paralichthys olivaceus. The parasite was first reported in the mid-1990s from the Sea of Japan and became epidemic within cultured and wild flounder populations after several years. Infected fish often suffer from severe anaemia and thus the parasite is thought to have played an important role in the recent depletion of flounder populations in some areas of Japan. However, the causal mechanism underlying the parasite epidemic and decreases in host populations is unclear because apparently N. hirame infection is not fatal to the host. Here, we tested the hypothesis that N. hirame indirectly reduces the survival of wild juvenile flounder by altering their behaviour and making them more susceptible to predation. We conducted a series of experiments to compare behaviours and predation susceptibility between experimentally infected juvenile P. olivaceus and uninfected fish. Results showed that N. hirame infection increases the activity level, alters diel activity and has negative effects on burrowing performance and swimming endurance. When juvenile flounder cohabitated with predators, the survival rate of infected juveniles was approximately 25% less than that of uninfected fish. We believe this is the first empirical evidence linking N. hirame infection to death of the host through predation. Consequences of N. hirame-induced behavioural change for the survival of juvenile flounder in the wild are discussed. We conclude that recent outbreaks of N. hirame are likely to have been a key factor in the decline of flounder populations in Japan.     

Manduca sexta prothoracicotropic hormone: evidence for a role beyond steroidogenesis

Prothoracicotropic hormone (PTTH) is a homodimeric brain peptide hormone that positively regulates the production of ecdysteroids by the prothoracic gland of Lepidoptera and probably other insects. PTTH was first purified from heads of adult domestic silkworms, Bombyx mori. Prothoracic glands of Bombyx and Manduca sexta undergo apoptosis well before the adult stage is reached, raising the recurring question of PTTH function at these later stages. Because Bombyx has been domesticated for thousands of years, the possibility exists that the presence of PTTH in adult animals is an accidental result of domestication for silk production. In contrast, Manduca has been raised in the laboratory for only five or six decades. The present study found that Manduca brains contain PTTH at all stages examined post‐prothoracic gland apoptosis, i.e., pharate adult and adult life, and that PTTH‐dependent changes in protein phosphorylation and protein synthesis were observed in several reproductive and reproduction‐associated organs. The data indicate that PTTH indeed plays a role in non‐steroidogenic tissues and suggest possible future avenues for determining which cellular processes are being so regulated. © 2009 Wiley Periodicals, Inc.     

The ITGAV rs3738919 variant and susceptibility to rheumatoid arthritis in four Caucasian sample sets

Introduction

Angiogenesis is an important process in the development of destructive synovial pannus in rheumatoid arthritis (RA). The ITGAV +gene encodes a cell cycle-associated antigen, integrin ανβ 3, which plays a role in RA angiogenesis. Previously, two independent studies identified an association between the major allele of the ITGAV single-nucleotide polymorphism (SNP) rs3738919 and RA. We therefore tested this association in an independent study using New Zealand (NZ) and Oxford (UK) RA case control samples.

Methods

We compared genotype frequencies in 740 NZ Caucasian RA patients and 553 controls genotyped for rs3738919, using a polymerase chain reaction-restriction fragment length polymorphism assay. A TaqMan genotyping SNP assay was used to type 713 Caucasian RA patients and 515 control samples from Oxford for the rs3738919 variant. Association of rs3738919 with RA was tested in these two sample sets using the chi-square goodness-of-fit test. The Mantel-Haenszel test was used to perform a meta-analysis, combining the genetic results from four independent Caucasian case control cohorts, consisting of 3,527 cases and 4,126 controls. Haplotype analysis was also performed using SNPs rs3911238, rs10174098 and rs3738919 in the Wellcome Trust Case Control Consortium, NZ and Oxford case control samples.

Results

We found no evidence for association between ITGAV and RA in either the NZ or Oxford sample set (odds ratio [OR] = 0.88, P_allelic = 0.11 and OR = 1.18, P_allelic = 0.07, respectively). Inclusion of these data in a meta-analysis (random effects) of four independent cohorts (3,527 cases and 4,126 controls) weakens support for the hypothesis that rs3738919 plays a role in the development of RA (OR_combined = 0.92, 95% confidence interval 0.80 to 1.07; P = 0.29). No consistent haplotype associations were evident.

Conclusions

Association of ITGAV SNP rs7378919 with RA was not replicated in NZ or Oxford case control sample sets. Meta-analysis of these and previously published data lends limited support for a role for the ITGAV in RA in Caucasians of European ancestry.     

Protein O-Mannosyltransferases B and C Support Hyphal Development and Differentiation in Aspergillus nidulans

Aspergillus nidulans possesses three pmt genes encoding protein O-d-mannosyltransferases (Pmt). Previously, we reported that PmtA, a member of the PMT2 subfamily, is involved in the proper maintenance of fungal morphology and formation of conidia (T. Oka, T. Hamaguchi, Y. Sameshima, M. Goto, and K. Furukawa, Microbiology 150:1973-1982, 2004). In the present paper, we describe the characterization of the pmtA paralogues pmtB and pmtC. PmtB and PmtC were classified as members of the PMT1 and PMT4 subfamilies, respectively. A pmtB disruptant showed wild-type (wt) colony formation at 30°C but slightly repressed growth at 42°C. Conidiation of the pmtB disruptant was reduced to approximately 50% of that of the wt strain; in addition, hyperbranching of hyphae indicated that PmtB is involved in polarity maintenance. A pmtA and pmtB double disruptant was viable but very slow growing, with morphological characteristics that were cumulative with respect to either single disruptant. Of the three single pmt mutants, the pmtC disruptant showed the highest growth repression; the hyphae were swollen and frequently branched, and the ability to form conidia under normal growth conditions was lost. Recovery from the aberrant hyphal structures occurred in the presence of osmotic stabilizer, implying that PmtC is responsible for the maintenance of cell wall integrity. Osmotic stabilization at 42°C further enabled the pmtC disruptant to form conidiophores and conidia, but they were abnormal and much fewer than those of the wt strain. Apart from the different, abnormal phenotypes, the three pmt disruptants exhibited differences in their sensitivities to antifungal reagents, mannosylation activities, and glycoprotein profiles, indicating that PmtA, PmtB, and PmtC perform unique functions during cell growth.Protein glycosylation, which is a major posttranslational modification, plays essential roles in eukaryotic cells from fungi to mammals (19). N-linked oligosaccharides in glycoproteins that share relatively common structures are structurally classified into high-mannose, complex, and hybrid types (3). O-linked oligosaccharides in glycoproteins are diverse with respect to their sugar components and the mode of sugar linkages among the eukaryotic organisms (8, 19). O mannosylation, which is commonly found in the glycoproteins of fungi, has been extensively studied in the budding yeast Saccharomyces cerevisiae (4, 21, 35). The initial reaction of mannose transfer to serine and threonine residues in proteins is catalyzed by protein O-d-mannosyltransferase (Pmt) in the endoplasmic reticulum (ER), where dolichyl phosphate-mannose is required as an immediate sugar donor (4). In the Golgi complex, O mannosylation in S. cerevisiae is linearly elongated by up to five mannose residues by mannosyltransferases (Mnt) that utilize GDP-mannose as the mannosyl donor. At least six Pmt-encoding genes (PMT1 to -6), three α-1,2-Mnt-encoding genes (KRE2, KTR1, and KTR3), and three α-1,3-Mnt-encoding genes (MNN1, MNT2, and MNT3) are known to be involved in O mannosylation in S. cerevisiae (21, 31, 45).The Pmt family of proteins can be classified into the PMT1, PMT2, and PMT4 subfamilies based on phylogeny (6). Proteins of the PMT1 subfamily form a heteromeric complex with proteins belonging to the PMT2 subfamily, and PMT4 subfamily proteins form a homomeric complex (7). Simultaneous disruptions of three different types of PMT genes were lethal (4), suggesting that each class provided a unique function for O mannosylation. Yeasts other than S. cerevisiae, such as Schizosaccharomyces pombe (38, 41), Candida albicans (29), and Cryptococcus neoformans (28), possess three to five pmt genes, which have been characterized. Several studies provide evidence that protein O mannosylation modulates the functions and stability of secretory proteins and thereby affects the growth and morphology of these yeasts. O mannosylation by Pmt2 in S. cerevisiae (ScPmt2) provides protection from ER-associated degradation and also functions as a fail-safe mechanism for ER-associated degradation (11, 13, 23). Likewise, in C. albicans, CaPmt1- and CaPmt4-mediated O mannosylation specifically protects CaSec20 from proteolytic degradation in the ER (40). Cell wall integrity is maintained in S. cerevisiae by increased stabilization and correct localization of the sensor proteins ScWsc and ScMid2 due to O mannosylation by ScPmt2 and ScPmt4 (20). Similarly, the stability and localization to the plasma membrane of axial budding factor ScAxl2/Bud10 is enhanced by ScPmt4-mediated O mannosylation, increasing its activity (32). ScPmt4-mediated O glycosylation also functions as a sorting determinant for cell surface delivery of ScFus1 (30). CaPmt4-mediated O glycosylation is required for environment-specific morphogenetic signaling and for the full virulence of C. albicans (29).With respect to filamentous fungi like Aspergillus that develop hyphae in a highly ordered manner, which then differentiate to form conidiospores, little is known about the function and synthetic pathway of the O-mannose-type oligosaccharides. O-Glycans in glycoproteins of Aspergillus include sugars other than mannose, and their structures have been determined (8). The initial mannosylation catalyzed by Pmts is found in Aspergillus and occurs as in yeasts (8).We characterized the pmtA gene of Aspergillus nidulans (AnpmtA), belonging to the PMT2 subfamily, and found that the mutant exhibited a fragile cell wall phenotype and alteration in the carbohydrate composition, with a reduction in the amount of skeletal polysaccharides in the cell wall (26, 33). Recently, the Afpmt1 gene belonging to the PMT1 family of Aspergillus fumigatus, a human pathogen, was characterized. AfPmt1 is crucial for cell wall integrity and conidium morphology (46).In this study, we characterize the pmtB and pmtC genes of A. nidulans to understand their contribution to the cell morphology of this filamentous fungus. We also demonstrate that the PmtA, PmtB, and PmtC proteins have distinct specificities for protein substrates and function differently during cell growth of filamentous fungi.     

A simple method for preserving environmental DNA in water samples at ambient temperature by addition of cationic surfactant

Environmental DNA (eDNA) analysis is a powerful tool within ecology for the study of the distribution or abundance of aquatic species, although the simplification of water sampling is required for enabling light and fast field sampling to expand further application of eDNA analysis. Here, certain candidate chemicals belonging to the group of cationic surfactants were examined for their effectiveness as preservatives for eDNA water samples by simply adding the chemicals to water samples to suppress the degradation of eDNA. The quaternary ammonium compound benzalkonium chloride (BAC) at a final concentration of 0.01% was effective to retain 92% of eDNA derived from the bluegill sunfish Lepomis macrochirus in an 8-h incubation test at ambient temperature, which assumed a transportation of water samples in 1-day field sampling during the daytime. Meanwhile, eDNA in water samples without BAC retained only 14% of the initial eDNA. Moreover, an additional long-term incubation test (up to 10 days) revealed BAC-treated samples retained ~70 and 50% of bluegill DNA compared to the initial amount after 1- and 10-day incubation at ambient temperature, respectively. Meanwhile, eDNA in naïve samples reduced to 20% after 1-day incubation and reached undetectable levels after 10 days. Up to now, many eDNA studies have adopted on-site filtration followed by filter fixation, which requires many pieces of equipment. Addition of BAC can protect eDNA in water samples with less effort and equipment resulting in an increase of measurement accuracy of the eDNA quantity and detection probability of rare species by preventing the disappearance of rare sequences in water samples.     

Polyamine Oxidase5 Regulates Arabidopsis Growth through Thermospermine Oxidase Activity

The major plant polyamines (PAs) are the tetraamines spermine (Spm) and thermospermine (T-Spm), the triamine spermidine, and the diamine putrescine. PA homeostasis is governed by the balance between biosynthesis and catabolism; the latter is catalyzed by polyamine oxidase (PAO). Arabidopsis (Arabidopsis thaliana) has five PAO genes, AtPAO1 to AtPAO5, and all encoded proteins have been biochemically characterized. All AtPAO enzymes function in the back-conversion of tetraamine to triamine and/or triamine to diamine, albeit with different PA specificities. Here, we demonstrate that AtPAO5 loss-of-function mutants (pao5) contain 2-fold higher T-Spm levels and exhibit delayed transition from vegetative to reproductive growth compared with that of wild-type plants. Although the wild type and pao5 are indistinguishable at the early seedling stage, externally supplied low-dose T-Spm, but not other PAs, inhibits aerial growth of pao5 mutants in a dose-dependent manner. Introduction of wild-type AtPAO5 into pao5 mutants rescues growth and reduces the T-Spm content, demonstrating that AtPAO5 is a T-Spm oxidase. Recombinant AtPAO5 catalyzes the conversion of T-Spm and Spm to triamine spermidine in vitro. AtPAO5 specificity for T-Spm in planta may be explained by coexpression with T-Spm synthase but not with Spm synthase. The pao5 mutant lacking T-Spm oxidation and the acl5 mutant lacking T-Spm synthesis both exhibit growth defects. This study indicates a crucial role for T-Spm in plant growth and development.Polyamines (PAs) are low-molecular mass aliphatic amines that are present in almost all living organisms. Cellular PA concentrations are governed primarily by the balance between biosynthesis and catabolism. In plants, the major PAs are the diamine putrescine (Put), the triamine spermidine (Spd), and the tetraamines spermine (Spm) and thermospermine (T-Spm; Kusano et al., 2008; Alcázar et al., 2010; Mattoo et al., 2010; Takahashi and Kakehi, 2010; Tiburcio et al., 2014). Put is synthesized from Orn by Orn decarboxylase and/or from Arg by three sequential reactions catalyzed by Arg decarboxylase (ADC), agmatine iminohydrolase, and N-carbamoylputrescine amidohydrolase. Arabidopsis (Arabidopsis thaliana) does not contain an ORNITHINE DECARBOXYLASE gene (Hanfrey et al., 2001) and synthesizes Put from Arg via the ADC pathway. Put is further converted to Spd via an aminopropyltransferase reaction catalyzed by spermidine synthase (SPDS). In this reaction, an aminopropyl residue is transferred to Put from decarboxylated S-adenosyl-Met, which is synthesized by S-adenosyl-Met decarboxylase (SAMDC; Kusano et al., 2008). Spd is then converted to Spm or T-Spm, reactions catalyzed in Arabidopsis by spermine synthase (SPMS; encoded by SPMS) or thermospermine synthase (encoded by Acaulis5 [ACL5]), respectively (Hanzawa et al., 2000; Knott et al., 2007; Kakehi et al., 2008; Naka et al., 2010). A recent review reports that T-Spm is ubiquitously present in the plant kingdom (Takano et al., 2012).The PA catabolic pathway has been extensively studied in mammals. Spm and Spd acetylation by Spd/Spm-N¹-acetyltransferase (Enzyme Commission no. 2.3.1.57) precedes the catabolism of PAs and is a rate-limiting step in the catabolic pathway (Wallace et al., 2003). A mammalian polyamine oxidase (PAO), which requires FAD as a cofactor, oxidizes N¹-acetyl Spm and N¹-acetyl Spd at the carbon on the exo-side of the N⁴-nitrogen to produce Spd and Put, respectively (Wang et al., 2001; Vujcic et al., 2003; Wu et al., 2003; Cona et al., 2006). Mammalian spermine oxidases (SMOs) perform oxidation of the carbon on the exo-side of the N⁴-nitrogen to produce Spd, 3-aminopropanal, and hydrogen peroxide (Vujcic et al., 2002; Cervelli et al., 2003; Wang et al., 2003). Thus, mammalian PAOs and SMOs are classified as back-conversion (BC)-type PAOs.In plants, Spm, T-Spm, and Spd are catabolized by PAO. Plant PAOs derived from maize (Zea mays) and barley (Hordeum vulgare) catalyze terminal catabolism (TC)-type reactions (Tavladoraki et al., 1998). TC-type PAOs oxidize the carbon at the endo-side of the N⁴-nitrogen of Spm and Spd to produce N-(3-aminopropyl)-4-aminobutanal and 4-aminobutanal, respectively, plus 1,3-diaminopropane and hydrogen peroxide (Cona et al., 2006; Angelini et al., 2008, 2010). The Arabidopsis genome contains five PAO genes, designated as AtPAO1 to AtPAO5. Four recombinant AtPAOs, AtPAO1 to AtPAO4, have been homogenously purified and characterized (Tavladoraki et al., 2006; Kamada-Nobusada et al., 2008; Moschou et al., 2008; Takahashi et al., 2010; Fincato et al., 2011, 2012). AtPAO1 to AtPAO4 possess activities that convert Spm (or T-Spm) to Spd, called partial BC, or they convert Spm (or T-Spm) first to Spd and subsequently to Put, called full BC. Ahou et al. (2014) report that recombinant AtPAO5 also catalyzes a BC-type reaction. Therefore, all Arabidopsis PAOs are BC-type enzymes (Kamada-Nobusada et al., 2008; Moschou et al., 2008; Takahashi et al., 2010; Fincato et al., 2011, 2012; Ahou et al., 2014). Four of the seven PAOs in rice (Oryza sativa; OsPAO1, OsPAO3, OsPAO4, and OsPAO5) catalyze BC-type reactions (Ono et al., 2012; Liu et al., 2014a), whereas OsPAO7 catalyzes a TC-type reaction (Liu et al., 2014b). OsPAO2 and OsPAO6 remain to be characterized, but may catalyze TC-type reactions based on their structural similarity with OsPAO7. Therefore, plants possess both TC-type and BC-type PAOs.PAs are involved in plant growth and development. Recent molecular genetic analyses in Arabidopsis indicate that metabolic blocks at the ADC, SPDS, or SAMDC steps lead to embryo lethality (Imai et al., 2004; Urano et al., 2005; Ge et al., 2006). Potato (Solanum tuberosum) plants with suppressed SAMDC expression display abnormal phenotypes (Kumar et al., 1996). It was also reported that hydrogen peroxide derived from PA catabolism affects root development and xylem differentiation (Tisi et al., 2011). These studies indicate that flux through metabolic and catabolic PA pathways is required for growth and development. The Arabidopsis acl5 mutant, which lacks T-Spm synthase activity, displays excessive differentiation of xylem tissues and a dwarf phenotype, especially in stems (Hanzawa et al., 2000; Kakehi et al., 2008, 2010). An allelic ACL5 mutant (thickvein [tkv]) exhibits a similar phenotype as that of acl5 (Clay and Nelson, 2005). These results indicate that T-Spm plays an important role in Arabidopsis xylem differentiation (Vera-Sirera et al., 2010; Takano et al., 2012).Here, we demonstrate that Arabidopsis pao5 mutants contain 2-fold higher T-Spm levels and exhibit aerial tissue growth retardation approximately 50 d after sowing compared with that of wild-type plants. Growth inhibition of pao5 stems and leaves at an early stage of development is induced by growth on media containing low T-Spm concentrations. Complementation of pao5 with AtPAO5 rescues T-Spm-induced growth inhibition. We confirm that recombinant AtPAO5 catalyzes BC of T-Spm (or Spm) to Spd. Our data strongly suggest that endogenous T-Spm levels in Arabidopsis are fine tuned, and that AtPAO5 regulates T-Spm homeostasis through a T-Spm oxidation pathway.