Synthesis of orthogonally protected (<Emphasis Type="Italic">3R</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)- and (<Emphasis Type="Italic">3S</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)-4,5-diamino-3-hydroxypentanoic acids

Summary.  The paper describes two methods of the synthesis of ethyl (3R,4S)- and (3S,4S)-4-[(benzyloxycarbonyl)amino]-5-[(tert-butyloxycarbonyl)amino]-3-hydroxypentanoates, useful for the syntheses of edeine analogs. Differently N-protected (S)-2,3-diaminopropanoic acid was used as a substrate in both procedures. The absolute configuration of newly generated asymmetric carbon atoms C-3 in β-hydroxy-γ,δ-diamino products was assigned by means of ¹H NMR spectroscopy after their transformation into corresponding piperidin-2-ones. Received May 24, 2002 Accepted October 10, 2002 Published online December 18, 2002 Acknowledgment The authors are indebted to the Faculty of Chemistry, Technical University of Gdańsk for financial support. Authors' address: Zbigniew Czajgucki, M. Sc., Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Technical University of Gdańsk, 11/12 Narutowicza St., 80-952 Gdańsk, Poland, Fax +48 58 347 11 44, E-mail: zmczaj@wp.pl     

Four new species of <Emphasis Type="Italic">Crinipellis </Emphasis>and <Emphasis Type="Italic">Marasmius </Emphasis>in eastern Honshu,Japan

 Four new species of Crinipellis and Marasmius (Agaricales, Basidiomycetes) in eastern Honshu, Japan, are described and illustrated: (1) Crinipellis conchata sp. nov. (section Excentricinae), forming a conchate pileus and a strongly excentric, short stipe, was found on a dead twig of Trachelospermum asiaticum in Mt. Takao, Tokyo; (2) Marasmius funalis sp. nov. (section Androsacei), forming a densely white-hispid, dark brown stipe bearing numerous setiform caulocystidia, was found on a dead twig of Cryptomeria japonica or on leaf litter in Tokyo and Kanagawa; (3) Marasmius maculosus sp. nov. (section Sicci), having a relatively large, reddish-brown pileus distinctly mottled with pale colored spots and Siccus-type cheilocystidia and pileipellis cells with relatively long setulae, was found on leaf litter in the lowland forest of Kanagawa and Chiba; and (4) Marasmius sasicola sp. nov. (section Marasmius), having a small, plicate-sulcate pileus, a filiform, wiry, blackish stipe, collariate lamellae, and Siccus-type cheilocystidia and pileipellis elements, was found on fallen dead leaves of grass bamboo in Kanagawa. Received: January 30, 2002 / Accepted: May 24, 2002     

Phylogeny of <Emphasis Type="Italic">Gunnera</Emphasis>

 The phylogeny of the genus Gunnera is investigated for the first time. Twelve species representing the six currently recognised subgenera are analysed. Two chloroplast DNA regions, the rbcL gene and the rps16 intron, together provide 46 informative characters out of 2335. A combined analysis of both genes gives four most parsimonious trees, firmly establishing the east South American G. herteri as sister group to the rest of the genus. The African G. perpensa is sister group to two well-supported clades, one including the South American subgenera Misandra and Panke, the other the Australian/New Zealand/Malayan species of subgenera Milligania and Pseudogunnera. Thus, South America is a composite area for Gunnera, showing up at two different levels in the cladogram. Our analysis supports a close biogeographic relationship between Australia and New Zealand. The evolution of some morphological characters is discussed. Lastly, the unusual structure of some of the rbcL sequences is reported. Received July 6, 2000 Accepted October 24, 2000     

<Emphasis Type="Italic">Pseudocercospora griseola</Emphasis> Causing Angular Leaf Spot on <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> Produces 1,8-Dihydroxynaphthalene-Melanin

Pseudocercospora griseola is the causal agent of angular leaf spot of common bean (ALS). It has undergone parallel coevolution with its host and two major groups have been defined, “Andean” (P. griseola f. griseola) and “Mesoamerican” (P. griseola f. mesoamericana). The aim of this study was to analyze the nature and the level of the dark pigment synthesized by the representatives of each group. After 21 days of incubation on potato dextrose agar medium, P. griseola f. griseola isolate S3b developed colonies with diameters of 17.5 ± 1.3 mm and concentric rings of pigmentation. Isolate T4 of P. griseola f. mesoamericana presented smaller colonies (9.9 ± 0.3 mm) with a uniform dark-gray color. Both isolates, S3b and T4, produced the same pigment, a 1,8-dihydroxynaphthalene-melanin, although different in quantity and structural features as suggested by the IR spectrum. The P. griseola f. griseola isolate S3b had a higher growth rate and melanin content as well as smaller sensitivity to melanin synthesis inhibitors compared to the isolate T4 of P. griseola f. mesoamericana. These results suggest a possible link between melanin and growth in P. griseola.     

Proteolysis of <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Saccharomycopsis fibuligera</Emphasis>: characterization of digestion products

α-Amylase from Saccharomycopsis fibuligera R-64 was successfully purified by butyl Toyopearl hydrophobic interaction chromatography, followed by Sephadex G-25 size exclusion and DEAE Toyopearl anion exchange chromatography. The enzyme has a molecular mass of 54 kDa, as judged by SDS PAGE analysis. Upon tryptic digestion, two major fragments with relative molecular masses of 39 kDa and 10 kDa, which resemble the A/B and C-terminal domains in the homologous Taka-amylase, were obtained and were successfully separated with the Sephadex G-50 size exclusion column. The 39-kDa fragment demonstrated a similar amylolytic activity to that of the undigested enzyme. However, it was found that the K _m value of the 39-kDa fragment was about two-times higher than that of the undigested enzyme. Moreover, thermostability studies showed a lower half-life time for the 39-kDa fragment. These findings suggest that the 39-kDa fragment is the catalytic domain, while the 10-kDa fragment is the C-terminal one, which plays a role in thermostability and starch binding. Although the undigested enzyme is able to act on raw starches at room temperature, with maize starches as the best substrate, neither the undigested enzyme nor the fragments adsorb the tested raw starches. These results propose Saccharomycopsis fibuligera α-amylase as a raw starch-digesting but not adsorbing amylase, with a similar domain organization to that of Taka-amylase A.     

Phylogenetic analysis of homologous proteins encoded by UL2 and UL23 genes of <Emphasis Type="Italic">Herpesviridae</Emphasis>

The proteins encoded by the Herpesviridae β-gene play a critical role in the replication stage of the virus. In this paper, phylogenetic analyses provided evidence that some β-gene products, such as UL2 and UL23 from HSV1, have their homologous genes in its family, and also exist in prokaryotic organisms, indicating that these viruses appear to have been assembled over evolutionary time by numerous independent events of horizontal gene transfer. Fundation item: National Natural Science Funds (30570081)     

Synthesis and use of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-di-Boc-glutamate γ-semialdehydes and related aldehydes

Summary.  This review article focuses on the synthesis and reactions of N,N-di-Boc glutamate and aspartate semialdehydes as well as related aldehydes. These building blocks are prepared according to various strategies from glutamic and aspartic acids and find interesting synthetic applications. In the first part, the methods for the synthesis of N,N-di-Boc-amino aldehydes are summarized. The applications of these chiral synthons for the synthesis of unnatural amino acids and other bioactive compounds are discussed in the second section. Received April 24, 2002 Accepted August 13, 2002 Published online January 30, 2003 Authors' address: Prof. Violetta Constantinou-Kokotou, Chemical Laboratories, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece, E-mail: vikon@aua.gr Abbreviations: AcNH-TEMPO, 4-acetamido-2,2,6,6-tetramethyl-1-piperidinyloxy free radical; AIBN, 2,2′-azobis(2-methylpropionitrile); Aliquat, methyltrioctylammonium chloride; Bn, benzyl; Boc, tert-butoxycarbonyl; Bu^t, tert-butyl; m-CPBA, 3-chloroperoxybenzoic acid; DAST, diethylaminosulfur trifluoride; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; (R,R)-(+)-DET, (R,R)-(+)-diethyltartrate; DIBALH, diisobutyl aluminium hydride; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; Et₃N, triethylamine; KHMDS, potassium bis(trimethylsilyl)amide; (S)-LLB, lanthanium-lithium-bis-metallic binaphthol catalyst; MsCl, methanesulfonyl chloride; NEM, N-ethylmorpholine; NMO, 4-methylmorpholine N-oxide; PPA, propylphosphonic acid anhydride; TBHP, tert-butyl hydroperoxide; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMSI, 1-(trimethylsilyl)imidazole; Trt, trityl.     

Chromosome phylogeny of <Emphasis Type="Italic">Zamia</Emphasis> and <Emphasis Type="Italic">Ceratozamia</Emphasis> by means of Robertsonian changes detected by fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization (FISH) technique of rDNA

 Appearance and location of 45S rDNA and 5S rDNA signals were compared in chromosomes of nine species of the aneuploid Zamia and their taxonomically and phylogenetically closely related Ceratozamia mexicana. The 45S rDNA signal was detected in the proximal region of six chromosomes in Zamia angustifolia, Z. integrifolia, Z. pumila and Z. pygmaea (all 2n=16); in the proximal region of 6–14 chromosomes in Z. furfuracea, Z. loddigesii, Z. skinneri and Z. vazquezii (all 2n=18); and on the proximal region of 20 chromosomes in Z. muricata (2n=23). The 5S rDNA signals were commonly seen near the terminal region of the short arm of two metacentric chromosomes in the four species with 2n=16 and Z. furfuracea, Z. loddigesii and Z. vazquezii with 2n=18. Other 5S rDNA signals were seen near the terminal region of two terminal-centromeric chromosomes in Z. skinneri and near the terminal region of a metacentric and a telocentric chromosomes in Z. muricata. In contrast, those with 45S and 5S rDNA signals were exhibited in chromosomes of Ceratozamia mexicana in a different manner from those in the nine species of Zamia; the 45S rDNA signal in the terminal region of four metacentric and two submetacentric chromosomes and the 5S rDNA signal near the proximal region of two metacentric chromosomes. Received November 1, 1999 Accepted January 10, 2001     

Overexpression of <Emphasis Type="Italic">WUSCHEL</Emphasis> in <Emphasis Type="Italic">C. chinense</Emphasis> causes ectopic morphogenesis

Capsicum chinense is a recalcitrant species for in vitro morphogenesis, and up to date there is no efficient system for genetic transformation and regeneration of this species via somatic embryogenesis. Here, we carried out an in vitro transformation of C. chinense via Agrobacterium tumefaciens co-cultivation with a system that expresses the heterologous gene WUSCHEL from Arabidopsis thaliana. WUSCHEL has been shown to promote the transition from vegetative to embryogenic state when overexpressed. We tested if the expression of WUSCHEL in C. chinense would promote an embryogenic response in this species. After 15 days of induction, the segments of transformed stems begun to form globular structures, suggesting that heterologus WUSCHEL was active and involved in the process of morphogenesis.     

Overexpression of the Chitosanase Gene in <Emphasis Type="Italic">Fusarium solani</Emphasis> via <Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-Mediated Transformation

To overexpress the chitosanase gene (csn) in F. solani, a vector based on pCAMBIA 1300 was constructed. The csn gene, which is under control of the Aspergillus nidulans gpdA promoter and A. nidulans trpC terminator, was introduced back into the F. solani genome by Agrobacterium tumefaciens-mediated transformation, and the herbicide-resistance gene bar from Streptomyces hygroscopicus was used as the selection marker. Transformants which showed a significant increase in chitosanase production (~2.1-fold than control) were obtained. Southern blot analysis indicated that most transformants had a single-copy T-DNA integration.     

Identification and Characterization of an Autolysin Gene, <Emphasis Type="Italic">atlh</Emphasis>, from <Emphasis Type="Italic">Streptococcus downei</Emphasis>

An autolysin gene, atlh, was identified and sequenced from Streptococcus downei MFe28 using degenerate polymerase chain reaction (PCR) and the gene-walking method. Atlh protein encoded by atlh is composed of 879 amino acids, with a molecular weight of 95,902.26. Atlh possesses four 15-amino-acid residue repeats in the putative cell-wall-binding domain and has a catalytic domain in the C-terminus. The deduced amino acid sequence of atlh showed homology to S. mutans autolysin AtlA (68.4% similarity). Inactivation of atlh resulted in elongated chain formation compared to the parent strain. Recombinant proteins Atlh and its derivatives were constructed and analyzed by zymography. Zymographic analysis revealed that the Asp-771 residue of Atlh was essential for lytic activity and that lytic activity was not diminished by the deletion of repetitive regions in the putative cell-wall-binding domain of Atlh. Biofilm assay showed that the wild-type strain formed glucose- and sucrose-dependent biofilms, the atlh mutant diminished this ability. These results suggest that Atlh is associated with cell separation and biofilm formation.     

Characterization of the nematocidal toxocyst in <Emphasis Type="Italic">Pleurotus</Emphasis> subgen. <Emphasis Type="Italic">Coremiopleurotus</Emphasis>

Toxocysts of the genus Pleurotus are blastoconidia-like ovoid structures surrounded by a liquid droplet containing a toxin that paralyzes nematodes. This study investigated toxocyst development using a strain S396 of Pleurotus cystidiosus subsp. abalonus (subgen. Coremiopleurotus). The surface of the liquid droplet was found to be an elastic envelope. When a nematode touches the toxocyst, the envelope adheres to the worm and bursts. Toxocysts are induced simultaneously with coremia formation in the absence of nematodes and developed only from aerial hyphae in which nuclear division had ceased. In the early stage of toxocyst development, liquid springs repeatedly from the tip of the sterigma-like stipe before ovoid (blastoconidium-like structure) formation. A certain substance in the liquid might polymerize to form the envelope while the ovoid simultaneously budded in the droplet. The nucleus tends to locate near the toxocyst, especially in early stage of toxocyst development. Each dikaryotic cell predominantly formed one or two toxocyst(s) while each monokaryotic cell predominantly formed one. In rare cases, a nucleus existed in the toxocyst, suggesting the possibility that the toxocyst is a vestigial blastoconidium.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation and rooting of <Emphasis Type="Italic">Acacia mangium</Emphasis> microshoots from juvenile and mature origins

Acacia mangium microshoots from juvenile and mature genotypes were micropropagated through a regular subculture regime for more than 3 yr in vitro. Average multiplication rates of 5.5 for the juvenile source and of 3.9 for the mature clone were obtained during this period on the 6-benzylaminopurine-enriched multiplication medium. Although the juvenile material displayed higher potential for axillary shoot and root formation than the mature clone overall, the differences were not statistically significant with noticeable variations in the course of time from one subculture to another. On specific rooting media, the juvenile material rooted overall in greater proportions than the mature material, notwithstanding noteworthy interactions between the age of the plant material and the various experimental factors tested, i.e. sucrose concentration, macrosalt formulation and light regime. The stimulating effect of darkness on juvenile plant material rooting rates was more obvious than for the mature clone, which responded more inconsistently. Addition of 4 μM indole-3-acetic acid, indole-3-butyrie acid, or 1-naphthaleneacetic acid in the rooting medium significantly increased the proportion of rooted microshoots of both origins. The rooting criteria observed were also prone to vary depending on the experimental date. The data indicate that rooting of juvenile and mature Acacia mangium materials have average rates of 90% and 77%, respectively. These are high enough to consides possible applications of these procedures toward operational activities.     

<Emphasis Type="Italic">Pseudolagarobasidium calcareum</Emphasis>: Japanese records and cultural characteristics

Pseudolagarobasidium calcareum (Basidiomycotina, Corticiaceae s.l.) is newly reported for Japan, and its cultural characteristics are described for the first time. Seven of nine Japanese and Taiwanese specimens examined produce arthroconidia associated with the basidiomata. Variability in color of the hymenial surface is also observed among the specimens. In culture, this species is characterized by fiber hyphae (quasi-binding hyphae) and arthroconidia. Mating tests reveal a multiallelic, bifactorial incompatibility system of this fungus and intercompatibility between Japanese and Taiwanese strains. This species resembles Pseudolagarobasidium subvinosum but differs in producing the fiber hyphae not only in culture but also in the basidiomata. Received: July 10, 2001 / Accepted: March 29, 2002     

Gene Expression,Intron Density,and Splice Site Strength in <Emphasis Type="Italic">Drosophila</Emphasis> and <Emphasis Type="Italic">Caenorhabditis</Emphasis>

In this paper we investigate the relationships among intron density (number of introns per kilobase of coding sequence), gene expression level, and strength of splicing signals in two species: Drosophila melanogaster and Caenorhabditis elegans. We report a negative correlation between intron density and gene expression levels, opposite to the effect previously observed in human. An increase in splice site strength has been observed in long introns in D. melanogaster. We show this is also true of C. elegans. We also examine the relationship between intron density and splice site strength. There is an increase in splice site strength as the intron structure becomes less dense. This could suggest that introns are not recognized in isolation but could function in a cooperative manner to ensure proper splicing. This effect remains if we control for the effects of alternative splicing on splice site strength. Reviewing Editor: Dr. Nicolas Galtier     

Isolation of <Emphasis Type="Italic">madA</Emphasis> homologs in <Emphasis Type="Italic">Pilobolus crystallinus</Emphasis>

Pilobolus crystallinus shows unique photoresponses at various growing stages. cDNAs for putative photoreceptors were cloned from this fungus. Three genes named pcmada1, pcmada2, and pcmada3 were identified from the PCR fragments, and amplified with degenerated primers for the LOV domain, which is conserved in many blue-light receptors. Deduced amino acid sequences for PCMADA1, PCMADA2, and PCMADA3 had one light-oxygen-voltage (LOV)-sensing and two PER-ARNT-SIM (PAS) domains. A zinc finger DNA-binding motif was conserved in the C-terminals of PCMADA1 and PCMADA3. However, PCMADA2 lacked the zinc finger motif. Expression of pcmada1 was suppressed by blue light whereas that of pcmada3 was promoted by blue-light irradiation.     

Comparative Molecular Genetic Analysis of β-Fructosidases of Yeasts <Emphasis Type="Italic">Saccharomyces</Emphasis>

To study the evolution of the polymeric β-fructosidase (invertase) genes (SUC) of yeasts Saccharomyces, new SUC gene of S. cariocanus was cloned and sequenced and the nucleotide and amino acid sequences were compared for all known β-fructosidases of Saccharomyces species. The proteins showed 90–97% homology. The most divergent was S. bayanus β-fructosidase. The results testified again to high conservation of yeast β-fructosidases. Transitions C-T prevail in the total spectrum of nucleotide substitutions observed in the coding regions of the SUC genes; most of these transitions are in the third codon position and cause no changes in the amino acid sequences of the encoded proteins. The six Saccharomyces species each carry one (probably, non-telomeric) β-fructosidase gene. SUC is on chromosome IX in S. cerevisiae, S. bayanus, S. kudriavzevii, S. mikatae, and S. paradoxus and in a translocation region on chromosome XV in S. cariocanus.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 3, 2005, pp. 413–419.Original Russian Text Copyright © 2005 by Korshunova, Naumova, Naumov.     

Plant litter feedback and population dynamics in an annual plant,<Emphasis Type="Italic"> Cardamine pensylvanica</Emphasis>

The presence of litter has the potential to alter the population dynamics of plants. In this paper, we explore the effects of litter on population dynamics using a simple experimental laboratory system with populations of the annual crucifer, Cardamine pensylvanica. Using a factorial experiment with four densities and three litter levels, we determined the effect of litter on biomass and plant fecundity, and the life stages responsible for these changes in yield. Although litter had significant effects on seed germination and on seedling survivorship, we show, using a population dynamics model, that these effects were not demographically significant. Rather, the potential effect of litter on population dynamics resulted almost entirely from its effect on biomass. Persistent litter suppressed plant biomass and apparently removed the direct density effect present in the absence of litter. Thus, litter changed the shape of the recruitment curve from slightly humped to asymptotic. In addition to changing the shape of the recruitment curve, litter reduced the carrying capacity of the populations. Thus, the population dynamics model indicated that not all statistically significant responses were dynamically significant. Given the potential complexity of litter effects, simple population models provide a powerful tool for understanding the potential consequences of short-term responses. Received: 8 September 1999 / Accepted: 5 April 2000