Production of β-glycosidases (linamarase and amygdalase) and pectolytic enzymes by Penicillium spp.

Fifty-eight strains, representing 31 species of Penicillium, were screened for extracellular -glycosidase (amygdalase/linamarase) and pectolytic (polygalacturonase, pectin lyase) enzymes. One strain each of P. turbatum, P. piceum and P. paxilli showed very high -glycosidase activity and slightly lower activities were found in P. crustosum, P. expansum, P. oxalicum and P. aurantiogriseum. Generally, maximum -glycosidase activity showed reached during the stationary phase of growth. The seven species with highest -glycosidase activity showed different patterns of pectolytic activities, indicating that different species or combinations of species could be selected for different potential applications.L. Brimer is with the Department of Pharmacology and Pathobiology, Royal Veterinary & Agricultural University, 13 Bulowsvej, DK 1870 Frederiksberg C, Denmark; A.R. Cicalini and F. Federici are with the Dipartimento Agrobiologia e Agrochimica, University of Tuscia, Via S.C. de Lellis, I-01100 Viterbo, Italy. M. Petruccioli is with the Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali, University of Basilicata, Via N. Sauro, 85, I-85100 Potenza, Italy.     

Ovary-ovule transmitting tract in Crocus (Iridaceae), structure and calcium distribution

 The structure of and the calcium distribution in the ovary-ovule transmitting tracts of Crocus were examined by using conventional and antimonate fixation methods for electron microscopy, and fluorescence microscopy as well. The ovarian locules are bordered with epidermal cells and transmitting cells. The epidermal cells are extensively cutinized, and the cytoplasm is rich in the organelles and vesicles prevalently engaged in thickening the exposed wall. The transmitting cells have uncutinized walls with numerous labyrinthine ingrowths, and the cytoplasm is rich in organelles and vesicles engaged in the secretion of the extracellular matrix (e.m.). The micropylar tissue of the ovule consists of largely vacuolated cells with scarcely structured cytoplasm, the e.m. appears to be of gametophytic origin. Both the ovary and ovule e.m. have a glycosidic and proteinic nature, and are rich in loosely bound Ca²⁺ ions. In the embryo sac, Ca²⁺ ions are concentrated in one synergid. The results are discussed in relation to the guided growth of pollen tubes. Received April 9, 2002; accepted July 9, 2002 Published online: November 28, 2002 Addresses of the authors: Giuseppe Chichiriccò (e-mail: Chichiri@univaq.it), Dipartimento di Scienze Ambientali, University of L'Aquila, Via Vetoio, I-67100 L'Aquila, Italy. Anna Maria Ragnelli, Pierpaolo Aimola, Dipartimento di Biologia di Base ed Applicata, University of L'Aquila, Via Vetoio, I-67100 L'Aquila, Italy.     

Synthesis of a versatile constrained analogue of dipeptide DG (Asp-Gly)

Summary. The synthesis of an orthogonally protected constrained analogue of dipeptide DG (Asp-Gly) is reported exploiting alkylation of a chiral lactam. The versatility of this analogue was proven by removal of t-Boc protecting group, followed by coupling under homogeneous conditions with t-Boc-Arg(Z₂)-Gly, to give a conformationally restricted analogue of RGDG tetrapeptide. Authors’ address: Mario Orena, Dipartimento di Scienze e Tecnologie Chimiche, Università Politecnica delle Marche, Via Brecce Bianche, I-60131 Ancona, Italy     

Improved processes for the production and isolation of dynemicin A and large-scale fermentation in a 10000-liter fermentor

Summary Supplementing the culture ofMicromonospora chersina sp. nov. No. M956-1 with NaI (0.5 mg/l) enhanced the production of dynemicin A by 35-fold in shake flask culture. Homogeneous dynemicin A was obtained from the whole broth extract by Dicalite chromatography, Sephadex LH-20 chromatography and vacuum liquid chromatography. Gram quantities of dynemicin A were obtained from the fermentation ofM. chersina sp. nov. No. M956-1 in a 10000-liter fermentor.     

Chlorophyll-proteins from maize seedlings grown under intermittent light conditions

We studied the organization of the antenna system of maize (Zea mays L.) seedlings grown under intermittent light conditions for 11 d. These plants had a higher chlorophyll-a/b ratio, a higher ratio of carotenoids to chlorophyll and a lower ratio of chlorophyll to protein than plants grown in continuous light. We found all chlorophyll-protein complexes of maize to be present. However, the minor chlorophyll a/b-proteins CP29 and CP26, and to a greater extent CP24 and the major light-harvesting complex II were reduced relative to the photosystem (PS) II core-complex. Also the chlorophyll a/b-antennae of PSI were reduced relative to the reaction-centre polypeptides. When isolated by flatbed isoelectrofocussing, the chlorophyll-a/b complexes of PSII showed a higher chlorophyll-a/b ratio and a lower ratio of chlorophyll to protein than the same complexes from continuous light; additionally, they bound more carotenoids per protein than the latter. Thus the altered organization of the photosynthetic apparatus of plants from intermittent light is caused by two different factors: (i) the altered stoichiometry of chlorophyll-binding proteins and (ii) a different ratio of pigment to protein within individual chlorophyll-proteins.Abbreviations Chl chlorophyll - CL continuous light - F fraction - HPLC high-performance liquid chromatography - IEF isoelectrofocussing - IL intermittent light - LHCII light-harvesting complex II - PAGE polyacrylamide-gel electrophoresis - Phe pheophytin - SDS sodium dodecyl sulfate This work was supported by the grant no. 4.7240.90 from the Italian Ministry of Agriculture and Forestry. We thank Drs. R. Barbato (Dipartimento di Biologia, Padua, Italy) and Olivier Vallon (Institut de Biologie Physico-Chimique, Paris, France) for their gifts of antibodies, Drs. R. Barbato and P. Dainese (Dipartimento di Biologia, Padua, Italy) for fruitful discussion and Prof. G. Gennari (Dipartimento di Chimica fisica, Padua, Italy) for his assistance in recording the excitation spectra. J.M. was supported by a Stipendium from the Deutsche Forschungsgemeinschaft, which is gratefully acknowledged.     

Chromosome endoreduplication as a factor of salt adaptation in Sorghum bicolor

Summary. Nuclear DNA amounts were measured by Feulgen cytophotometry in Sorghum bicolor cv. 610 plants early exposed to 150 mM NaCl, a treatment known to induce an increased tolerance to salinity in plants carrying this genotype. In salt-treated plants, the percentages of 8C, 16C, and 32C nuclei in roots in the primary state of growth were 21.9%, 13.3%, and 4.3%, respectively. By contrast, in nonsalinized plants, only 3.5% of the nuclei had an 8C content and no higher DNA contents were observed. The salt treatment induced chromosome endoreduplication during the differentiation of cells in the root cortex, where 41.2% of the cells displayed a DNA content higher than 4C (versus 1.3% in control plants). No enhancement of endopolyploidy was observed in cells of the root vascular cylinder or the leaves of the salt-treated plants. In another S. bicolor genotype (DK 34-Alabama), noncompetent for salt adaptation, the same NaCl treatment did not induce chromosome endoreduplication in root cortex cells. Endopolyploidy may be considered as a part of the adaptive response of S. bicolor competent genotypes to salinity. Correspondence and reprints: Dipartimento di Biologia Cellulare e Ambientale, Università di Perugia, Via A. Pascoli, 06123 Perugia, Italy.     

Rapid clonal propagation of three mulberries,Morus cathayana HemsL,M. lhou Koiz. andM. serrata Roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees

High-frequency bud break and multiple shoots were induced in apical shoot buds and nodal explants ofMorus cathayana, M. lhou andM. serrata on Murashige and Skoog (MS) medium containing 0.5–1.0 mg/l 6-benzylaminopurine (BAP). Addition of gibberellic acid (0.4 mg/l) along with BAP induced faster bud break both in apical shoot buds and nodal explants and also enhanced the frequency of bud break in all three species. Shoot culture initiation was greatly influenced by explant type, explant age and explanting season. The shoots were successfully rooted on half-strength MS medium containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/l. The plantlets were successfully acclimated and eventually established in soil.Abbreviations BAP 6-Benzylaminopurine - GA ₃ Gibberellic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid - Kn Kinetin - MS Murashige and Skoog (1962) medium - NAA 1-Naphthalene acetic acid     

Gene variation and gene flow inOrchis morio (Orchidaceae) from Italy

Data are presented on genetic variation at 27 enzyme loci of the Green-Winged orchid,Orchis morio, in 18 population samples from Italy. The existence in Italy of two subspecies, i.e. subspp.morio andpicta, is not supported by allozyme data. No genetic heterogeneity was found betweenmorio-like andpicta-like samples and specimens. Moreover, morphological transition between the two forms was observed in different Italian populations. The parameters of genetic variability estimated forO. morio populations are consistent with those found among monocotyledon plants, and among those outcrossing, animal-pollinated and with wind-dispersed seeds. Genetic diversity of ItalianO. morio is mostly within populations. Correspondingly, low values of interpopulational genetic distance were found. This appears to be due to high levels of gene flow, which were estimated with different methods. The lack ofO. longicornu from Italian samples, as well as of any hybrid withO. morio (F₁, backcrossed or recombinant individuals) is demonstrated on the basis of genetic data. It is concluded that recurrent reports ofO. longicornu from Italy are due to confusion withO. morio or with otherOrchis species.     

Growth and survival ofAzospirillum brasilense andArthrobacter giacomelloi in binary continuous culture

Summary Azospirillum brasilense andArthrobacter giacomelloi were grown in single and mixed succinate-limited continuous cultures at a partial oxygen pressure of 0.01atm. Growth, viability and survival during nutrient starvation were examined at various dilution rates. At D=0.05 h^–1, K_s values for succinate consumed were calculated.Arthrobacter giacomelloi viability was inversely related to dilution rate whereasAzo. brasilense was directly related. Slightly lower values of viability were obtained in mixed culture, but the ratio between the microorganisms was constant. The survival ofArth. giacomelloi in single culture decreased with increasing growth rate while survival ofAzo. brasilense was directly related to dilution rate. Acetylene reduction activity was generally very low in both single and mixed cultures. Respiration rate was also determined and the mixed culture showed an oxygen uptake rate higher than that of single cultures.Research work supported by CNR, Italy. Special grant I.P.R.A. Sub-project 1. Paper N. 317.     

Suitability of lipid materials for culture ofMalassezia as evaluated from its cellular fatty acid composition

Malassezia is a facultative or obligatory lipophilic yeast. We devised new lipid-supplemented media suitable for the culture ofMalassezia. Malassezia furfur andM. pachydermatis grew well on both solid and liquid media supplemented with creaming powder preparations which are commercially available at moderate prices. Striking differences were found between the cellular fatty acid compositions ofM. furfur grown on media supplemented with creaming powder and that grown on media with conventional olive oil.Malassezia furfur grown on media with olive oil had nearly the same fatty acid composition as olive oil, with C18:1 amounting to 80%, while that grown on media supplemented with creaming powder had C16, C18:1 and C18:2 as the principal components. The use of these supplementary lipids appeared not to inhibit the normal synthesis of fatty acid inM. furfur. For the culture ofM. pachydermatis, media supplemented with creaming powder were also found more suitable than lipid-free media. The media devised are considered excellent, because they appear to provide a more natural growth environment forMalassezia.     

Effects of relative humidity and temperature conditions on pollen fluorochromatic reaction of Rosmarinus officinalis L. (Lamiaceae)

Summary. Mediterranean ecosystems are characterized by seasonal and annual fluctuations in humidity and temperature which are considered limiting factors for plant growth and might have played a key role in the selection of species that compose the present vegetation. After anther release, pollen is generally exposed to various changes of temperature and humidity conditions, therefore its viability and consequently successful fruit set are strongly affected by these environmental parameters. The aim of this research was to study the effect of different combinations of humidity and temperature on pollen membrane integrity of Rosmarinus officinalis L. in order to investigate possible relations between pollen features and climatic conditions during flowering. This species is an evergreen perennial shrub, occurring among the sclerophyllous vegetation of Mediterranean maquis. In many areas of Southern Italy, it shows a flowering period starting from the beginning of winter and spreading throughout spring months. The analysis of data showed that duration of pollen viability of R. officinalis is enhanced by the co-occurrence of low temperature and high humidity. Moreover, these conditions are able to newly raise the viability of pollen kept at higher temperatures and lower humidity. These observations indicate that reactivation of the pollen membrane depends on low temperature apart from high humidity. Therefore “vernalization” of rosemary pollen enhances its viability, supporting that pollen behavior is adapted to winter condition and allows flowering in winter and early spring. Correspondence and reprints: Laboratorio di Botanica ed Ecologia Riproduttiva, Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici, Naples, Italy.     

In vitro propagation ofMedicago andLotus species by node culture

Summary Nodes ofMedicago sativa, Lotus corniculatus, Lotus tenuis, andLotus pedunculatus were cultured on MS basal media with different growth regulators. InM. sativa each node produced one shoot and the apical dominance was unaffected by high levels of cytokinins, and subsequent cycles of culture. Shoot development was stimulated by the presence ofN ⁶-isopentenyl-adenine in the culture medium and was dependent on the genotype of the explant. Shoot development was not affected by the original position of the node on the plant nor by the plant age. Shoots rooted in MS medium gelled with starch and containing 2 mg·liter⁻¹ indol-3-acetic acid. In the threeLotus species, node culture was a more effective technique than inM. sativa. The number of shoots per node increased with the time of culture and with the presence of 0.05 mg·liter⁻¹ of 6-benzylaminopurine. The highest number of shoots derived from one node was achieved inL. pedunculatus and inL. tenuis by culturing single nodes, whereas inL. corniculatus stem segments had to be totally covered by the medium for success. Rooting was easily achieved in MS medium with or without auxins.     

Extracellular proteolytic activity ofCryptococcus neoformans

Eight strains ofCryptococcus neoformans var.neoformans isolated from AIDS patients in the Infectious Disease Institute, University of Turin, Italy, were examined for growth and extracellular proteolytic activity in culture with solid and liquid media. All of the strains grew well on Yeast Carbon Base (YCB) agar medium supplemented with both 0.1% (w/v) bovine serum albumin (BSA) and 0.01% (w/v) polypeptone (Pp), and produced a clear proteolytic zone around their colonies, whereas they exhibited less growth and proteolytic activity on YCB medium supplemented with BSA alone. Strain #8 with a strong proteolytic activity was cultured in three different liquid media. Its growth was limited in YCB medium supplemented with 0.1% BSA, but was moderate in that with 0.01% Pp. Enhanced growth was supported by the addition of both BSA and Pp to the YCB medium. The relative value of the final cellular yields obtained with the above YCB-0.1% BSA, YCB-0.01% Pp and YCB-0.1% BSA-0.01% Pp media was approximately 1:10:20. In the culture with YCB medium containing both BSA and Pp, a rapid decrease in the amount of BSA was demonstrated by a spectrophotometric assay and gel electrophoresis of the culture supernatant after the log-to-stationary phase. The proteolytic activity in the culture supernatant became detectable after the log phase when tested with skim milk agarose plates. These results allowed us to conclude thatCr. neoformans var.neoformans is able to secrete protease and to utilize protein as a source of nitrogen.     

Phage specificity and lipopolysaccarides of stem- and root-nodulating bacteria (Azorhizobium caulinodans, Sinorhizobium spp., and Rhizobium spp.) of Sesbania spp

Phage susceptibility pattern and its correlation with lipopolysaccharide (LPS) and plasmid profiles may help in understanding the phenotypic and genotypic diversity among highly promiscuous group of rhizobia nodulating Sesbania spp.; 43 phages were from two stem-nodulating bacteria of S. rostrata and 16 phages were from root-nodulating bacteria of S. sesban, S. aegyptica and S. rostrata. Phage susceptibility pattern of 38 Sesbania nodulating bacteria was correlated with their LPS rather than plasmid profiles. Different species of bacteria (A. caulinodans- ORS571, SRS1-3 and Sinorhizobium saheli- SRR907, SRR912) showing distinct LPS subtypes were susceptible to different group of phages. Phages could also discriminate the strains of Si. saheli (SSR312, SAR610) possessing distinct LPS subtypes. Phages of Si. meliloti (SSR302) were strain-specific. All the strains of R. huautlense having incomplete LPS (insignificant O-chain) were phage-resistant. In in vitro assay, 100% of the phages were adsorbed to LPS of indicator bacterium or its closely related strain(s) only. These observations suggest the significance of LPS in phage specificity of Sesbania nodulating rhizobia. Highly specific phages may serve as biological marker for monitoring the susceptible bacterial strains in culture collections and environment.     

Active MHC class Ib genes in rat are pseudogenes in the mouse

The most telomeric class I region of the MHC in rat and mouse is the M region, which contains about 20 class I genes or gene fragments. The central part carries three class I genes—M4, M5, and M6—which are orthologous between the two species. M4 and M6 are pseudogenes in the mouse but transcribed, intact genes in the rat. To analyze the pseudogene status for the mouse genes in more detail, we have sequenced the respective exons in multiple representative haplotypes. The stop codons are conserved in all mouse strains analyzed, and, consistent with the pseudogene status, all strains show additional insertions and deletions, taking the genes further away from functionality. Thus, M4 and M6 indeed have a split status. They are silent in the mouse but intact in the closely related rodent, the rat.GenBank accession numbers: AF057065 to AF057072 (exon 3 of H2-M4 of reported mouse strains), AF057976 to AF057985 (exon 3 of RT1.M4 of reported rat strains), AF058923 and AF058924 (exon 2 of RT1.M4 of strains PVG and BN), AY286080 to AY286092 (exon 4 of H2-M6 of reported mouse stains), and AY303772 (full-length genomic sequence of RT1.M6-1^l)     

Spirostomum spp. (Ciliophora, Protista), a suitable system for endocytobiosis research

Summary. Among ciliate genera, only Paramecium and Euplotes species have been studied extensively as host organisms of bacterial endocytobionts. In this article, we show that members of the genus Spirostomum may also serve as a suitable system for endocytobiosis research. Two strains of Spirostomum minus (Heterotrichea, Ciliophora) collected in Germany and Italy, respectively, were found to harbor different types of bacterial infections. Bacteria of various sizes and shapes were observed in the cytoplasm or in the nuclei of the ciliates. The bacteria in the cytoplasm were either surrounded by a peribacterial membrane or lay naked. One of the bacterial species was found in the vicinity of the contractile fibrillar system (myonemes) of the ciliates. In rare cases, another type of bacteria was observed associated with mitochondria. The macronuclei of both the Italian and the German strains were crowded with endocytobionts. The endonuclear bacteria in the two S. minus strains differed with respect to their cytoplasmic structures but they were of similar size and both were rod shaped. According to the results of in situ hybridization, the endonuclear bacteria of the Italian strain belong to the subgroup of alphaproteobacteria, whereas the bacteria associated with the fibrillar system appeared to be gram-positive bacteria with high G+C content. While both the German and the Italian strains were found to permanently maintain their endocytobionts, they were at least partly colonized by different bacteria. This is taken as an indication that geographically separated populations of ciliates may be stably infected by different endocytobionts, possibly due to different ecological conditions. For S. minus and S. ambiguum a total of 7 different bacterial endocytobionts have now been recorded. We recommend the members of the genus Spirostomum as a suitable system for endocytobiosis research. Correspondence and reprints (present address): Laboratorio di Protistologia, Dipartimento di Etologia, Ecologia ed Evoluzione, Università di Pisa, Via A. Volta 4, 56126 Pisa, Italy.     

Cage culture ofKappaphycus alvarezii var.tambalang (Gigartinales,Rhodophyceae)

Kappaphycus alvarezii var.tambalang was cultured in a 3 × 3 m bamboo raft installed inside a 4 × 4 m floating net cage ofLates calcarifer (sea bass) broodstock at SEAFDEC Sub-station Igang, Guimaras, Philippines, from December 1989 to May 1990. Growth and production ofK. alvarezii var.tambalang was influenced by the culture months. The highest growth rate and production were recorded in January and May, respectively, while the lowest growth rate and production were observed in March. Average growth rate (wet weight) ranged from 3.72 to 7.17% day^-1, while production ranged from 575.5 to 2377 g m^-1 line^-1. A total production of approximately 123 t (fresh) or 37 t (dried) ha^-1 in the 5-month harvest season was produced from this culture system. Cultivation ofK. alvarezii var.tambalang in cages is possible, which indicates that seaweeds can be grown with carnivorous finfish, a practice which is still untapped.     

Dominant mutant alleles of yeast protein kinase geneCDC15 suppress thelte1 defect in termination of M phase and genetically interact withCDC14

LTE1 encodes a homolog of GDP-GTP exchange factors for the Ras superfamily and is required at low temperatures for cell cycle progression at the stage of the termination of M phase inSaccharomyces cerevisiae. We isolated extragenic suppressors which suppress the cold sensitivity oflte1 cells and confer a temperature-sensitive phenotype on cells. Cells mutant for the suppressor alone were arrested at telophase at non-permissive temperatures and the terminal phenotype was almost identical to that oflte1 cells at non-permissive temperatures. Genetic analysis revealed that the suppressor is allelic toCDC15, which encodes a protein kinase. Thecdc15 mutations thus isolated were recessive with regard to the temperature-sensitive phenotype and were dominant with respect to suppression oflte1. We isolatedCDC14 as a low-copy-number suppressor ofcdc15-rlt1.CDC14 encodes a phosphotyrosine phosphatase (PTPase) and is essential for termination of M phase. An extra copy ofCDC14 suppressed the temperature sensitivity ofcdc15-rlt1 cells, but not that ofcdc15-1 cells. In addition, some residues that are essential for the Cdc14 PTPase activity were found to be non-essential for the suppression. These results strongly indicate that Cdc14 possesses dual functions; PTPase activity is needed for one function but not for the other. We postulate that the cooperative action of Cdc14 and Cdc15 plays an essential role in the termination of M phase.     

Differentiation ofMelampsora rust species on willows in Japan using PCR-RFLP analysis of ITS regions of ribosomal DNA

To develop a reliable method for identifyingMelampsora species parasitic on willows in Japan, we differentiated 10Melampsora species by PCR-RFLP analysis. Internal transcribed, spacer (ITS) regions, including 5.8S ribosomal DNA, of 63 collections of 10Melampsora species and 4 collections of unidentified species were amplified by PCR. The fragments from the 67 collections varied in size (approximately 880 bp, 860 bp and 840 bp). The restriction sites in the amplified DNA fragments were mapped after the RFLP analysis using four restriction enzymes,Dra I,EcoRI,SspI andTaqI. All the collections were divided into 11 RFLP types. In the 6 species,M. caprearum, M. epiphylla, M. kamikotica, M. larici-urbaniana, M. microsora andM. yezoensis, the RFLP type was species-specific. The RFLP type ofM. chelidonii-pierotii andM. coleosporioides was identical. The collections ofM. epitea were separated into three RFLP types. One of these three types was identical with the type ofM. humilis. It is suggested that the PCR-RFLP analysis of ITS regions is a useful and reliable method for species identification ofMelampsora. Contribution No. 131, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.