Pectinolytic yeasts from cold environments: novel findings of Guehomyces pullulans,Cystofilobasidium infirmominiatum and Cryptococcus adeliensis producing pectinases

One hundred and three yeasts isolated from soil samples from King George Island and Tierra del Fuego province were screened in relation with their capability to produce pectinolytic enzymes. Although all the yeasts showed well-developed colonies at 20 °C, only eight showed a clear halo around the colony, indicative of pectin degradation. A secondary screening demonstrated that only four yeasts were capable to produce pectinases at low temperatures (8 °C). It could be seen that the selected yeasts were able to grow and produce high levels of polygalacturonase activity when submerged fermentations were performed using pectin-containing fruit wastes as substrates. None of the strains produced neither lyase nor rhamnogalacturonan hydrolase activities. Regarding pectin esterase activity, it was only produced in lower amounts by G. pullulans 8E (0.022 U ml⁻¹). A TLC analysis of the substrate cleavage pattern of the pectinolytic systems was consistent with an endo-type activity. The clarification of apple juice was only accomplished by G. pullulans pectinolytic system, with a clarification of 80% (%T₆₅₀) using 4 U/ml of enzyme at 20 °C. As far as we concern this work describes for the first time the production of pectinases by the cold-adapted yeasts species Cystofilobasidium infirmominiatum, Cryptococcus adeliensis and G. pullulans.

     

Biosynthesis of the xanthophyll plectaniaxanthin as a stress response in the red yeast Dioszegia (Tremellales, Heterobasidiomycetes, Fungi)

Carotenoid biosynthesis was examined in a phylloplane yeast identified by ITS, 18S and 28S rDNA analysis as a Dioszegia sp. close to D. takashimae. In well-aerated flask or fermentor cultures, this strain produced essentially a single pigment confirmed as the xanthophyll plectaniaxanthin by NMR analysis, at concentrations of 103-175 microgg(-1) biomass dry weight. Detailed studies showed increases in plectaniaxanthin concentrations in the presence of 5 mM hydrogen peroxide (1.8-fold), 50 and 100 microM duroquinone (3.1- and 3.7-fold, respectively), and 2% ethanol (4.9-fold). Whereas oxidative stress is known to enhance the biosynthesis of torularhodin or astaxanthin in other red yeasts where they are associated with an antioxidant function, this is the first report implicating plectaniaxanthin in a similar role. At reduced aeration, biosynthesis of plectaniaxanthin was suppressed and its putative precursor gamma-carotene accumulated. The carotenoid cyclase inhibitor nicotine (5-20 mM) inhibited plectaniaxanthin formation, with lycopene accumulating stoichiometrically. Hydroxy groups at C-1' and C-2' therefore seem to be introduced late in plectaniaxanthin biosynthesis, following cyclization of the beta-ionone ring.     

C(15) Acetogenins from the red alga Laurencia obtusa.

Four C(15) acetogenins, 13-epilaurencienyne (3Z) (1), 13-epipinnatifidenyne (3E) (2), (3E, 6S(*), 7R(*), 9S(*), 10S(*), 12R(*))-9-chloro-13-bromo-6:12-epoxy-7, 10-diacetoxypentadec-3-en-1-yne (3), (3Z, 6S(*), 7R(*), 9S(*), 10S(*), 12R(*))-9-chloro-13-bromo-6:12-epoxy-7, 10-diacetoxypentadec-3-en-1-yne (4), along with the known 13-epilaurencienyne (3E) (5), have been isolated from the organic extract of the red alga Laurencia obtusa, collected in the Aegean Sea, Greece. The structures of the new natural products, as well as their relative stereochemistry, were established by means of spectral data analysis, including 2D NMR spectroscopic experiments. Some of the new metabolites exhibited significant insecticidal activity.     

A single five-step desaturase is involved in the carotenoid biosynthesis pathway to beta-carotene and torulene in Neurospora crassa

Phytoene desaturase Al-1 from Neurospora crassa was expressed in Escherichia coli and an active enzyme was isolated which catalyzed the stepwise introduction of up to five double bonds into the substrate phytoene. The major reaction products were 3, 4-didehydrolycopene and lycopene. Several of the desaturation intermediates, zeta-carotene, neurosporene, and lycopene, were also accepted as a substrate by Al-1. In contrast to the structurally related bacterial enzymes, the cofactor involved in the dehydrogenation reaction was NAD for Al-1. In situ competition with a neurosporene- and lycopene-converting hydratase and cyclase indicated that these enzymes can divert intermediates of the desaturation sequence. Based on the in vitro and in vivo results, the organization of the phytoene desaturase from N. crassa was proposed as an assembly of identical protein units which are responsible for the multistep reaction. However, the spatial arrangement should be loose enough to allow an exchange of individual intermediates in both directions in and out of this complex. Since gamma-carotene is not accepted as a substrate by Al-1, the formation of torulene must proceed exclusively by the cyclization of 3,4-didehydrolycopene.     

Sulfated xylomannans isolated from red seaweeds Chondrophycus papillosus and C. flagelliferus (Ceramiales) from Brazil

Sulfated xylomannans were isolated from two species of genus Chondrophycus by aqueous extraction followed by KCl fractionation. Structural determination of the native, desulfated and Smith-degraded KCl-precipitated polysaccharides carried out by composition and methylation analysis and NMR spectroscopy (1D and 2D experiments) showed the following general structure: [see text] These xylomannans present different degrees of branching (15-25%) by beta-D-Xylp (70-80%) and beta-D-Manp-2-S (20-30%) and molecular weights (33-222kDa). This is the first report of the presence of a sulfated xylomannan in species of order Ceramiales.     

Fungi from the A. O. Garrett Herbarium,University of Utah (UT)

The University of Utah donated fungi from the A. O. Garrett Herbarium to the New York Botanical Garden (NY) in 1982. Most specimens are members either of the Ustilaginales or Uredinales (Basidiomycotina). Biographical data are given for some of the most important regional collectors represented: Albert O. Garrett, George L. Zundel, Seville Flowers, Marcus E. Jones, and Patrick J. O'Gara. Other contributors are listed in the appendix.     

Torularhodin and torulene are the major contributors to the carotenoid pool of marine Rhodosporidium babjevae (Golubev)

A carotenoid-producing yeast strain, isolated from the sub-arctic, marine copepod Calanus finmarchicus, was identified as Rhodosporidium babjevae (Golubev) according to morphological and biochemical characteristics and phylogenetic inference from the small-subunit ribosomal RNA gene sequence. The total carotenoids content varied with cultivation conditions in the range 66–117 μg per g dry weight. The carotenoid pool, here determined for the first time, was dominated by torularhodin and torulene, which collectively constituted 75–91% of total carotenoids under various regimes of growth. β-Carotene varied in the range 5–23%. A high-peptone/low-yeast extract (weight ratio 38:1) marine growth medium favoured the production of torularhodin, the carotenoid at highest oxidation level, with an average of 63% of total carotenoids. In standard yeast medium (YM; ratio 1.7:1), torularhodin averaged 44%, with increased proportions of the carotenes, torulene and β-carotene. The anticipated metabolic precursor γ-carotene (β,ψ-carotene) constituted a minor fraction (≤8%) under all conditions of growth.     

The role of proteins in C(3) plants prior to their recruitment into the C(4) pathway

Our most productive crops and native vegetation use a modified version of photosynthesis known as the C(4) pathway. Leaves of C(4) crops have increased nitrogen and water use efficiencies compared with C(3) species. Although the modifications to leaves of C(4) plants are complex, their faster growth led to the proposal that C(4) photosynthesis should be installed in C(3) crops in order to increase yield potential. Typically, a limited set of proteins become restricted to mesophyll or bundle sheath cells, and this allows CO(2) to be concentrated around the primary carboxylase RuBisCO. The role that these proteins play in C(3) species prior to their recruitment into the C(4) pathway is addressed here. Understanding the role of these proteins in C(3) plants is likely to be of use in predicting how the metabolism of a C(3) leaf will alter as components of the C(4) pathway are introduced as part of efforts to install characteristics of C(4) photosynthesis in leaves of C(3) crops.     

Leaf anatomical characters in relation to the C3 and C4 photosynthetic pathway in Cyperus (Cyperaceae)

Leaf anatomical characters of twelve species from the genus Cyperus, a genus known to contain species with both C₃ and C₄ plants, have been investigated. We investigated and established the usefulness of all‐inclusive functional leaf anatomical characters for identifying the photosynthetic pathways of these species. The species investigated were C. articulatus L., C. compressus L., C. difformis L., C. dilatatus Schum. & Thonn., C. distans L., C. esculentus L., C. haspan L., C. imbricatus Retz., C. iria L., C. rotundus L., C. sphacelatus Rottb. and C. tenuiculmis (Boeck.) Hooper, collected from locations in southwestern Nigeria. Standard anatomical procedures for examining epidermal and cross sections of leaves were employed. Our data suggested that a combination of characters, such as the occurrence of Kranz tissue, maximum cell distant count, maximum lateral cell count, interveinal distance, and to some extent leaf and mesophyll thickness, provide a reliable basis for the assessment of the photosynthetic pathways of the investigated species as compared to the isolated characters used previously. The study indicate that C. difformis and C. haspan are C₃ species while the rest follow the C₄ photosynthetic pathway. A salient feature of this study is the identification of C. dilatatus as belonging to the C₄ group.     

Studies in Heterobasidiomycetes, Part 31*). On the Anther Smuts of Silene vuigaris (Moench) Garcke

Microbotryum silenes-inflatae (DC) G. Deml & Oberw. and Microbotryum violaceoirregulare (Brandenburger & Schwinn) G. Deml & Oberw., both parasitic in the anthers of Silene vulgaris (Moench) Garcke, are compared by their morphology, karyology, and some cultural characteristics. The infection of their hosts is similar in both species, but causes different sizes of the flowers and different color of the spore mass. The teliospore initials of that smuts are produced in dikaryotic sporogenous hyphae. During gelatinization of the hyphal coat, surface ornamentations on the teliospores are formed. Mature teliospores are monokaryotic, and presumably diploid, reticulate in Microbotryum silenes-inflatae and echinulate in M. violaceo-irregulare. Teliospores of both species germinate without a resting periode. Germination results in a commonly three-celled promycelium, which in M. silenes-inflatae separates from the producing teliospore, while in M. violaceo-irregulare it remains on the teliospore during basidiospore formation. The basidiospores are monokaryotic and propagate by budding. Monokaryotic strains were isolated and characterized by standard yeast identification tests and by some enzymatic activities on solid media. While no differences in the enzymatic activities can be found, the species differ in their utilization of carbon sources.     

Torulene and torularhodin,protects human prostate stromal cells from hydrogen peroxide-induced oxidative stress damage through the regulation of Bcl-2/Bax mediated apoptosis

The current study was designed to elucidate the cytoprotective effects and possible mechanisms of torulene and torularhodin on hydrogen peroxide (H₂O₂)-induced oxidative stress damage in human prostate stromal cells (WPMY-1). After treated with H₂O₂, a notable decrease was appeared in cell viability, yet the decrease was attenuated when cells were pretreated with torulene and torularhodin (0.5–10?μM) as evaluated by WST-1 assay. Pretreatment with these two carotenoids significantly attenuated H₂O₂-induced apoptosis in WPMY-1 cells through the inhibition of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) overproduction, as well as the activation of the activities in catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Finally, pretreatment of cells with carotenoids resulted in the regulation of the mRNA and protein expression of Bcl-2 and Bax in H₂O₂-exposed prostate stromal cells. The present results indicate that both torulene and torularhodin can protect human prostate stromal cells from oxidative stress damage via Bcl-2/Bax mediated apoptosis.     

Detection of short-chain carbonyl products of lipid peroxidation from malaria-parasite (Plasmodium vinckei)-infected red blood cells exposed to oxidative stress.

Reversed-phase h.p.l.c. was used to detect 2,4-dinitrophenylhydrazine-reactive carbonyl products, which excludes malonaldehyde, in malaria-parasite (Plasmodium vinckei)-infected murine red blood cells (RBCs). A number of alkanals, 4-hydroxyalk-2-enals and alka-2,4-dienals were tentatively identified by comparison with authentic standards. The formation of 4-hydroxynon-2-enal, deca-2,4-dienal and hexanal was greater in P. vinckei-infected RBCs than in their uninfected counterparts and was increased by the presence of t-butyl hydroperoxide. Several of these aldehydes have previously been shown to be toxic to various types of cells, including P. falciparum, in vitro. The iron chelator desferrioxamine and the free-radical scavenger butylated hydroxyanisole inhibited the formation of these aldehydes. These experiments demonstrate that products of lipid peroxidation other than malonaldehyde are formed during the exposure of malaria-infected RBCs in vitro to drugs that generate reactive oxygen species and have anti-parasitic activity. The formation of products of this type during the natural course of malaria infection may have implications for the mechanisms underlying intra-RBC parasite death and the tissue damage associated with the disease.     

Progamotaenia ruficola sp. n. (Cestoda: Anoplocephalidae) from the red kangaroo, Macropus rufus (Marsupialia)

Progamotaenia ruficola sp. n. is described from the red kangaroo (Macropus rufus) from New South Wales, Australia. It is distinguished from other similar species having paired, nondiverticulate uteri in each prolottis: Progamotaenia festiva (Rudolphi 1819), P. diaphana (Zschokke 1907), and P. macropodis Beveridge 1976, by the extremely broad, straight-edged velum, the lack of an external seminal vesicle, the cirrus armature, the number of testes, the lack of vaginal atrophy following insemination, and the morphology of the egg.     

C-banding patterns in chromosomes and sperm of Strophosoma capitatum (De Geer, 1775) (Coleoptera: Curculionidae, Brachyderinae)

An analysis was made of the C-banded karyotype of Strophosoma capitatum (Deg.). The results indicate that the chromosome number is 2n = 22 and n Male = 10 + Xyp. The examined karyotype shows a pericentromeric position of constitutive heterochromatin in all autosomes. The shorter arm of the X chromosome is heterochromatic while the y chromosome is wholly euchromatic. Successive stages of spermatogenesis were analysed.     

Contribution of polyol pathway to arteriolar dysfunction in hyperglycemia. Role of oxidative stress, reduced NO, and enhanced PGH(2)/TXA(2) mediation

Hyperglycemia increases glucose metabolism via the polyol pathway, which results in elevations of intracellular sorbitol concentration. Thus we hypothesized that elevated level of sorbitol contributes to the development of hyperglycemia-induced dysfunction of microvessels. In isolated, pressurized (80 mmHg) rat gracilis muscle arterioles (approximately 150 microm), high glucose treatment (25 mM) induced reduction in flow-dependent dilation (from maximum of 39 +/- 2% to 15 +/- 1%), which was significantly mitigated by an aldose reductase inhibitor, zopolrestat (maximum 27 +/- 2%). Increasing doses of sorbitol (10(-10)-10(-4) M) elicited dose-dependent constrictions (maximum 22 +/- 3%), which were abolished by endothelium removal, a prostaglandin H(2)/thromboxane A(2) (PGH(2)/TXA(2)) receptor (TP) antagonist SQ-29548, or superoxide dismutase (SOD) plus catalase (CAT). Incubation of arterioles with sorbitol (10(-7) M) reduced flow-dependent dilations (from maximum of 39 +/- 2% to 20 +/- 1.5%), which was not further affected by inhibition of nitric oxide synthase by N(omega)-nitro-l-arginine methyl ester but was prevented by SOD plus CAT and mitigated by SQ-29548. Nitric oxide donor sodium nitroprusside-induced (10(-9)-10(-6) M) dilations were also decreased in a SQ-29548 and SOD plus CAT-reversible manner, whereas adenosine dilations were not affected by sorbitol exposure. Sorbitol significantly increased arterial superoxide production detected by lucigenin-enhanced chemiluminescence, which was inhibited by SOD plus CAT. Sorbitol treatment also increased arterial formation of 3-nitrotyrosine. We suggest that hyperglycemia by elevating intracellular sorbitol induces oxidative stress, which interferes with nitric oxide bioavailability and promotes PGH(2)/TXA(2) release, both of which affect regulation of vasomotor responses of arterioles. Thus increased activity of the polyol pathway may contribute to the development of microvascular dysfunction in diabetes mellitus.     

Development of microsatellite markers for the red tide‐forming harmful species Chattonella antiqua,C. marina,and C. ovata (Raphidophyceae)

We have developed 11 microsatellite markers that are specific to Chattonella antiqua, C. marina, and C. ovata, the red tide‐forming harmful phytoplanktons. The 11 loci were amplified in the three species. The number of alleles per locus ranged from 5 to 16. The three species shared most microsatellite regions, although the genetic differences in specific loci were detected among them. These markers of the Chattonella species will be beneficial for biogeographical, detailed taxonomic, studies.     

Onchocercosis in red deer (Cervus elaphus) from Spain.

Onchocercosis, caused by Onchocerca flexuosa, was observed in red deer (Cervus elaphus) from Spain for the first time. Adult specimens of P. flexuosa were found in nodules in subcutaneous tissues in 42 of 125 (33%) red deer between October 1994 and September 1995; intensity of infection +/- SD was 3.93 +/- 5.26 nodules per infected host. A clear seasonal pattern in the distribution of nodules was observed, with higher values of prevalence and intensity in fall and winter in contrast to spring and summer. Significant differences were found among age groups in prevalence, but not in the mean intensity. No differences in infection were apparent between male and female adult red deer.     

Metamorphosis season from megalopa to the first crab stage in snow crab Chionoecetes opilio and red snow crab C. japonicus (Crustacea,Decapoda, Majoidea) in the Sea of Japan,estimated from captive culture

ABSTRACT

Metamorphosis season of megalopae to the first crab stage in snow crab Chionoecetes opilio and red snow crab C. japonicus was inferred by culturing wild-born megalopae collected from the Sea of Japan. Metamorphosis occurred from late June to late July (mainly in July) in snow crab, and from early July to early October (mainly from August to September) in red snow crab. The number of days required from the time of collection to metamorphosis was less than the intermoult period previously reported for snow crab megalopae. However, the developmental period of the megalopae was estimated as substantially longer in red snow crab than in snow crab. Previous studies have shown that the hatching season and the period of the zoeal stage in both two species are similar. These results suggest that a different metamorphosis season between the two species would be due to a difference in their megalopal intermoult period.