Metabolomics and differential gene expression in anthocyanin chemo-varietal forms of Perilla frutescens

We have investigated metabolite profiles and gene expression in two chemo-varietal forms, red and green forms, of Perilla frutescens var. crispa. Striking difference in anthocyanin content was observed between the red and green forms. Anthocyanin, mainly malonylshisonin, was highly accumulated in the leaves of the red form but not in the green form. Less obvious differences were also observed in the stems. However, there was no remarkable difference in the contents and patterns of flavones and primary metabolites such as inorganic anions, organic anions and amino acids. These results suggest that only the regulation of anthocyanin production, but not that of other metabolites, differs in red and green forms. Microscopic observation and immunohistochemical studies indicated that the epidermal cells of leaves and stems are the sites of accumulation of anthocyanins and localization of anthocyanidin synthase protein. By differential display of mRNA from the leaves of red and green forms, we could identify several genes encoding anthocyanin-biosynthetic enzymes and presumptive regulatory proteins. The possible regulatory network leading to differential anthocyanin accumulation in a form-specific manner is discussed.     

Cryptococcus neoformans and Cryptococcus gattii Isolated from the Excreta of Psittaciformes in a Southern Brazilian Zoological Garden

Cryptococcus neoformans, a major pathogen in immunocompromised patients, is a ubiquitous free-living fungus that can be isolated from soils, avian excreta and plant material. To further study potential saprophytic sources of this yeast in the Southern Brazilian State Rio Grande do Sul, we analyzed fecal samples from 59 species of captive birds kept in cages at a local Zoological Garden, belonging to 12 different orders. Thirty-eight environmental isolates of C. neoformans were obtained only from Psittaciformes (Psittacidae, Cacatuidae and Psittacula). Their variety and serotype were determined, and the genetic structure of the isolates was analyzed by use of the simple repetitive microsatellite specific primer M13 and the minisatellite specific primer (GACA)₄ as single primers in the PCR. The varieties were confirmed by pulsed-field gel electrophoresis (PFGE). Thirty-three isolates (87%) were from the var. grubii, serotype A, molecular type VNI and five (13%) were Cryptococcus gattii, serotype B, molecular type VGI. All the isolates were mating type α. Isolates were screened for some potential virulence factors. Quantitative urease production by the environmental isolates belonging to the C. gattii was similar to the values usually obtained for clinical ones.     

Growth of Cryptococcus neoformans in a thiamine-free medium

The growth of Cryptococcus neoformans in a minimal liquid synthetic medium with or without thiamine (10 g/ml) was investigated. In these media the presence or absence of thiamine had no effect on the development of C. neoformans. To check these results, we performed a series of experiments on a solid form of the minimal synthetic medium. In this study a series of six serial transfers were carried out to starve the cells of nutrients that may have been carried over from their growth on rich media. In each of the transfers on the solid synthetic medium, C. neoformans showed a similar and scarce growth. This finding indicates that C. neoformans could be autotrophic in respect to thiamine.     

Structural studies of the capsular polysaccharide of a non-neoformans Cryptococcus species identified as C. laurentii, which was reclassified as Cryptococcus flavescens, from a patient with AIDS

Cryptococcus flavescens, a strain originally identified as C. laurentii, was isolated from the cerebrospinal fluid of an AIDS patient, and the soluble capsular polysaccharide of the yeast was investigated. Glucuronoxylomannan (GXM) was obtained from C. flavescens under conditions similar to those used to obtain C. neoformans polysaccharide. However, the GXM differed from C. neoformans polysaccharide in the decreased O-acetyl group content. The structure of GXM was determined by methylation analysis, partial acid hydrolysis, NMR analyses, and controlled Smith degradation. These analyses indicated that GXM has the following structure: an alpha-(1-->3)-D-mannan backbone with side chains of beta-D-glucuronic acid residues bound to the C-2 position of the mannose residue. The C-6 position of the mannose is substituted with D-man-beta-(1-->4)-D-xyl-beta-(1--> disaccharide. Furthermore, the existence of side chains containing more than two xylose residues was suggested. This mannosylxylose side chain is a novel structure in polysaccharides of C. neoformans and other Cryptococcus species.     

Trypanosoma lewisi-induced immunosuppression: the effects on alveolar macrophage activities against Cryptococcus neoformans

The immunosuppressive effect of Trypanosoma lewisi infection on alveolar macrophage (AM) activities against Cryptococcus neoformans was studied in an animal model. Two groups of rats were treated with T. lewisi and killed after 4 (4d-rats) and 7 days (7d-rats), respectively. A third group not given T. lewisi, served as control. AM were challenged in vitro with C. neoformans. Phagocytosis was assessed with a fluorescence method. Superoxide anion production was evaluated with the nitroblue tetrazolium (NBT) test. The survival of cryptococci was estimated by counting colony-forming units. The numbers of detached AM from culture plates were determined using a Bürker chamber. The NBT response, adhesion to plate surface and killing activity, but not the phagocytosis of AM from 4d-rats were significantly impaired compared to control or 7d-rats. Thus, T. lewisi causes transitory immunosuppressive effects on AM activities. This rapid T. lewisi immunosuppression model may be useful to study new approaches to anticryptococcal therapy.     

Effects of temporal heterogeneity of water supply on the growth of Perilla frutescens depend on plant density

Background and Aims

Plants respond to the spatial and temporal heterogeneity of a resource supply. However, their responses will depend on intraspecific competition for resource acquisition. Although plants are subject to various intensities of intraspecific competition, most studies of resource heterogeneity have been carried out under a single density so that the effects of intraspecific competition on plant responses to resource heterogeneity are largely unknown.

Methods

A growth experiment was performed to investigate plant responses to the temporal heterogeneity of water supply and nutrient levels under multiple plant densities. The annual plant Perilla frutescens was grown using different combinations of frequency of water supply, nutrient level and density, while providing the same total amount of water under all conditions. The effects of the treatments on biomass, allocation to roots and intensity of competition were analysed after 48 d.

Key Results

Biomass and allocation to roots were larger under homogeneous than under heterogeneous water supply, and the effects of water heterogeneity were greater at high density than at low density. The effects of water heterogeneity were greater at high nutrient level than at low level for biomass, while the effects were greater at low nutrient level than high level for allocation to roots. Competition was severer under homogeneous than under heterogeneous water supply.

Conclusions

Competition for water probably makes plants more sensitive to the water heterogeneity. In addition, the intensity of intraspecific competition can be affected by the temporal patterns of water supply. Because both resource heterogeneity and intraspecific competition affect resource acquisition and growth of plants, their interactive effects should be evaluated more carefully under future studies.     

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).     

Isolation, characterization and evolution of a new thermophilic Bacillus licheniformis for lactic acid production in mineral salts medium

The high fermentation cost of lactic acid is a barrier for polylactic acid (PLA) to compete with the petrochemical derived plastics. In order to lower the cost of lactic acid, the industry needs a microorganism that can ferment various sugars at high temperature (50 °C) and at the same time using low cost mineral salts (MS) medium. One such bacterium, BL1, was isolated at 50 °C and identified as Bacillus licheniformis. BL1 can ferment glucose to optically pure l-lactate with a maximum specific productivity of 7.8 g/h l in LB medium and 0.7 g/h l in MS medium at 50 °C. BL1 can also consume 10% and 15% glucose in 20 and 48 h, respectively. After serial transfer of BL1 and BL2 in different concentrations of xylose and MS medium respectively, the final mutant BL3 could efficiently ferment glucose and xylose with specific productivity of 1.9 g/h l and 1.2 g/h l in strict MS medium.     

Synthesis,molecular docking and antibacterial evaluation of new 1,4-naphthoquinone derivatives contains carbazole-6,11-dione moiety

A new series of new 1,4-naphthoquinone derivatives containing carbazole-6,11-dione moiety, which has not been reported yet, has been synthesized from 1,4-naphthoquinone and 4-aminophenylsulfone involving a Michael addition, benzoylation, and Pd-catalyzed coupling. This set of compounds has been evaluated for in vitro antibacterial studies against different Gram-positive and Gram-negative bacteria, and most of the synthesized compounds exhibited good antibacterial activity and the minimum inhibitory concentrations (MICs) are compared with the standard drugs used. Compound 7 exhibited good antibacterial activity among all the molecules studied with the best MIC of 2.1 μg/mL against Bacillus subtilis. To understand the molecular interactions with targeted proteins, the molecular docking of all the synthesized compounds were carried out; between 14 molecules docked, compound 7 was the one with the best glide and E model score of −7.73 and −95.37, respectively. In all docked molecules, compound 5 exhibited least glide and E model score of −4.55 and −101.56, respectively.

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Evaluation of CHROMagar™ StrepB: A new chromogenic agar medium for aerobic detection of Group B Streptococci in perinatal samples

A selective and chromogenic medium, the CHROMagar™ StrepB agar (CHROM-B) designed for aerobic isolation of Group B Streptococci (GBS) in pregnancy-related specimens, was evaluated in a two-Phase study. CHROM-B was evaluated against CPS3 during the first Phase and against Granada afterwards. It was compared to blood agar plates (COH) and to colimycin nalidixic agar plates (CNA) over both Phases. The study which included 1356 samples, yielded 124 GBS. CHROM-B was significantly more sensitive than COH (76.6% vs 53.2% on d1 and 92.7% vs 64.5% on d2; p < 0.001 for both). CHROM-B yielded positive results sooner than CNA. CPS3 under-performed, partly because of microbiota overgrowth and partly because it did not produce a single and unique colour from the GBS colonies. CHROM-B produced its unique GBS-expected colour sooner than Granada yielding a significantly sooner result for 10% (6/60; p < 0.025). Every 124 GBS could grow typical colonies on CHROM-B and False Negatives were only due to paucimicrobial samples. Granada failed to produce the expected colour from one non-haemolytic GBS. We conclude that CHROMagar™ StrepB performed significantly better, irrespective of the haemolytic properties of GBS strains, and significantly sooner than COH, CNA, CPS3 and Granada.     

Morphological and molecular evidence of three species of pikes Esox spp. (Actinopterygii,Esocidae) in France,including the description of a new species

This integrative taxonomy study of French pikes compares morphological characters and molecular sequence data (mitochondrial COI and nuclear Plagl2 genes). In addition to the expected E. lucius, DNA sequences and morphology both support a new species in France, E. aquitanicus sp. nov. from the Charente to the Adour drainages. It is characterized by a color pattern of sides with narrow 1–1.5-scale-wide oblique vertical bands, conferring it a marbled coat, a snout only 0.9 times larger than the postorbital length, an anal fin basis 1.1–1.2 times larger than the caudal peduncle length, 101 to 121 lateral scales, 53 to 57 vertebrae, as well as 24 diagnostic sites in the COI gene and 3 in the Plagl2 gene. Partial COI sequences (131 bp) from modern and historical specimens indicate also the presence of E. cisalpinus and E. lucius during the 19th century in Lake Geneva. Morphological and molecular data points to a possible hybridization between E. lucius with both other local pike species, representing a risk for them. Their endangerment status should be evaluated rapidly in order to take conservation measures.     

The isolation,characterization and antifungal susceptibilities of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> from bird excreta in Klang Valley,Malaysia

The occurrence of Cryptococcus neoformans in bird excreta in Klang valley, Malaysia was determined in this study. Of 544 samples of bird excreta collected from a local zoo, pet shops and public areas, 20 strains of C. neoformans were isolated. All C. neoformans strains were serotype A and thus identified as C. neoformans variety grubii. All did not produce color changes on canavanine–glycine–bromothymol blue agar. All were of α-mating types, as determined by a pheromone-specific PCR assay. The antifungal susceptibility testing using agar diffusion method Neo-sensitabs showed that all were susceptible to amphotericin B, fluconazole and itraconazole.     

Presidential address: rediscovering parasites using molecular tools--towards revising the taxonomy of Echinococcus, Giardia and Cryptosporidium

Molecular biology has provided parasitologists with a fantastic variety of techniques that have had a major impact on research into parasites and parasitism. Molecular tools have revealed the extent and nature of genetic diversity in parasites and this information has made a significant contribution to studies on the population genetics and evolutionary biology of parasites. Similarly, epidemiology has benefited enormously from the application of molecular tools in terms of studying parasite life cycles and transmission, and in the development of specific and sensitive methods for diagnosis and surveillance. However, the theme I wish to develop in this paper is concerned with the contribution molecular tools have made to parasite taxonomy and systematics, and in particular, the fact that in many cases molecular tools are validating the proposals made many years ago by taxonomists and biologists which were discounted or not fully accepted at the time. To do this I have chosen four examples (Echinococcus, Entamoeba, Giardia, Cryptosporidium) where recent research involving molecular characterisation has confirmed observations made many years ago and has resulted in a need to revise the taxonomy of different groups of parasites.     

Molecular phylogeny of Phoma and allied anamorph genera: Towards a reclassification of the Phoma complex

The present generic concept of Phoma is broadly defined, with nine sections being recognised based on morphological characters. Teleomorph states of Phoma have been described in the genera Didymella, Leptosphaeria, Pleospora and Mycosphaerella, indicating that Phoma anamorphs represent a polyphyletic group. In an attempt to delineate generic boundaries, representative strains of the various Phoma sections and allied coelomycetous genera were included for study. Sequence data of the 18S nrDNA (SSU) and the 28S nrDNA (LSU) regions of 18 Phoma strains included were compared with those of representative strains of 39 allied anamorph genera, including Ascochyta, Coniothyrium, Deuterophoma, Microsphaeropsis, Pleurophoma, Pyrenochaeta, and 11 teleomorph genera. The type species of the Phoma sections Phoma, Phyllostictoides, Sclerophomella, Macrospora and Peyronellaea grouped in a subclade in the Pleosporales with the type species of Ascochyta and Microsphaeropsis. The new family Didymellaceae is proposed to accommodate these Phoma sections and related anamorph genera. The present study demonstrated that Phoma radicina, the type species of Phoma sect. Paraphoma and Phoma heteromorphospora, the type species of Phoma sect. Heterospora can be assigned to the Phaeosphaeriaceae and Leptosphaeriaceae respectively.     

Morphological and molecular characterization of a new microsporidian species from the predatory mite Metaseiulus occidentalis (Nesbitt) (Acari,Phytoseiidae)

A new microsporidian species is described from the predatory mite Metaseiulus (formerly Typhlodromus or Galendromus) occidentalis (Nesbitt) (Acari, Phytoseiidae). The ultrastructure of this new species is presented together with the first molecular characterization for a microsporidium of mites. All stages of this new microsporidium are haplokaryotic and develop in direct contact with the host-cell cytoplasm. Sporogony is disporoblastic and spores are formed in eggs, immature stages, and adults of M. occidentalis. There are two morphological classes of spores, one with a short polar filament (3-5 coils) that measured 2.53 x 1.68 microm and one with a longer polar filament (8-9 coils) that measured 3.14 x 1.77 microm. Horizontal transmission of this new species occurs by cannibalism of eggs and other stages and perhaps involves the spores with the long polar filament. Spores with the short polar filament may play a role in autoinfection and vertical (transovarial) transmission that is highly efficient in transferring the microsporidium from adults to progeny. Analysis of the small subunit ribosomal DNA indicated that this species from M. occidentalis is most closely related to the Nosema/Vairimorpha clade of microsporidia. A conflict between the morphological and molecular data is discussed. The species is compared to previously described microsporidia of arachnids resulting in creation of Oligosporidium occidentalis n. sp. in the family Unikaryonidae.     

Zoanthids (Cnidaria: Hexacorallia: Zoantharia) from shallow waters of the southern Chilean fjord region, with descriptions of a new genus and two new species

The taxonomy of the order Zoantharia (=Zoanthidea=Zoanthiniaria) is greatly hampered by the paucity of diagnostic morphological features. To facilitate discrimination between similar zoanthids, a combination of morphological and molecular analyses is applied here. The three most abundant zoanthid species in shallow waters of the southern Chilean fjord region are described. Comparison with other zoanthids using molecular markers reveals that two of them are new to science; these are described as Mesozoanthus fossii gen. n., sp. n. and Epizoanthus fiordicus sp. n. Their representatives grow on rocky substratum and do not live in symbiosis with demosponges. In the less abundant M. fossii, animals are greyish in colour and resemble members of Parazoanthus in growth form. Individual polyps can be up to 35 mm long. The more abundant E. fiordicus are also greyish; the polyps arise from thin stolons and reach only 12 mm in length. The third species studied is Parazoanthus elongatus McMurrich, 1904. For these three Chilean zoanthid species, in-situ photographs are presented as well as information on distribution, habitat and associated species. Establishment of the Mesozoanthus gen. n. is of particular importance to taxonomy in the chaotic suborder Macrocnemina.