Fungal endophyte diversity in tundra grasses increases by grazing

Fungal endophytes are species rich and ubiquitous, yet, apart from the genus Epichloë, their ecology is largely unknown. Here we explore how herbivores affect the diversity of fungal endophytes in tundra grasslands. We assess both hyphal morphological and taxonomic diversity in grass individuals.By microscopic examination we identified endophytes to be present in all sampled grass individuals whereas identification to taxonomic units were only achieved in a subset of the individuals using laser micro dissection pressure catapulting and culturing for endophyte isolation. Hyphal morphological diversity was significantly higher in grasses exposed to grazing, along with 45 % more taxonomic units achieved.Our results suggest that grazing is an important mediator of fungal endophyte diversity in tundra grasslands. Furthermore, we suggest laser dissection of stained endophytes as a method for further exploring the ecological role of fungal endophytes.     

Entamoeba histolytica: flavins in axenic organisms.

The individual flavin species of axenic Entamoeba histolytica were assayed: separated riboflavin was assayed by the lumiflavin method; flavin-adenine dinucleotide (FAD), by an enzymatic method; flavin mononucleotide (FMN) was calculated from the difference, total flavin minus FAD and riboflavin. The amount of flavin in micrograms per grams fresh cells follows: total flavin, 7.6 ± 0.9 calculated as riboflavin; riboflavin, 1.6 ± 0.7; FMN, 6.6 ± 0.5; and FAD, 1.2 ± 0.1. Recalculated to nanomoles per milligrams total amebal protein these values were: total flavin, 0.21; riboflavin, 0.04; FMN, 0.15; and FAD, 0.02. The identity of each flavin was confirmed by a paper chromatographic method. Analyses on Panmede, the main source of flavins in the TP-S-1 medium, indicate that it contains all three forms of flavin. Its contribution to growth medium in micrograms per milliliters: riboflavin, 2.1 ± 0.3; FMN, 0.6 ± 0.1; and FAD, 0.4 ± 0.1. The in vivo biosynthesis of FMN and FAD from riboflavin by E. histolytica is demonstrated. A new and convenient method was found to separate riboflavin from flavin nucleotides in tissue extracts.     

Selenium deficiency in Crustacea

Summary The effect of selenium deprivation onDaphnia magna was examined under controlled rearing conditions in a synthetic culture medium. After three generations, fertility was significantly reduced in deprived (Se^–) animals. Growth and mortality of parent daphnids and development of parthenogenetic eggs were not affected during this period. In the fourth generation Se^– daphnids rejected parts of their second antennae. At the ultrastructural level antennal muscle tissue was severely affected. Animals deprived of selenium had mitochondria and sarcoplasmic reticulum with myelin-like alterations. Giant lysosomes were present and complete lysis of muscle fibrils was observed in antennal muscle cells. These alterations are characteristic features of peroxidic damage in tissues. This interpretation is consistent with the function of selenium as a constituent of the enzyme glutathione peroxidase which protects cells from peroxidation. Selenium should be included in synthetic culture media for daphnids.Abbreviations GSH-Px selenium dependent glutathione peroxidase - Se⁺/Se^– selenium supplemented (control)/selenium deprived animals - SOD Superoxide dismutase - SR sarcoplasmic reticulum     

Phenotypic and molecular analysis of dominant occurring antibiotic active-producing Streptomyces soil flora in Northern Jordan

This investigation aimed to determine the relatedness of dominant occurring soil Streptomyces spp. in Northern Jordan based on their RAPD-PCR fingerprints, and to compare RAPD technique with the conventional phenotypic characterization of Streptomyces isolates. Fifty-eight white and gray color-bearing aerial mycelia antibiotic active-producing Streptomyces soil isolates along with three reference strains were genetically analyzed by RAPD-PCR. Polymorphisms between the isolates showed 1 to 10 bands per isolate and ranged from 200 to 3200 bp in size. Results revealed one common band of ~600 bp shared by ~85% of the isolates, and the observation of bands specific to some reference strains and some soil isolates. When RAPD patterns were analyzed with the UPGMA, results revealed clustering the tested isolates into two equal main super clusters (50% each). Super cluster I appeared to be homogenous and include the three reference strains. However, super cluster II was heterogeneous and but not including any of the reference strains. The association of the antibiotic activity of the dominant white and gray aerial mycelium-bearing Streptomyces isolates to RAPD clustering is reported for the first time, and the RAPD-PCR fingerprints generated here deserve to be cloned, characterized and sequenced in future as Streptomyces species-specific DNA markers. The more random primers used in the analysis may add to RAPD technique a cost-effective, fast, precise result, and less labor work solution for analyzing the similarities and differences among the Streptomyces isolates.     

Characterization of a co-culture system of neurons and hepatocytes

A co-culture system of cerebellar granule cells (glutamatergic neurons) and hepatocytes has been developed. Petri dishes divided in halves by a temporary septum were coated with poly-L-lysine and cerebellar granule cells plated in one of the compartments. Five days later hepatocytes were plated in the other compartment and after 2 days the septum was removed and the two cell types shared the same culture medium for a period of 5 days. During this period of time cultures of neurons and hepatocytes kept separately or in co-culture exhibited identical characteristics with regard to activities of pyruvate kinase and glucokinase (hepatocytes), aspartate aminotransferase (neurons) as well as evoked transmitter release (neurons) and content of cytochrome P-450 (hepatocytes). The results show that it is possible to maintain neurons and hepatocytes in co-culture sharing the same culture medium for a prolonged period of time. Such a system may serve as a pharmacological model to study interactions between liver and brain cells with regard to neuroactive drugs.     

The influence of paternal species on the origin of callus in anther culture of Solanum hybrids

Summary Stamen culture of several Solanum species and interspecific hybrids was performed and each stamen was scored for presence and origin of callus. Each species and hybrid has a characteristic callus growth pattern — either no growth (0), callus growth from the filament (F), or callus growth from the anther (A). Characteristic growth types of the interspecific hybrids indicate that callus growth takes precedence over no growth. Hybrids between F and A species exhibit mainly paternal passage of either characteristic callus type. Possible explanations for this pattern of inheritance are male plastome factors or imprinting of paternal genes. The latter explanation is presently preferred.Paper No. 2084 from the Laboratory of Genetics. Research supported by the College of Agricultural and Life Sciences, a gift from Frito-Lay, Inc., and grants from NIH (GM15422) and the International Potato Center.     

Changes in ion composition and hexitol content of differentPlantago species under the influence of salt stress

Summary The effect of salinity on the ionic balance and the hexitol content of the halophyteP. maritima L. and the nonhalophytesP. major L. ssp.major, P. lanceolata L., andP. media L. was studied in a culture experiment. In response to salt application the nonhalophilous species increased their internal electrolyte content to a considerably greater extent at high transpiration thanP. maritima. Excessive uptake of Cl and SO₄ following salt-treatment was in most cases compensated by a decline in the total organic anion content in all species under investigation. Na was strongly accumulated in the shoots ofP. maritima when subjected to salt-stress, while the nonhalophytic species tended to exclude this ion from leaf tissue enhancing Mg-uptake for charge balance. Acyclic polyhydric alcohols (sorbitol and mannitol), the dominant soluble carbohydrates in allPlantago species studied, increased, with one exception, in all plants under saline conditions. The results indicate marked physiological differences between the halophyteP. maritima and the nonhalophytic members of the genusPlantago in their reaction to salinity being perhaps partially responsible for differences in the degree of salt tolerance.     

Primary sequence differences between Chido and Rodgers variants of tryptic C4d of the human complement system

The initiation sites of the Galleria mellonella L. nuclear polyhedrosis virus (G.m. NPV) DNA replication were revealed. For this purpose SCLd 135 cells permitting the G.m. NPV productive reproduction were transformed by the recombinant plasmids containing the viral genome individual fragments in pRSF 2124 and pBR 322 vectors. It was revealed that 2 of the 32 recombinant plasmids can autonomously replicate in the eucaryotic cells. According to the Maxam-Gilbert method the DNA G.m. NPV fragment (1300 bp) primary structure of pHBR plasmid was determined. The structure analysis revealed the typical regulator signals as in the replicons. The possible regulation mechanism of the DNA G.m. NPV synthesis initiation was supposed.     

Thiol antioxidants protect human lens epithelial (HLE B-3) cells against tert-butyl hydroperoxide-induced oxidative damage and cytotoxicity

Oxidative damage to lens epithelial cells plays an important role in the development of age-related cataract, and the health of the lens has important implications for overall ocular health. As a result, there is a need for effective therapeutic agents that prevent oxidative damage to the lens. Thiol antioxidants such as tiopronin or N-(2-mercaptopropionyl)glycine (MPG), N-acetylcysteine amide (NACA), N-acetylcysteine (NAC), and exogenous glutathione (GSH) may be promising candidates for this purpose, but their ability to protect lens epithelial cells is not well understood. The effectiveness of these compounds was compared by exposing human lens epithelial cells (HLE B-3) to the chemical oxidant tert-butyl hydroperoxide (tBHP) and treating the cells with each of the antioxidant compounds. MTT cell viability, apoptosis, reactive oxygen species (ROS), and levels of intracellular GSH, the most important antioxidant in the lens, were measured after treatment. All four compounds provided some degree of protection against tBHP-induced oxidative stress and cytotoxicity. Cells treated with NACA exhibited the highest viability after exposure to tBHP, as well as decreased ROS and increased intracellular GSH. Exogenous GSH also preserved viability and increased intracellular GSH levels. MPG scavenged significant amounts of ROS, and NAC increased intracellular GSH levels. Our results suggest that both scavenging ROS and increasing GSH may be necessary for effective protection of lens epithelial cells. Further, the compounds tested may be useful for the development of therapeutic strategies that aim to prevent oxidative damage to the lens.     

Genetic characterization of chytrids isolated from larval amphibians collected in central and east Texas

Chytridiomycosis, an emerging infectious disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), has caused amphibian population declines worldwide. Bd was first described in the 1990s and there are still geographic gaps in the genetic analysis of this globally distributed pathogen. Relatively few genetic studies have focused on regions where Bd exhibits low virulence, potentially creating a bias in our current knowledge of the pathogen's genetic diversity. Disease-associated declines have not been recorded in Texas (USA), yet Bd has been detected on amphibians in the state. These strains have not been isolated and characterized genetically; therefore, we isolated, cultured, and genotyped Bd from central Texas and compared isolates to a panel of previously genotyped strains distributed across the Western Hemisphere. We also isolated other chytrids from east Texas not known to infect amphibians. To identify larval amphibian hosts, we sequenced part of the COI gene. Among 37 Bd isolates from Texas, we detected 19 unique multi-locus genotypes, but found no genetic structure associated with host species, Texas localities, or across North America. Isolates from central Texas exhibit high diversity and genetically cluster with BdGPL isolates from the western U.S. that have caused amphibian population declines. This study genetically characterizes isolates of Bd from the south central U.S. and adds to the global knowledge of Bd genotypes.