Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C, with a t ₅₀ of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase).     

Streptomyces sp. ASBV‐1 reduces aflatoxin accumulation by Aspergillus parasiticus in peanut grains

Aims: To evaluate the ability of Streptomyces sp. (strain ASBV‐1) to restrict aflatoxin accumulation in peanut grains. Methods and Results: In the control of many phytopathogenic fungi the Streptomyces sp. ASBV‐1 strain showed promise. An inhibitory test using this strain and A. parasiticus was conducted in peanut grains to evaluate the effects of this interaction on spore viability and aflatoxin accumulation. In some treatments the Streptomyces sp ASBV‐1 strain reduced the viability of A. parasiticus spores by c. 85%, and inhibited aflatoxin accumulation in peanut grains. The values of these reductions ranged from 63 to 98% and from 67% to 96% for aflatoxins B₁ and G₁, respectively. Conclusions: It was demonstrated that Streptomyces sp. ASBV‐1 is able to colonize peanut grains and thus inhibit the spore viability of A. parasiticus, as well as reducing aflatoxin production. Significance and Impact of the Study: The positive finding for aflatoxin accumulation reduction in peanut grains seems promising and suggests a wider use of this actinobacteria in biological control programmes.     

Gill infection of Leporinus macrocephalus Garavello & Britski, 1988 (Osteichthyes: Anostomidae) by Henneguya leporinicola n. sp. (Myxozoa: Myxobolidae). Description, histopathology and treatment

Piau?us (Leporinus macrocephalus), were raised in 300 m2 ponds (density of 10 fish/m2) presenting asphyxia signals and daily mortality of 27 fishes. Specimens with 8-cm total body length, were collected for necropsy. Mucus of body surface and pieces of organs were collected and examined microscopically, in wet mounts, stained or in histological sections. The smears examination showed the presence of several spores in the secondary lamellae of the gill filaments, identified as Henneguya leporinicola n.sp (Myxozoa: Myxobolidae). Histopathological study showed epithelial hyperplasia and fulfilling of the spaces between the secondary lamellae, congestion and telangiectasia sinusoidal. It was also observed hyperplasia of the goblet cells and several cysts of parasite with 70.3 microns diameter. Such cysts were situated among the secondary lamellae, covered or not by the hyperplasic epithelium. With this diagnostic, three applications of formalin solution 10 ml/m3 were carried out. Fifteen days after that, fish were examined again to ascertain whether the treatment was efficient on disease caused by the protozoa. The tissue alterations present in the gills after the treatment were just a moderate sinusoidal congestion and a slight epithelial hyperplasia on the base of the secondary lamellae.     

Additional observations on the gross morphology and microstructure of Baccaconularia Hughes, Gunderson et Weedon, 2000, a Cambrian (Furongian) conulariid from the north-central USA

Baccaconularia Hughes, Gunderson et Weedon, 2000, from the Furongian Series (Cambrian System) of the north-central USA, has been interpreted as a conulariid cnidarian, based on a suite of gross morphological similarities shared only with other post-Cambrian genera currently assigned to this group. Closely spaced, squarish to subrectangular facial nodes of Baccaconularia are aligned in distinct longitudinal files. Nodes also display a subtler, more or less rectilinear transverse alignment, though this pattern commonly is disrupted by offset parallel to the longitudinal files. In their shape and pattern of arrangement, the nodes of Baccaconularia are most similar to the squarish to elongate nodes of Pseudoconularia Bouček, 1939. Longitudinal node files of Baccaconularia may also be compared with the longitudinal facial ridges of Conularia cambria Walcott, 1890 from the Furongian of Wisconsin. Apical angles of Baccaconularia range from approximately 13° to 14.5°. Scanning electron imaging of B. cf. robinsoni shows that its thin, phosphatic skeleton is finely lamellar, with the thickness of individual lamellae measuring approximately 1 μm. The skeleton also exhibits microscopic circular pores and crater-like pits that range from approximately 5 to 10 μm in diameter. These pores and pits are similar in size, geometry, areal density and pattern of arrangement to those of many post-Cambrian conulariids. Microscopic circular pores are documented here for the first time in the genus Archaeoconularia Bouček, 1939 from the Upper Ordovician of the Czech Republic. Although the origin of the pores and pits is open to alternative interpretations, the discovery of these features and fine lamination in Baccaconularia strengthens the argument that this genus is a Cambrian conulariid.     

An uncommon H3/Ser10 phosphorylation pattern in Cestrum strigilatum (Solanaceae), a species with B chromosomes

Cestrum strigilatum (Solanaceae) is a South American shrub with B chromosomes. Bs show a univalent behavior when a single B is present, have non-Mendelian segregation, and are poor in genes and rich in repetitive DNA. In this study, the histone H3 at serine 10 (H3/Ser10) phosphorylation pattern was investigated during mitosis and meiosis of C. strigilatum collected from the wild and was compared in A and B chromosomes. The results revealed that H3/Ser10 phosphorylation of A chromosomes occurred only in the pericentromeric region in both mitosis and meiosis, whereas in the B univalent, phosphorylation appeared in almost the whole extent of the chromosome, except in the terminal portion of the long arm. In meiosis II, the phosphorylation of A chromosomes was similar to that in the first division of meiosis, but the Bs did not show H3/Ser10 phosphorylation. Our results suggest that phosphorylation at the pericentromeric region may be associated with chromosome motility during cell divisions and with the cohesion of B chromatids in a univalent structure in meiosis I.     

Cold activity of Beauveria and Metarhizium, and thermotolerance of Beauveria

Heat and cold are environmental abiotic factors that restrict the use of entomopathogenic fungi as agents for biological control of insects. The thermotolerance and cold activity of 60 entomopathogenic fungal isolates, including five species of Beauveria and one isolate of Engyodontium albus (=Beauveria alba) were examined as to tolerance of temperatures that might be encountered during field use. In addition, cold activity of eight Metarhizium spp. isolates was evaluated. The isolates were from various geographic regions, arthropod hosts or substrates. High variability in conidial thermotolerance was found among the Beauveria spp. isolates after exposure to 45 °C for 2 h, as evidenced by low (0-20%), medium (20-60%), or high germination (60-80%). The thermal death point (0% germination) for three rather thermotolerant B. bassiana isolates (CG 138, GHA and ARSEF 252) was 46 °C for 6 h. At low temperatures (5 °C), with few exceptions (e.g. CG 66, UFPE 479, CG 227, CG 02), most of the B. bassiana isolates germinated well (ca. 100%). On the other hand, only one isolate of Metarhizium sp. was cold-active (i.e. ARSEF 4343 from Macquarie Island, 54.4°S, Australia). This probably is a M. frigidum isolate. The E. albus isolate (UFPE 3138) was the most susceptible isolate to both heat and cold stress. Isolates ARSEF 252 and GHA of B. bassiana, on the other hand, presented exceptionally high thermotolerance and cold activity. Some isolates with high cold activity, however, were thermosensitive (e.g. ARSEF 1682) and others with high thermotolerance had low cold activity (e.g. CG 227). An attempt to correlate the latitude of origin with thermotolerance or cold activity indicated that B. bassiana isolates from higher latitudes were more cold-active than isolates from nearer the equator, but there was not a similar correlation for heat.     

Enhancing effect of a protein from <Emphasis Type="Italic">Lonomia obliqua</Emphasis> hemolymph on recombinant protein production

Gene expression in animal cells allows large scale production of proteins used for either structure and function studies or therapeutic purposes. Maximizing recombinant protein production is necessary to optimize cell growth and protein expression. Some studies have demonstrated the presence of pharmacologically active substances in insect hemolymph. In this work, we have identified and purified a protein from Lonomia obliqua hemolymph able to increase the production of the rabies virus glycoprotein, expressed in Drosophila melanogaster S2 cells, by about 59%.     

Haematological and biochemical reference intervals for farmed channel catfish

The reference intervals for biochemical variables and red blood cell indices of healthy intensively bred channel catfish Ictalurus punctatus were determined. The blood variables were determined using standardized clinical methods. The reference intervals (25th and 75th percentiles) were established using a non-parametric method. Reference intervals for plasma glucose, serum total protein, sodium, potassium, calcium, magnesium, chloride concentration, primary and secondary red blood cell indices were established. The haematological and biochemical reference intervals established may allow important clinical decisions about channel catfish.     

Evolutionary History of the PER3 Variable Number of Tandem Repeats (VNTR): Idiosyncratic Aspect of Primate Molecular Circadian Clock

The PER3 gene is one of the clock genes, which function in the core mammalian molecular circadian system. A variable number of tandem repeats (VNTR) locus in the 18th exon of this gene has been strongly associated to circadian rhythm phenotypes and sleep organization in humans, but it has not been identified in other mammals except primates. To better understand the evolution and the placement of the PER3 VNTR in a phylogenetical context, the present study enlarges the investigation about the presence and the structure of this variable region in a large sample of primate species and other mammals. The analysis of the results has revealed that the PER3 VNTR occurs exclusively in simiiforme primates and that the number of copies of the primitive unit ranges from 2 to 11 across different primate species. Two transposable elements surrounding the 18th exon of PER3 were found in primates with published genome sequences, including the tarsiiforme Tarsius syrichta, which lacks the VNTR. These results suggest that this VNTR may have evolved in a common ancestor of the simiiforme branch and that the evolutionary copy number differentiation of this VNTR may be associated with primate simiiformes sleep and circadian phenotype patterns.