Potential use of nylon scouring pad cubes attachment method for pectinase production by Aspergillus niger HFD5A-1

This study investigated the novel use of scouring pad cubes as a support matrix for immobilization of fungal cell to enhance the pectinase production. Nylon scouring pad cubes were used for immobilized Aspergillus niger HFD5A-1 cells for pectinase production in flask submerge fermentation system. The enzyme activity of immobilized cell in scouring pad cubes gave higher activity compared to free cells. Various physical parameters for culture condition were studied to evaluate its effects on pectinase production. The maximum enzyme activity obtained was 11.05 U/mL on the 6th day of cultivation after using the optimized parameters of 6 scouring pad cubes, 1 × 10⁷ spores/mL of inoculum size, agitation speed of 150 rpm and incubated at 30 °C. The use of nylon scouring pad cubes gave an increment of about 335.0% of pectinase production (11.05 U/mL) compared to free cells (2.54 U/mL). The results therefore show scouring pad cubes could be a favorable carrier to immobilize the fungal cells for higher enzyme production in submerged fermentation.     

Novel assays for proteases using green fluorescent protein-tagged substrate immobilized on a membrane disk

Green fluorescent protein (GFP) is very stable for various proteases. Using this property, three protease assay methods designated the disk separation assay for remaining GFP (DSAR), the disk separation assay for liberated GFP (DSAL), and the homogeneous assay for fluorescence concentrated on membrane (HAFCOM) were developed. These methods employ a nylon membrane designated “Cleave-Checker” on which GFP-SpA_B (domain B in staphylococcal protein A) is immobilized. The SpA_B region was used as a substrate for the protease, and the isolation of GFP from the membrane generated by the digestion of the SpA_B region was detected. In DSAR, it was possible to detect solution of at least 25 ng/ml trypsin or proteinase K by visual observation. The most important feature of DSAR is that the detection of the protease is possible only under UV light. In contrast, DSAL is suitable for a highly sensitive assay. The assay ranges of DSAL were 1.6 to 100 ng/ml in trypsin and 1.6 to 400 ng/ml in proteinase K. HAFCOM does not require bound/free (B/F) separation; thus, the procedure is simpler than that with DSAL and the reproducibility is high. The assay ranges of HAFCOM were 25 to 400 ng/ml in trypsin and 12.5 to 200 ng/ml in proteinase K. The Cleave-Checker used for these methods was stable in a dry state, and long-term preservation for at least several months was possible.     

Echinococcus granulosus: cellular territories and morphological regions in mature protoscoleces

Basic aspects of the generation, structure and function of Echinococcus granulosus protoscoleces are unknown. We review the work done on the structure and ultrastructure of the E. granulosus protoscolex and provide new data together with a comprehensive view of this form of the parasite. The surface, as observed by scanning electron microscopy, tightly correlates with five cellular territories characterized in the interior using light and transmission electron microscopy as well as a histochemical technique. Three of these territories are surrounded by a basal lamina that is also present in the internal side of the tegument, suggesting a complex internal organization. These cellular territories correlate with the expression of specific genes and the regionalization of DNA synthesis in protoscoleces. Additionally, a proposal to explain movements of the body of this form of the parasite in relation to the neck or to the germinal layer of the hydatid cyst is provided.     

The effect of out-wintering pad design on dirtiness score, somatic cell score and mastitis incidence in dairy cows

This study aimed to compare three woodchip out-wintering pad (OWP) designs, and indoor cubicle housing with regard to cow dirtiness scores during the winter housing period, and udder health during both the winter period and the following lactation, for spring-calving dairy cows. The treatments were: an uncovered (UP) and covered (CP) OWP with a concrete feed apron; an uncovered OWP with self-feed silage pit provided directly on the woodchips (SP); and indoor cubicle housing (IC). Data were compared during 2 years: year 1 was a case study while year 2 was an experimental study. In year 1, treatments were UP (space allowance = 12 m2/cow), CP (6 m2/cow) and IC. In year 2, all three OWP designs (12 m2/cow) were compared with IC. Animals were assigned to treatments at the end of lactation in the autumn, and remained there while dry until calving the following spring. Subsequently, all cows were at pasture during lactation. Outcome measures for analysis were cow dirtiness score, somatic cell score (SCS) and incidence of clinical mastitis during the dry period and during lactation. Quarter milk samples were also taken at drying off, calving and 3 weeks post partum both years, and at approximately 113 days in milk in year 2. Samples were analysed for presence of mastitis-causing agents and SCS was determined. Sub-clinical mastitis was diagnosed when cows had an SCS greater than 200 000, or California mastitis test greater than 1 in at least one quarter. In year 1, cows in CP were dirtier than cows in the other two treatments. These animals also had the highest SCS during lactation and tended to have more mastitis-causing agents isolated from quarter milk samples. In year 2, when all cows were stocked at the same density, cows in the sheltered OWP (i.e. CP) had similar dirtiness scores to cows in cubicles and significantly lower dirtiness scores than cows in the unsheltered OWP designs, i.e. UP and SP. However, there were no effects on SCS or quarter sample results. Cleaning of OWP's stocked at 12 m2/cow reduced cow dirtiness scores. However, cleaning of CP in year 1 when cows were stocked at 6 m2/cow had no effect on dirtiness scores. We conclude that dry cows stocked at 12 m2/cow on OWP's are unlikely to have udder health problems in the subsequent lactation. Furthermore, provision of shelter and cleaning of the woodchips are management factors that help to keep cows clean on OWP's.     

THE IMPACT OF STORM-FLOW ON RIVER BIOFILM ARCHITECTURE1

The impact of storm-flow on river biofilm architecture was investigated using transmission (TEM) and scanning (SEM) electron microscopy. TEM resin substrata were colonized under light-grown (LG) or dark-grown (DG) conditions for 33 weeks in the Clywedog River, North Wales, prior to exposure to ambient-flow (approx. 60 cm·s^?1) or storm-flow (approx. 235 cm·s^?1+ river sediment) in a laboratory flume. Line transect methodology was used to quantify information from TEM ultrathin sections of LG material. In the LG ambient-flow biofilm, bacteria were more abundant directly adjacent to the substratum and were noticeably denser directly under the adnate diatom Cocconeis. Higher in the biofilm, the bacteria were loosely dispersed in the matrix between other cells. Cyanobacteria occurred most frequently as single cells but were also found in large “palisade” formations adjacent to the substratum. Significant horizontal and vertical nearest-neighbor associations were noted for both bacteria and cyanobacteria. Cells of Cocconeis were common adjacent to the substratum, providing shelter to, and often elevated upon, an “organic pad” of bacteria, cyanobacteria, and densely staining exopolysaccharide. Cyanobacteria and Cocconeis were resistant to removal by storm-flow, but Cocconeis frustules were sometimes damaged. Bacteria in the LG storm-flow samples were less common adjacent to the substratum and were sometimes more dispersed higher in the biofilm than in ambient-flow samples. We suggest that storm-flow hydrodynamic forces may redistribute bacteria adjacent to the substratum into higher areas of the biofilm. In addition, bacteria and the exopolysaccharide matrix were sometimes removed down to the substratum by storm-flow, unless beneath Cocconeis. The DG biofilm consisted almost entirely of bacteria. Storm-flow only removed surface growth from DG biofilms, and SEM revealed peritrich stalk abrasion and “blow-down.” Pre-disturbance biofilm architecture appears to influence the form of destruction. We suggest that the “microcosms” of Cocconeis and their underlying cells not only serve as an inoculum to recolonize the surface when conditions permit but enhance immigration by interrupting flow patterns across the surface.     

褐飞虱翅型分化的组织学研究

利用石蜡切片的方法,观察并比较了褐飞虱Nilaparvata lugens (Stal)长翅型和短翅型纯系各龄若虫及成虫的翅芽和间接飞行肌的发育情况。发现在4龄的第8h以后间接飞行肌就开始分化,长翅型若虫的间接飞行肌正常发育而短翅型若虫间接飞行肌的发育则被抑制。在5龄初始时方可明显观察到翅芽分化,短翅型若虫前翅芽细胞增殖速度明显慢于长翅型,并且其后翅芽停止发育。本文还比较了长翅型雌、雄性个体之间间接飞行肌的发育情况。     

Fungal adhesion pad formation and penetration of root cuticle in early stage mycorrhizas ofPicea abies andLaccaria amethystea

Summary Primary events during the establishment of the fungus-root symbiosis in ectomycorrhizas are still little understood. No attention has been paid so far to the adhesion of hyphae to the root cuticle and penetration of this barrier, although the importance of the cuticle has been shown for pathogen-plant interactions. Early developmental stages of in vitro mycorrhization ofLaccaria amethystea onPicea abies after short periods of incubation in growth chambers under elevated CO₂ concentrations were studied by light and transmission electron microscopy. No structural changes in mycorrhization related to elevated CO₂ were found, but fine roots and mycorrhizas developed faster. Adhesion pad formation was observed at hyphal tips in contact with the root cuticle. The adhesion pad was connected to the outer cell wall layer of the hypha and reacted positively to the Swift reaction for cysteine rich proteins. Although the reaction cannot be considered as totally specific, findings are discussed in respect to hydrophobins, which have recently been found to be expressed during early steps in ectomycorrhizal development. The root cuticle was dissolved and penetrated by fungal tips of the fingerlike branching mycelium attached to the root surface. The findings are compared with well documented pathogenic fungus-plant interactions at the cuticle. The possibility of restriction of hyphal attack to that part of the cuticle covering cell junctions is discussed.     

Microstructure of the adhesive pad in the millipede Orthomorphella pekuensis (Polydesmida: Paradoxosomatidae)

Secondary sexual structures in millipedes have evolved to improve the efficiency with which spermatophores are transferred from males to females. Here, we demonstrate the microstructural characteristics of the sexual dimorphism in the leg adhesive pads of the millipede Orthomorphella pekuensis, using field emission scanning electron microscopy. The adult male of this millipede has developed typical adhesive pads on the legs as a secondary sexual character: these help the male hold on to the female during mating. The adhesive pads are located on the ventral surface of the tarsus in the form of numerous filamentous bristles with horizontal striation. There are approximately 300 bristles, each with an average length of approximately 60 µm, on each tarsus. Each bristle has a round socket on the surface of the tarsus and an acuminate tip. The striation appears only on the distal half of each bristle, typically.