A Comparison of Techniques Useful for Preparing Nematodes for Scanning Electron Microscopy

Second-stage juveniles of Meloidogyne incognita were prepared by several different techniques for scanning electron microscopy (SEM). Sequential fixation in the cold (4-8 C) was superior to rapid fixation at room temperature, glutaraldehyde and glutaraldehyde-formalin were better fixatives than formalin alone, and critical point drying with carbon dioxide or Freon gave similar results that were only slightly better than air drying with Freon. Freeze drying sequentially fixed nematodes from 100% ethanol in liquid propane produced the best preserved specimens with the fewest artifacts. Specimens of various free-living and plant-parasitic nematodes were prepared for SEM by freeze drying. This technique was adequate for most genera but unsatisfactory for a few. Although each genus may require a different procedure for optimum preservation of detail, sequential fixation with glutaraldehyde and freeze drying are comparable and often superior to commonly used techniques for preparing nematodes for SEM.     

Plasmodium yoelii: novel rhoptry proteins identified within the body of merozoite rhoptries in rodent Plasmodium malaria

The biogenesis, organization and function of the rhoptries are not well understood. Antisera were prepared to synthetic peptides prepared as multiple antigenic peptides (MAPs) obtained from a Plasmodium yoelii merozoite rhoptry proteome analysis. The antisera were used in immunofluorescence and immunoelectron microscopy of schizont-infected erythrocytes. Twenty-seven novel rhoptry proteins representing proteases, metabolic enzymes, secreted proteins and hypothetical proteins, were identified in the body of the rhoptries by immunoelectron microscopy. The merozoite rhoptries contain a heterogeneous mixture of proteins that may initiate host cell invasion and establish intracellular parasite development.     

Cryofracturing for Scanning Electron Microscope Observations of Internal Structures of Nematodes

Nematodes were prepared for scanning electron microscopy by cryofracturing in ethanol and then by critical-point drying in carbon dioxide. Cross sections of Caenorhabditis briggsae and Xiphinema americanum showed the arrangement of the intestine, ovaries, muscle cells, and some layers of the cuticle. The technique is complementary to transmission electron microscopy and facilitates the interpretation of results from thin sections.     

ent-Verticillane-type diterpenoids from the Japanese liverwort Jackiella javanica

Three ent-verticillane diterpenoids and two ent-sesquiterpenoids were isolated from the Japanese liverwort Jackiella javanica Schiffn. together with five known ent-verticillane and three ent-kaurane diterpenoids, and three sesquiterpenoids. Five ent-verticillane epoxides were synthetically prepared from ent-verticillols action to clarify the absolute configuration of natural ent-9,10-epoxyverticillol. Their structures were established by extensive NMR spectroscopic and X-ray crystallographic analyses.     

Effective separation of peptides using highly dense thermo-responsive polymer brush-grafted porous polystyrene beads

For the development of well-defined highly dense thermo-responsive polymer grafted surface as an improved stationary phase for thermo-responsive chromatography, poly(N-isopropylacrylamide) (PIPAAm) brush-grafted porous polystyrene beads were prepared by surface-initiated atom transfer radical polymerization (ATRP). The PIPAAm grafted region of polystyrene beads was adjusted by the addition of isooctane as a poor solvent for polystyrene upon the reaction of ATRP initiator immobilization. Using a thermo-responsive HPLC column containing the prepared beads with PIPAAm brush grafted on the inside pores nearby the outer surfaces, angiotensin subtypes were effectively separated with aqueous mobile phase, because the densely grafted PIPAAm on nearby the outer surface effectively interacted with the peptides hydrophobically. Retention of basic peptide was achieved by the beads with basic mobile phase. These results indicated that the prepared beads with grafted PIPAAm nearby the outer surface became an effective chromatographic stationary phase for retaining basic peptides using wide pH range of mobile phase.     

Synthesis of (+)-(2R,3S,4R)-2,3,4-trihydroxycyclohexanone from D-glucose

We have described the synthesis of (+)-(2R,3S,4R)-2,3,4-trihydroxycyclohexanone by the reduction of a keto-conduritol derivative, the latter being prepared in five steps from (-)-(2S,3R,4S,5S)-2,3,4-tribenzyloxy-5-hydroxycyclohexanone, which is in turn readily synthesized from D-glucose.     

Development of the Handy Bio-Strand and its application to genotyping of OPRM1 (A118G)

We previously developed a three-dimensional microarray system, the Bio-Strand, which exhibits advantages in automated DNA analysis in combination with our Magtration Technology. In the current study, we have developed a compact system for the Bio-Strand, the Handy Bio-Strand, which consists of several tools for the preparation of Bio-Strand Tip, hybridization, and detection. Using the Handy Bio-Strand, we performed single nucleotide polymorphism (SNP) genotyping of OPRM1 (A118G) by allele-specific oligonucleotide competitive hybridization (ASOCH). DNA fragments containing SNP sites were amplified from genomic DNA by PCR and then were fixed on a microporous nylon thread. Thus, prepared Bio-Strand Tip was hybridized with allele-specific Cy5 probes (<15mer), on which the SNP site was designed to be located in the center. By optimizing the amount of competitors, the selectivity of Cy5 probes increased without a drastic signal decrease. OPRM1 (A118G) genotypes of 23 human genomes prepared from whole blood samples were determined by ASOCH using the Handy Bio-Strand. The results were perfectly consistent with those determined by PCR direct sequencing. ASOCH using the Handy Bio-Strand would be a very simple and reliable method for SNP genotyping for small laboratories and hospitals.     

A general route to pendant C-glycosyl 1,2- and 1,3-diamines

Practical and convenient preparations of C-glycosyl 1,2- and 1,3-alkanediamines are described. Two 1,2-ethylenediamine derivatives were synthesized from acetylated allyl alpha-C-glycosyl compounds via dibromination, azidation, carbohydrate deprotection, and azide reduction. Four 1,3-propanediamine derivatives were prepared from acetylated sugar halides via C-glycosylation with sodiomalononitrile, followed by the reduction of the nitrile moieties and the deacetylation of the carbohydrate moiety. These 1,3-propanediamine derivatives have the beta-anomeric configurations. The methods reported here serve as general routes to access carbohydrate-diamine conjugates with C-glycosyl linkages.     

Indium-promoted Barbier-type allylations in aqueous media: a convenient approach to 4-C-branched monosaccharides and (1-->4)-C-disaccharides

Starting from methyl 6-bromo-4,6-dideoxy-alpha-D-threo-4-enopyranoside, 4-C-branched sugars have been prepared through indium-promoted Barbier-type allylation of various aldehydes in aqueous media followed by hydroboration of the resulting double bond. The intermediate unsaturated monosaccharides were shown to rearrange in acidic media to give 4-C-acetyl-5-C-alkyl pyranose compounds. From beta-1-formyl sugars the corresponding beta-(1-->4)-C-disaccharides were obtained.     

Trypanosoma gambiense and T. equiperdum: characterization of variant specific antigens

It has been shown that by a simple procedure the variant specific protective antigen can be isolated from both T. gambiense and T. equiperdum. The protection afforded mice by this antigen is relatively long term and a high percentage of mice has complete protection. It has been suggested that the antigens are highly antigenic and although antigenically unique for each relapse strain, they have one or more common physical characteristic. It is therefore hypothesized that a multivalent vaccine might be prepared by similar procedures for protection against trypanosomiasis.     

Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells

Lipid rafts are defined as cholesterol and sphingolipid enriched domains in biological membranes. Their role in signalling and other cellular processes is widely accepted but the methodology used for their biochemical isolation and characterization remains controversial. Raft-like membranes from rat submandibular glands were isolated by two different protocols commonly described in the literature; one protocol was based on selective solubilization by Triton X-100 at low temperature and the other protocol consisted in extensive sonication. In both cases a low density vesicular fraction was obtained after ultracentrifugation in a sucrose density gradient. These fractions contained about 20% of total cholesterol but less than 8% of total proteins, and were more rigid than bulk membranes. Fatty acid analyses revealed a similar composition of raft-like membranes isolated by the two different methods, which was characterized by an enrichment in saturated fatty acids in detriment of polyunsaturated acids when compared with the whole cell membranes. Protein profile of detergent resistant membranes or raft-like membranes prepared by sonication was assessed by silver staining after SDS-PAGE and by MALDI-TOF. Both analyses provided evidence of a different protein composition of the Triton X-100 and sonication preparations. Immunoblot experiments revealed that raft-like membranes prepared by detergent extraction or sonication were free of Golgi apparatus or endoplasmic reticulum protein markers (β-COP and calnexin, respectively) and that they were not substantially contaminated by transferrin receptor (a non-raft protein). While caveolin-1 was highly enriched in raft-like membranes prepared by the two methods, the P2X₇ receptor was enriched in raft-like membrane fractions prepared by sonication, but almost undetectable in the detergent resistant membranes. It can be concluded that both methods can be used to obtain raft-like membranes, but that detergent may affect protein interactions responsible for their association with different membrane domains.     

Biotransformations of ent-18-acetoxy-6-ketomanoyl oxides epimers at C-13 with filamentous fungi

Two ent-18-acetoxy-6-oxomanoyl oxides, epimers at C-13, have been prepared from ent-6alpha,8alpha,18-trihydroxylabda-13(16),14-diene (andalusol), isolated from Sideritis foetens, by means of several chemical pathways and a regioselective acylation with Candida cylindracea lipase (CCL). Biotransformation of these 13-epimeric ent-manoyl oxides by Fusarium moniliforme and Neurospora crassa produced mainly ent-1beta- or ent-11alpha-hydroxylations, as well as their deacetylated derivatives, in both epimers. In addition, with the 13-epi substrate N. crassa originated other minor hydroxylations by the ent-alpha face at C-1 or at C-12, whereas an ent-11beta-hydroxyl group, probably originated by reduction of an 11-oxo derivative also isolated, was achieved with the 13-normal substrate.     

The application of phenylmethanethiol and benzenethiol derivatives as odorless organosulfur reagents in the synthesis of thiosugars and thioglycosides

p-octyloxyphenylmethanethiol and p-dodecylbenzenethiol were prepared as new odorless organosulfur reagents. Thiosugars and thioglycosides were synthesized using these reagents without encountering any malodorous procedures.     

Determination of the composition of the oligosaccharide phosphate fraction of Pichia (Hansenula) holstii NRRL Y-2448 phosphomannan by capillary electrophoresis and HPLC

The promising new anticancer agent, PI-88, is prepared by the sulfonation of the oligosaccharide phosphate fraction of the extracellular phosphomannan produced by the yeast Pichia (Hansenula) holstii NRRL Y-2448. The composition of the oligosaccharide phosphate fraction was determined by capillary electrophoresis (CE) with indirect UV detection using 6 mM potassium sorbate at pH 10.3 as the background electrolyte. Further confirmation of the composition was obtained by HPLC analysis of a sample dephosphorylated by treatment with alkaline phosphatase. The structure of the hexasaccharide component has been determined by isolation and NMR spectroscopic analysis of its dephosphorylated derivative. Additionally, the structure of a second, previously undetected tetrasaccharide component (a hexosamine) has been determined by isolation and NMR spectroscopic analysis of the acetate of its dephosphorylated derivative. It is demonstrated that CE is an ideal method for the quality control of the oligosaccharide phosphate fraction for use in the production of PI-88.     

Preparation of the methyl ester of hyaluronan and its enzymatic degradation

A methyl ester of hyaluronan in which the carboxyl groups were fully esterified was prepared using trimethylsilyl diazomethane. This derivative, while not depolymerized by hyaluronan lyases or hyaluronan hydrolases, was a substrate for both chondroitin ACI lyase (EC 4.2.2.5) from Flavobacterium heparinum and chondroitin ACII lyase (EC 4.2.2.5) from Arthrobacter aurescens. The major product isolated in these depolymerization reactions was methyl alpha-L-threo-hex-4-enepyranosyluronate-(1-->3)-2-acetamido-2-deoxy-alpha,beta-D-glucopyranoside as determined by 1H NMR spectroscopy and MALDITOF mass spectrometry.     

Expression of a glucocorticoid receptor (DlGR1) in several tissues of the teleost fish Dicentrarchus labrax

Since glucocorticoids have a role in maintaining the homeostatic status in fish, in the present paper mRNA expression (in situ hybridization) and tissue immunohistochemical localization of a glucocorticoid receptor (DlGR1) in several Dicentrarchus labrax organs are reported. Riboprobe and specific antibodies were prepared by using the DlGR1 that has been previously cloned and sequenced from peritoneal cavity leukocytes. Both mRNA and receptor were identified in head kidney, spleen, gills, intestine, heart and liver tissues. The functional roles of DlGR1 localization are discussed.     

Stability of diacylglycerol acyltransferase in dehydrated bovine muscle tissue

Meaningful estimates of diacylglycerol acyltransferase (EC 2.3.1.20) activity in different tissue samples require effective, unbiased methods of sample storage. Samples of the pars costalis diaphragmatis muscle (skirt muscle of the diaphragm) were obtained from 18- to 20-month-old cattle and assayed for microsomal protein content and diacylglycerol acyltransferase activity after having been stored under various conditions as dissected tissue or microsomes prepared from dissected tissue. There was relative enrichment of diacylglycerol acyltransferase specific activity (p<0.05) when samples prepared from the pars costalis diaphragmatis muscle were dehydrated and stored for 2 weeks, as compared to the control condition (in which the microsome fraction was prepared from fresh pars costalis diaphragmatis muscle and assayed immediately). The results suggested that dehydration was an effective method of storage for bovine muscle samples destined for estimation of the microsomal diacylglycerol acyltransferase activity. The dehydration approach for preparing samples for analysis of diacylglycerol acyltransferase activity might also prove useful to investigators who are interested in obtaining reliable estimates of the activity of other enzymes in tissue samples.     

Spawn viability in edible mushrooms after freezing in liquid nitrogen without a cryoprotectant

Five strains of edible mushrooms (Lentinula boryana, Lentinula edodes, Pleurotus djamor, Pleurotus pulmonarius, and Volvariella volvacea) were studied. Spawn were prepared from sorghum seeds and then incubated for 14 days under optimum conditions for each species. Once covered by mycelia, the sorghum seeds were placed in polycarbonate vials for freezing in liquid nitrogen. The effect of adding a cryoprotective solution before freezing (either 10% glycerol v/v or 5% dimethylsulfoxide v/v) was evaluated as a function of mycelial growth and percent viability. Three main treatments were undertaken: (1) freezing with a glycerol or dimethylsulfoxide cryoprotectant, (2) freezing with water and (3) freezing without cryoprotectant or water. Samples were maintained frozen for a week, after which time they were thawed (10 min at 30 degrees C) and the seeds placed in Petri dishes with a culture medium. A recovery rate of 96.8% was obtained for the total number of samples summed over all strains and treatments. In contrast, 99.2% of the samples frozen without cryoprotectant were recovered. The recovery of frozen mycelia was delayed with respect to a control group, which was not frozen. However, no difference was observed in percent recovery and mycelial diameter when a new series of spawn was prepared from mycelia that had been previously frozen. Results obtained from this experiment demonstrate that an adequate recovery of mycelia can be obtained without using a cryoprotectant. This capacity might enable large quantities of commercial mushroom strains to be handled at reduced production costs. It is suggested that the mycelia survived freezing without cryoprotectants because they were embedded and protected within the sorghum seeds used to elaborate the spawn.     

8-Hydroxy-(+)-delta-cadinene is a precursor to hemigossypol in Gossypium hirsutum

[(3)H](+)-delta-Cadinene and its 8-hydroxy derivative, prepared from (1RS)-[1-(3)H]FPP by the action of one and two recombinant enzymes, respectively, were infiltrated into cotyledons of bacterial blight-resistant cotton plants as they biosynthesized sesquiterpene phytoalexins in response to infection by Xanthomonas campestris pv. malvacearum. Following both treatments, tritium appeared in the HPLC fraction that contained hemigossypol. Hemigossypol was isolated from the cotyledons that had been treated with [(3)H](+)-8-hydroxy-delta-cadinene and was trimethylsilylated and purified. In two experiments, specific radioactivity of the hemigossypol derivative indicated that 5% and 10%, respectively, of the [(3)H](+)-8-hydroxy-delta-cadinene had been converted to hemigossypol.     

Effects of KHEYLRFamide and KNEFIRFamide on cyclic adenosine monophosphate levels in Ascaris suum somatic muscle

KHEYLRF-NH(2) (AF2) is a FMRFamide-related peptide (FaRP) present in parasitic and free-living nematodes. At concentrations as low as 10 pM, AF2 induces a biphasic tension response, consisting of a transient relaxation followed by profound excitation, in neuromuscular strips prepared from Ascaris suum. In the present study, the effects of AF2 on cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inositol-1,4,5-triphosphate (IP(3)) levels were measured following muscle tension recordings from 2 cm neuromuscular strips prepared from adult A. suum. AF2 induced a concentration- and time-dependent increase in cAMP, beginning at 1 nM; cAMP levels increased by 84-fold following 1 h exposure to 1 microM AF2. cGMP and IP(3) levels were unaffected by AF2 at concentrations 相似文献