A method for separation of trehalose from insect hemolymph

A method was developed for the determination of trehalose levels in insect hemolymph. The disaccharide is first purified by gel-permeation chromatography and then quantitated by the anthrone colorimetric procedure. The concentration of trehalose in hemolymph from eleven species is reported. The method is applicable to determinations in other tissues and organisms as well.     

Effect of cryoprotectants on the activity of hemolymph nucleating agents in physical solutions

The ability of 11 different organic solutes in physical solution to mask the effect of nucleating agents from hemolymph of freezing tolerant insects was tested. The masking effect was tested by measuring the supercooling points of samples with various solute concentrations, with and without hemolymph. Hemolymph was obtained from freeze-tolerant Eleodes blanchardi tenebrionid beetles.The depressive effect of the solutes on the supercooling points was nearly equivalent to the corresponding melting point depression, indicating that the depression was due only to the colligative properties of the solutes. Thus, no ability for nucleator masking was demonstrated.     

A rapid method for quantitative determination of L-tryptophan

Summary A modified colorimetric technique automatized by an Alpkem micro-continuous flow analyser was described for estimating the concentration of L-tryptophan in fermentation broth. This approach provided a convenient alternative to HPLC for L-tryptophan estimation and may help to avoid the time-consuming and laborious screening work encountered in the strain improvement programme.     

A target protease activity of serpins in insect hemolymph

Two zymogens of the serine enzymes (prophenoloxidase activating enzyme and BAEEase, an enzyme hydrolyzing ethyl ester), which are thought to be components of prophenoloxidase cascade in silkworm (Bombyx mori) plasma, were activated through the action of microbial cell wall components. The two active enzymes of the zymogens were studied with regard to the regulation of their activities by two endogenous serpins (silkworm anti-trypsin and silkworm anti-chymotrypsin).

BAEEase activity was shown to be inactivated by silkworm antitrypsin, whereas the inactivation of prophenoloxidase activating enzyme by either of silkworm antitrypsin and silkworm antichymotrypsin could not be demonstrated under the experimental conditions.     

A method for estimation of population densities of ice nucleating active Pseudomonas syringae in buds and leaves of mango

Active ice nucleation strains of Pseudomonas syringae pv. syringae have been associated with a necrotic disease in mango trees growing in Málaga (southern Spain). In this paper a simple multiple-tube test is described to estimate the number of active ice nucleation bacteria associated with plant tissues and, also from suspensions of isolated bacterial strains. This method is based on the most probable number technique developed for microbiological analysis of water. The tube test presented a higher detection sensitivity of active ice nuclei than the traditional drop-freezing test, because a larger amount of plant material could be analysed routinely. Both methods demonstrated a similar accuracy. A high correlation was obtained between the tube test-estimated number of ice nuclei and populations of Ps. syringae -like organisms enumerated on King's agar B.     

Purification and characterization of an insect hemolymph lipoprotein ice nucleator: evidence for the importance of phosphatidylinositol and apolipoprotein in the ice nucleator activity

Summary A lipoprotein with ice nucleator activity was purified from the hemolymph of the freezetolerant larvae of the craneflyTipula trivittata. Characterization of this lipoprotein ice nucleator (LPIN) showed that it differed from other previously described insect hemolymph lipoproteins which lack ice nucleator activity, by the presence of phosphatidylinositol (PI) at 11.0% by weight of the total phospholipid content. The potential roles of PI and other lipoprotein components in the ice nucleating activity were examined using various phospholipases, proteases, LPIN antibodies, borate compounds and various lipid-protein reconstitutions. It was found that phosphatidylinositol specific phospholipase C was the most effective phospholipase in eliminating the activity of the LPIN. Borate compounds effectively depressed activity. Treatment of the LPIN with protease also eliminated ice nucleator activity but the binding of LPIN specific antibody did not. Reconstitutions consisting of the native LPIN lipids, PI specific phospholipase-treated native LPIN lipids, or pure standard phospholipids with the apolipoproteins of the LPIN andManduca sexta larval lipoproteins gave evidence that both the apolipoproteins of the LPIN and PI are necessary for the ice nucleating activity.Abbreviations LPIN polyclonal antibodies to lipoprotein ice nucleator - ANOVA analysis of variance - Apo-I apolipoprotein I - Apo-II apolipoprotein II - LPIN lipoprotein ice nucleator - PAGE polyacrylamide gel electrophoresis - PAS Periodoacetate-Schiff's base - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - SCP supercooling point (ice nucleation temperature) - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - TLC thin layer chromatography     

High ice nucleating ability in plant leaves

A high ice nucleating ability in leaves of Veronica persicaand Buxus microphylla was observed on analyzing the frequencydistribution of their freezing temperatures. The most efficientice nucleators in Veronica leaves exist in structures withinthe leaf blades (mesophyll), and those of Buxus in structureswithin the midrib (vascular tissue). Dissolved or suspendedleaf structures were much less effective. (Received March 16, 1973; )     

The qualitative and quantitative analysis of insect hemolymph sugars by high performance thin-layer chromatography

• 1.1. A procedure is described for the analysis of sugars in insect hemolymph by high performance thin-layer chromatography.
• 2.2. Densitometric scanning of spots after plate developments shows linear responses with most sugars in an analytical range of 125ng-2.0 /gmg; detection limits range from 30/2–60 ng.
• 3.3. Quantitative measurements of trehalose, glucose and fructose can be made on hemolymph sample volumes of less than one microliter, allowing blood sugar analysis of individual insects.
• 4.4. Hemolymph sugar determinations on six species of insects agree well with values reported in the literature, but mean values for a species often showed higher variability because samples were taken from individuals.

     

A simple method for the quantitative determination of muramic acid

A simple method for microdetermination of muramic acid is elaborated. The method is based on the degradation of muramic acid to lactic acid, followed by degradation of the latter to acetaldehyde which can be determined colorimetrically with p-hydroxydiphenyl (PHD). A linear relationship exists between the concentration of muramic acid (up to 20 μg), and absorbance at 560 nm. Substances usually present in the hydrolysates of bacterial cell wall peptidoglycan do not interfere in the determination.     

A highly sensitive method for quantitative determination of abscisic Acid

An abscisic acid derivative was formed by reaction with pentafluorobenzyl bromide which allowed highly sensitive detection by gas-liquid chromatography with electron capture detection. In comparison to the methyl ester derivative, the pentafluorobenzyl derivative of abscisic acid was four times more sensitive to electron capture detection and was stable at room temperature in the presence of ultraviolet light. Derivatization was rapid and the molecular weight of the new compound was confirmed by gas-liquid chromatography-mass spectrometry.     

A method for the quantitative determination of neutral glycosphingolipids in urine sediment

A method is described for the isolation and quantitation of six neutral glycosyl ceramides from human urinary sediment. Total lipids were extracted from sediments of 24-hr urine collections, and the glycosyl ceramides were isolated by silicic acid column chromatography followed by thin-layer chromatography. Methanolysis of the individual glycosyl ceramides yielded methyl glycosides which were quantitated as the trimethylsilyl ethers by gas-liquid chromatography. By this technique, the submicromolar concentrations of six glycosyl ceramides in normal subjects and in individuals with Fabry's disease, an hereditary glycosphingolipid storage disease, were determined. Trihexosyl ceramide (galactosyl-galactosylglucosyl ceramide) and a digalactosyl ceramide accumulated in the urinary sediment of patients with Fabry's disease.     

A simple method for quantitative determination of polysaccharides in fungal cell walls

A simple and reliable method for quantitative determination of cell wall polymers in fungal cell with an s.e.m. of 5% is described. This protocol is based on the hydrolysis by sulfuric acid of beta-glucan, mannan, galactomannan and chitin present at different levels in the wall of yeasts and filamentous fungi into their corresponding monomers glucose, mannose, galactose and glucosamine. The released monosaccharides are subsequently separated and quantified by high-performance ionic chromatography coupled to pulse amperometry detection, with a detection limit of 1.0 mug ml(-1). This procedure is well suited to screening a large collection of yeast mutants or to evaluating effects of environmental conditions on cell wall polysaccharide content. This procedure is also applicable to other fungal species, including Schizosaccharomyces pombe, Candida albicans and Aspergillus fumigatus. Results can be obtained in 3 d.     

A sensitive method for detection and quantitative determination of pyrrolizidine alkaloids

A sensitive and rapid method for detection and quantitative determination of pyrrolizidine alkaloids is presented. It is based on stoichiometric reaction of protonated alkaloids with methyl orange, followed by a release of the latter from the complex. The color intensity of the dye is assessed visually or spectrophotometrically. The method easily detects alkaloids at a concentration of 0.5 μg/ml. The sensitivity of the quantitative assay ranges from 0.006 to 0.045 μmol/ml (from about 2 to 15μg/ml). The method proved to be especially useful during extraction, purification, and separation of minor pyrrolizidine alkaloids.