Sensitive detection of transglycosylating activity of xyloglucan endotransglycosylase/hydrolase (XTH) after isoelectric focusing in polyacrylamide gels.

The paper describes a sensitive and rapid zymogram technique for detection of transglycosylating activity (XET) of xyloglucan endotransglycosylase/hydrolase (XTH; EC 2.4.1.207) in polyacrylamide isoelectric focusing gels. After the electrophoresis, the separating gel was overlaid and incubated with an agarose detection gel containing XET substrates: tamarind-seed xyloglucan as the glycosyl donor and sulphorhodamine-labeled xyloglucan-derived oligosaccharides (XGO-SRs) as the glycosyl acceptors. The transglycosylation catalyzed by XTH caused incorporation of the fluorescent label into the high-M(r) polysaccharide. Selective removal of unreacted XGO-SRs from the agarose replicas by washing with organic solvents revealed the zones corresponding to XET activity as bright pink fluorescent spots under UV-light. The method appears suitable for a number of purposes such as analysis of the isoenzyme composition of XTHs with XET activity in crude extracts from various plants and plant organs, monitoring the enzyme expression at various stages of plant development and/or for checking enzyme purity in the course of its isolation procedure.     

Trapping sand flies (Diptera: Psychodidae) in the Emilia-Romagna region of northern Italy.

The efficiency and practicality of two trapping methods for adult Phlebotomine sand flies in two areas of the Emilia-Romagna region (Italy) were evaluated. Suction traps (CO2) and sticky traps (ST) were used to collect sand flies every two weeks, from June to September, 1999, from 16:00 to 07:00. Two CO2 traps were activated at the same time for each area (one with light and one without light), whereas 38 (four with light and 34 without lights) and 48 (four with light and 44 without) sticky traps were activated in Borghi and Longiano, respectively. An Index of Apparent Abundance (IAA) was calculated for each trap type and area. A total of 2,253 sand flies was trapped over the four-month period, with 1,765 collected from Borghi and 488 from Longiano. Phlebotomus perfiliewi was the most abundant species collected, comprising 99.6% and 84.6% of the total flies trapped in Borghi and Longiano, respectively. Other species were also collected within the two areas (Phlebotomus perniciosus and Phlebotomus mascittii) but were not considered for further analyses due to low catches. Significantly more specimens were caught using CO2 than sticky traps and the addition of a light source also improved the catches, however, a significantly greater number of female specimens were collected by a CO2 trap without a light source. Phlebotomus perfiliewi thus appears to show a photophobic reaction in the case of females when trapped using CO2/light attractants.     

Pyruvate metabolism in germinating seeds during natural anaerobiosis

Lactate as well as ethanol is formed in seeds of soybean, maize, pea, bean, lentil and broad-bean in the course of germination during the so-called natural anaerobiosis. After 0 to 30 h of germination a concentration peak of lactate appears. Maximum in ethanol content is found after 40 h. The amount of ethanol is higher big more than one order of magnitude as compared to the amount of lactate. Both products of anaerobiosis occur in germinating seeds irrespective of the type of reserve substances. In contrast to alcohol dehydrogenase lactate dehydrogenase (EC 1. 1. 1. 27) is present in the dry seeds too. Its activity decreases during the first 12 h of germination. It is in this stage that its substrate, lactate, is usually present at a maximal concentration. During the later stages of germination the amount of lactate decreases and enzyme activity rises. There exists a reciprocal relationship between enzyme activity and substrate concentration. In the case of alcohol dehydrogenases (EC 1. 1. 1. 1) the maximum concentration of ethanol precedes the peak of enzyme activity.     

In Vitro and In Vivo Studies of Streptomycin-Dependent Cholera Vibrios

Streptomycin-dependent cholera vibrio strains were derived from Inaba, Ogawa, and NAG vibrios by the method of Mel. These phenotypes grew more slowly and attacked fermentable substances after a longer period of time than the streptomycin-sensitive parent strains. Rabbits injected with streptomycin-sensitive strains and their streptomycin-dependent forms showed homologous agglutinin production. Patas monkeys fed with 10(9) streptomycin-dependent strains shed them for 1 to 2 days without ill effect, whereas the same number of streptomycin-independent organisms caused disease. The possibility of the application of multiple doses of streptomycin-dependent organisms in oral immunization against cholera was considered.     

Changes in proton-magnetic-resonance spectra during aminolysis and enzymic hydrolysis of cephalosporins

1. Changes in proton-magnetic-resonance spectra were followed during the reaction of cephalosporins, deacetylcephalosporins, deacetoxycephalosporins and a Δ²-cephalosporin with ND₃ in D₂O. 2. Changes in proton-magnetic-resonance spectra were also followed during the hydrolysis of a cephalosporin and a deacetylcephalosporin in D₂O with a β-lactamase. 3. Structures for the reaction products are proposed. 4. The signals obtained after aminolysis of the β-lactam ring of a cephalosporin indicate that the reaction is accompanied by expulsion of the acetoxy group as acetate, formation of a double bond in the Δ⁴-position and the appearance of an exocyclic methylene group. 5. Aminolysis of deacetyl- and deacetoxycephalosporins can occur without immediate structural changes in the dihydrothiazine ring. 6. In contrast, the ring structure of the first product of enzymic hydrolysis of a deacetylcephalosporin is apparently identical with that of the product of aminolysis of the cephalosporin itself.     

Automatic collection of samples from suspension cell cultures for investigations of prolonged metabolic processes

A glc method has been described for the quantitative analysis of tobacco (and other plant tissue) for esculetin (as free esculetin and the glucosides esculin or cichoriin) which used tritium-foil electron-capture detection of as little as 15 ng esculetin. Polyphenols were directly extracted from plant material, using n-propanol, and the esculetin glucoside was hydrolyzed with n-propanol saturated with HCl gas to produce free esculetin. After silylation, the TMS-esculetin was separated on 10% OV-101, with caffeic acid n-butyl ester as the internal standard.