Simultaneous analysis of lysine, Nepsilon-carboxymethyllysine and lysinoalanine from proteins

Protein quality was assayed by simultaneous measurement of lysine (Lys), carboxymethyllysine (CML) and lysinoalanine (LAL). GC-FID analysis of N-tert-butyl dimethylsilyl (tBDMSi) derivatives of these amino acids was undertaken. tBDMSi derivates were separated on a CP-SIL 5CB commercially fused silica capillary column (25 m x 0.25 mm i.d., 0.25 microm film thickness) employing a thermal gradient programmed from 200 to 300 degrees C. The identity of tBDMSi derivatives of Lys, CML and LAL was established by GC-MS while FID detection was employed for quantification. Analytical parameters such as linearity (lysine 350-4200 microM, LAL 3-81 microM, CML 16-172 microM), precision (1-13% variation coefficients), accuracy (85-108% average recovery) and limits of detection (lysine 0.4 mg/100 g protein, LAL 5.0 mg/100 g protein, CML 3.4 mg/100 g protein) and quantification (lysine 1.4 mg/100g protein, LAL 15.2 mg/100 g protein, CML 11.2 mg/100 g protein) were determined for validation of the analytical approach. Model systems and real foods have been studied. Kinetic of CML formation from different food proteins (BSA, soy protein, casein and gluten) was performed employing model systems. Carboxymethylation rate depended on the source of protein. Maillard reaction progressed to advanced stages damaging the protein quality of stored infant foods, soy drinks, boiled eggs and dry powdered crepes. CML values ranged from 62 to 440 mg/100 g protein were measured. LAL was also formed during boiling eggs (21-68 mg/100g protein) indicating additional damage by crosslinking reaction. In agreement, lysine content was affected by both food processing and storage.     

Rapid clonal analysis of recurrent tuberculosis by direct MIRU-VNTR typing on stored isolates

Background  

The application of molecular tools to the analysis of tuberculosis has revealed examples of clonal complexity, such as exogenous reinfection, coinfection, microevolution or compartmentalization. The detection of clonal heterogeneity by standard genotyping approaches is laborious and often requires expertise. This restricts the rapid availability of Mycobacterium tuberculosis (MTB) genotypes for clinical or therapeutic decision-making. A new PCR-based technique, MIRU-VNTR, has made it possible to genotype MTB in a time frame close to real-time fingerprinting. Our purpose was to evaluate the capacity of this technique to provide clinicians with a rapid discrimination between reactivation and exogenous reinfection and whether MIRU-VNTR makes it possible to obtain data directly from stored MTB isolates from recurrent episodes.     

Development, longevity and reproduction of Trissolcus basalis (Wollaston) and Telenomus podisi Ashmead (Hymenoptera: Scelionidae) in natural conditions during autumn and winter, in southern Paraná, Brazil

The development, longevity and reproduction of Trissolcus basalis (Wollaston) and Telenomus podisi Ashmead were investigated under natural conditions during autumn and winter from 1999 to 2001, in Southern Paraná State, Brazil, in order to understand how these parasitoids overwinter in subtropical regions. Adults of T. basalis e T. podisi emerged during autumn and winter from eggs parasitized between April and June. Adult longevity in natural conditions was higher than eight and seven months, for T. basalis and T. podisi respectively, showing that both species overwinter in the adult stage. Percentage survival of T. basalis and T. podisi females during autumn and winter was higher than 85% and mortality increased significantly in spring. Females maintained for six months in natural conditions parasitized E. heros eggs after transference to 25 degrees C, and the presence of females in the offspring showed that mating occurred during autumn and winter. Temperature during immature development influenced significantly the adult performance, regulating the longevity and reproductive capacity after dormancy. Female parasitoids that developed in higher temperatures lived longer and parasitized more host eggs after transference to 25 degrees C than females that developed under colder conditions.     

Insecticidal action of Bauhinia monandra leaf lectin (BmoLL) against Anagasta kuehniella (Lepidoptera: Pyralidae), Zabrotes subfasciatus and Callosobruchus maculatus (Coleoptera: Bruchidae)

Bruchid beetle larvae cause major losses in grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil (Callosobruchus maculatus) and the Mexican bean weevil (Zabrotes subfasciatus), are pests that damage stored seeds. The Mediterranean flour moth (Anagasta kuehniella) is of major economic importance as a flour and grain feeder; it is often a severe pest in flour mills. Plant lectins have been implicated as antibiosis factors against insects. Bauhinia monandra leaf lectin (BmoLL) was tested for anti-insect activity against C. maculatus, Z. subfasciatus and A. kuehniella larvae. BmoLL produced ca. 50% mortality to Z. subfaciatus and C. maculatus when incorporated into an artificial diet at a level of 0.5% and 0.3% (w/w), respectively. BmooLL up to 1% did not significantly decrease the survival of A. kuehniella larvae, but produced a decrease of 40% in weight. Affinity chromatography showed that BmoLL bound to midgut proteins of the insect C. maculatus. 33 kDa subunit BmoLL was not digested by midgut preparations of these bruchids. BmoLL-fed C. maculatus larvae increased the digestion of potato starch by 25% compared with the control. The transformation of the genes coding for this lectin could be useful in the development of insect resistance in important agricultural crops.     

A Tolerant Behavior in Salt-Sensitive Tomato Plants can be Mimicked by Chemical Stimuli

Lycopersicon esculentum plants exhibit increased salt stress tolerance following treatment with adipic acid monoethylester and 1,3-diaminepropane (DAAME), known as an inducer of resistance against biotic stress in tomato and pepper. For an efficient water and nutrient uptake, plants should adapt their water potential to compensate a decrease in water soil potential produced by salt stress. DAAME-treated plants showed a faster and stronger water potential reduction and an enhanced proline accumulation. Salinity-induced oxidative stress was also ameliorated by DAAME treatments. Oxidative membrane damage and ethylene emission were both reduced in DAAME-treated plants. This effect is probably a consequence of an increase of both non-enzymatic antioxidant activity as well as peroxidase activity. DAAME-mediated tolerance resulted in an unaltered photosynthetic rate and a stimulation of the decrease in transpiration under stress conditions without a cost in growth due to salt stress. The reduction in transpiration rate was concomitant with a reduction in phytotoxic Na⁺ and Cl⁻ accumulation under saline stress. Interestingly, the ABA deficient tomato mutant sitiens was insensitive to DAAME-induced tolerance following NaCl stress exposure. Additionally, DAAME treatments increased the ABA content of leaves, therefore, an intact ABA signalling pathway seems to be important to express DAAME-induced salt tolerance. Here, we show a possibility of enhance tomato stress tolerance by chemical induction of the major plant defences against salt stress. DAAME-induced tolerance against salt stress could be complementary to or share elements with induced resistance against biotic stress. This might be the reason for the observed wide spectrum of effectiveness of this compound.Key Words: adipic acid monoethyl ester, 1,3-diaminepropane, Lycopersicon esculentum, salt stress, oxidative stress, ethylene, chemical induced tolerance     

Stand microclimate and physiological activity of tree leaves in an oak-hornbeam forest

Summary In an uneven-aged, multi-species oak-hornbeam forest at Báb, SW Slovakia (former IBP Forest Research Site), a series of micrometeorological and ecophysiological measurements started in 1985. The aims of the work are to improve understanding of physiological processes (photosynthesis, respiration, and transpiration) of adult trees and stand microclimate, to collect data for simulation of the canopy (stand) photosynthesis and for ecological synthesis of the functioning of the forest ecosystem. In this paper, photosynthetically active radiation (PAR), air temperature (AT) and relative humidity (RH), wind speed (WS), and CO₂ concentration ([CO₂]) in and above the forest are characterized for the fully leaved season, using diurnal courses, vertical profiles and isodiagrams (isopleths). Approximately 50% of incident PAR was absorbed by the upper 4–5 m layer of leaves and only approximately 5% or less penetrated to the forest floor. Vertical gradients of AT and RH were generally low, but large differences in diurnal ranges of AT and RH were observed between vertical levels. The upper leaf canopy greatly reduced WS, and at a height of about 14 m above the ground it was close to zero. The highest diurnal [CO₂] maximum and variations occurred at 1 m above the ground, and the lowest above the forest. In good light conditions in the forest, the entire leaf canopy (overstorey and understorey canopy) is a large sink of CO₂. At night the forest stand is a source of CO₂, the largest internal source being the soil and forest floor.     

Single and combined effects of carbamazepine and vinpocetine on depolarization-induced changes in Na+, Ca2+ and glutamate release in hippocampal isolated nerve endings

The single and combined effects of carbamazepine and vinpocetine on the release of the excitatory amino acid neurotransmitter glutamate, on the rise in internal Na+ (Na(i), as determined with SBFI), and on the rise in internal Ca2+ (Ca(i), as determined with fura-2) induced by an increased permeability of presynaptic Na+ channels, with veratridine, or by an increased permeability of presynaptic Ca2+ channels with high K+, were investigated in isolated hippocampal nerve endings. The present study shows that carbamazepine and vinpocetine, both inhibit dose dependently the release of preloaded [3H]Glu induced by veratridine. However, carbamazepine is two orders of magnitude less potent than vinpocetine. The calculated IC(50)'s for carbamazepine and vinpocetine to inhibit veratridine-induced [3H]Glu release are 200 and 2 microM, respectively. Consistently 150 microM carbamazepine and 1.5 microM vinpocetine reduce the veratridine-induced rise in Na(i) in a similar extent. The single effects of carbamazepine and of vinpocetine on the presynaptic Na+ channel mediated responses, namely the rise in Na(i) and the release of Glu induced by veratridine, are additive. Responses that depend on the entrance of external Ca2+ via presynaptic Ca2+ channels, such as the release of [3H]Glu and the rise in Ca(i) induced by high K+, are insensitive to 300 microM carbamazepine and slightly reduced by 5 microM vinpocetine. It is concluded that the additive effects of carbamazepine, which is one of the most common antiepileptic drugs, and vinpocetine that besides its known neuroprotective action and antiepileptic potential is a memory enhancer, may perhaps be advantageous in the treatment of epileptic patients.     

Identification of two new intimin types in atypical enteropathogenic Escherichia coli.

Stool specimens of patients with diarrhea or other gastrointestinal alterations who were admitted to Xeral-Calde Hospital (Lugo, Spain) were analyzed for the prevalence of typical and atypical enteropathogenic Escherichia coli (EPEC). Atypical EPEC strains (eae+ bfp-) were detected in 105 (5.2%) of 2015 patients, whereas typical EPEC strains (eae+ bfp+) were identified in only five (0.2%) patients. Atypical EPEC strains were (after Salmonella) the second most frequently recovered enteropathogenic bacteria. In this study, 110 EPEC strains were characterized. The strains belonged to 43 O serogroups and 69 O:H serotypes, including 44 new serotypes not previously reported among human EPEC. However, 29% were of one of three serogroups (O26, O51, and O145) and 33% belonged to eight serotypes (O10:H-, O26:H11, O26:H-, O51:H49, O123:H19, O128:H2, O145:H28, and O145:H-). Only 14 (13%) could be assigned to classical EPEC serotypes. Fifteen intimin types, namely, alpha1 (6 strains), alpha2 (4 strains), beta1 (34 strains), xiR/b2 (6 strains), gamma1 (13 strains), gamma2/q (16 strains), delta/k (5 strains), epsilon1 (9 strains), nuR/e2 (5 strains), zeta (6 strains), iota1 (1 strain), muR/iota2 (1 strain), nuB (1 strain), xiB (1 strain), and o (2 strains), were detected among the 110 EPEC strains, but none of the strains was positive for intimin types mu1, mu2, lambda, or muB. In addition, in atypical EPEC strains of serotypes O10:H-, O84:H-, and O129:H-, two new intimin genes (eae-nuB and eae-o) were identified. These genes showed less than 95% nucleotide sequence identity with existing intimin types. Phylogenetic analysis revealed six groups of closely related intimin genes: (i) alpha1, alpha2, zeta, nuB, and o; (ii) iota1 and muR/iota2; (iii) beta1, xiR/beta2B, delta/beta2O, and kappa; (iv) epsilon1, xiB, eta1,eta2, and nuR/epsilon2; (v) gamma1, muB, gamma2, and theta; and (vi) lambda. These results indicate that atypical EPEC strains belonging to large number of serotypes and with different intimin types might be frequently isolated from human clinical stool samples in Spain.     

Role of caveolae in Leishmania chagasi phagocytosis and intracellular survival in macrophages

Caveolae are membrane microdomains enriched in cholesterol, ganglioside M1 (GM1) and caveolin-1. We explored whether caveolae facilitate the entry of Leishmania chagasi into murine macrophages. Transient depletion of macrophage membrane cholesterol by 1 h exposure to methyl-beta-cyclodextrin (MbetaCD) impaired the phagocytosis of non-opsonized and serum-opsonized virulent L. chagasi. In contrast, MbetaCD did not affect the phagocytosis of opsonized attenuated L. chagasi. As early as 5 min after phagocytosis, virulent L. chagasi colocalized with the caveolae markers GM1 and caveolin-1, and colocalization continued for over 48 h. We explored the kinetics of lysosome fusion. Whereas fluorescent-labelled dextran entered macrophage lysosomes by 30 min after addition, localization of L. chagasi in lysosomes was delayed for 24-48 h after phagocytosis. However, after transient depletion of cholesterol from macrophage membrane with MbetaCD, the proportion of L. chagasi-containing phagosomes that fused with lysosomes increased significantly. Furthermore, intracellular replication was impaired in parasites entering after transient cholesterol depletion, even though lipid microdomains were restored by 4 h after treatment. These observations suggest that virulent L. chagasi localize in caveolae during phagocytosis by host macrophages, and that cholesterol-containing macrophage membrane domains, such as caveolae, target parasites to a pathway that promotes delay of lysosome fusion and intracellular survival.