A Preliminary Investigation on the Chemical Composition of the Cell Surface of Five Enteropathogenic Escherichia coli Serotypes

The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy. Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins. Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues. The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100oC for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon. Phosphate, total sugar and protein contents were determined. Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose. Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction. Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction.

Fatty Acid and Sterol Composition of Three Phytomonas Species

Fatty acid and sterol analysis were performed on Phytomonas serpens and Phytomonas sp. grown in chemically defined and complex medium, and P. françai cultivated in complex medium. The three species of the genus Phytomonas had qualitatively identical fatty acid patterns. Oleic, linoleic, and linolenic were the major unsaturated fatty acids. Miristic and stearic were the major saturated fatty acids. Ergosterol was the only sterol isolated from Phytmonas sp. and P. serpens grown in a sterol-free medium, indicating that it was synthesized de novo. When P. françai that does not grow in defined medium was cultivated in a complex medium, cholesterol was the only sterol detected. The fatty acids and sterol isolated from Phytomonas sp. and P. serpens grown in a chemically defined lipid-free medium indicated that they were able to biosynthesize fatty acids and ergosterol from acetate or from acetate precursors such as glucose or threonine.

Biochemical characterization of a trypanosomatid isolated from the plant Amaranthus retroflexus

A protozoan flagelate has recently been isolated from Amaranthus retroflexus. This plant grows near economically important crops in southeastern Spain, which are known to be parasitized by Phytomonas spp. The present study focuses on the characterization of the energy metabolism of this new isolate. These flagellates utilize glucose efficiently as their primary energy source, although they are unable to completely degrade it. They excrete ethanol, acetate, glycine, and succinate in lower amount, as well as ammonium. The presence of glycosomes was indicated by the early enzymes of the glycolytic pathway, one enzyme of the glycerol pathway (glycerol kinase), and malate dehydrogenase. No evidence of a fully functional citric-acid cycle was found. In the absence of catalase activity, these flagellates showed significant superoxide dismutase activity located in the glycosomal and cytosolic fractions. These trypanosomes, despite being morphologically and metabolically similar to other Phytomonas isolated from the same area, showed significant differences, suggesting that they are phylogenetically different species.

Composition of fingermark residue: a qualitative and quantitative review

This article describes the composition of fingermark residue as being a complex system with numerous compounds coming from different sources and evolving over time from the initial composition (corresponding to the composition right after deposition) to the aged composition (corresponding to the evolution of the initial composition over time). This complex system will additionally vary due to effects of numerous influence factors grouped in five different classes: the donor characteristics, the deposition conditions, the substrate nature, the environmental conditions and the applied enhancement techniques. The initial and aged compositions as well as the influence factors are thus considered in this article to provide a qualitative and quantitative review of all compounds identified in fingermark residue up to now. The analytical techniques used to obtain these data are also enumerated. This review highlights the fact that despite the numerous analytical processes that have already been proposed and tested to elucidate fingermark composition, advanced knowledge is still missing. Thus, there is a real need to conduct future research on the composition of fingermark residue, focusing particularly on quantitative measurements, aging kinetics and effects of influence factors. The results of future research are particularly important for advances in fingermark enhancement and dating technique developments.

The composition of mineral waters sourced from Europe and North America in respect to bone health: composition of mineral water optimal for bone.

The consumption of mineral waters is increasing in industrialised countries. High intakes of Ca and other alkalising cations as well as a low acid intake are beneficial to bone. We examined which components of mineral waters are conditioning their Ca content and their alkalinising power, in order to define the optimal profile. European mineral waters were randomly selected on the Internet: 100 waters with less than 200 mg Ca/l (9.98 mEq/l) and fifty with more than 200 mg/l, all with complete data for SO4, P, Cl, Na, K, Mg and Ca, and most also for HCO3. For comparison, forty North American mineral waters were randomly chosen. The potential renal acid load (PRAL) was calculated for each mineral water. North American waters did not reveal significant results because of their low mineralisation. We performed correlations between all eight components in order to explore the properties of the mineral waters. In the European waters, twenty-six out of twenty-eight correlations showed a P value of <or= 0.01. In waters with PRAL >0 (acidifying waters), PRAL was positively correlated with SO4, Ca, K and Mg (P < 0.001). In those with PRAL < 0 (alkalinising waters), PRAL was negatively correlated with HCO3, Na, Mg, Ca, K, Cl and SO4 (P < 0.001). SO4 and HCO3 were not found together in high quantities in the same water for geochemical reasons. A high Ca content is associated with either a high SO4 or a high HCO3 content. SO4 theoretically increases Ca excretion, while HCO3 and low PRAL values are associated with positive effects on bone. Therefore, the best waters for bone health are rich in both HCO3 and Ca, and by consequence low in SO4.

Biochemical analysis of female mice urine with reference to endocrine function: a key tool for estrus detection.

Species-specific chemical signals released through urine, sweat, saliva and feces are involved in communication between animals. Urinary biochemical constituents along with pheromones may contribute to variation across reproductive cycles and facilitate to estrus detection. Hence, the present study was designed to analyze such biochemical profiles, such as proteins, carbohydrates, lipids, fatty acids, in response with steroid hormones such as estradiol and progesterone. The experimental groups were normal, prepubertal, ovariectomized, and ovariectomized with estrogentreated female mice. In normal mice, the protein and lipid concentrations in urine were significantly higher in proestrus and estrus phases and the quantity of fatty acids was also comparatively higher in estrus. Furthermore, certain fatty acids, namely tridecanoic, palmitic and oleic acids, were present during proestrus and estrus phases, but were exclusively absent in ovariectomized mice. However, the carbohydrate level was equally maintained throughout the four phases of estrous cycle. For successful communication, higher concentrations of protein and specific fatty acids in estrus are directly involved. The significant increase in estradiol at estrus and progesterone at metestrus seems to be of greater importance in the expression pattern of biochemical constituents and may play a notable role in estrous cycle regulation. Thus, we conclude that the variations observed in the concentration of the biochemical constituents depend on the phase of the reproductive cycle as well as hormonal status of animals. The appearance of protein and specific fatty acids during estrus phase raises the possibility to use these as a urinary indicators for estrus detection.

The Holiness Code between D and P. Some Comments on the Function and Significance of Leviticus 17-26 in the Composition of the Torah.

(Résumé de l'ouvrage) Das Deuteronomium nimmt sowohl in der Literaturgeschichte der alttestamentlichen Geschichtsbücher Josua bis Könige eine Schlüsselstellung ein als auch für die Entstehung des Pentateuchs. Wie lassen sich diese beiden Funktionen vereinbaren? Mit der Verhältnisbestimmung haben sich namhafte Wissenschafter der Arbeitsgruppe »Biblical and Ancient Near Eastern Law« im Rahmen der Internationalen Treffen der Society of Biblical Literature in Berlin (2002) und Cambridge (2003) befasst. Der Band präsentiert die neuesten Forschungsergebnisse. Er enthält Vorträge von E. Otto, K. Schmid, H.-C. Schmitt, T. Römer, W.M. Schniedewind, G.N. Knoppers, R. Achenbach, M.M. Zahn und C. Nihan.

Expression, purification and biochemical characterization of recombinant Ca-dependent protein kinase 2 of the malaria parasite Plasmodium falciparum.

Calcium-dependent protein kinases (CDPKs) are serine/threonine kinases that react in response to calcium which functions as a trigger for several mechanisms in plants and invertebrates, but not in mammals. Recent structural studies have defined the role of calcium in the activation of CDPKs and have elucidated the important structural changes caused by calcium in order to allow the kinase domain of CDPK to bind and phosphorylate the substrate. However, the role of autophosphorylation in CDPKs is still not fully understood. In Plasmodium falciparum, seven CDPKs have been identified by sequence comparison, and four of them have been characterized and assigned to play a role in parasite motility, gametogenesis and egress from red blood cells. Although PfCDPK2 was already discovered in 1997, little is known about this enzyme and its metabolic role. In this work, we have expressed and purified PfCDPK2 at high purity in its unphosphorylated form and characterized its biochemical properties. Moreover, propositions about putative substrates in P. falciparum are made based on the analysis of the phosphorylation sites on the artificial substrate myelin basic protein (MBP).

Clonotype selection and composition of human CD8 T cells specific for persistent herpes viruses varies with differentiation but is stable over time.

Protection from reactivation of persistent herpes virus infection is mediated by Ag-specific CD8 T cell responses, which are highly regulated by still poorly understood mechanisms. In this study, we analyzed differentiation and clonotypic dynamics of EBV- and CMV-specific T cells from healthy adults. Although these T lymphocytes included all subsets, from early-differentiated (EM/CD28(pos)) to late-differentiated (EMRA/CD28(neg)) stages, they varied in the sizes/proportions of these subsets. In-depth clonal composition analyses revealed TCR repertoires, which were highly restricted for CMV- and relatively diverse for EBV-specific cells. Virtually all virus-specific clonotypes identified in the EMRA/CD28(neg) subset were also found within the pool of less differentiated "memory" cells. However, striking differences in the patterns of dominance were observed among these subsets, because some clonotypes were selected with differentiation while others were not. Late-differentiated CMV-specific clonotypes were mostly characterized by TCR with lower dependency on CD8 coreceptor interaction. Yet all clonotypes displayed similar functional avidities, suggesting a compensatory role of CD8 in the clonotypes of lower TCR avidity. Importantly, clonotype selection and composition of each virus-specific subset upon differentiation was highly preserved over time, with the presence of the same dominant clonotypes at specific differentiation stages within a period of 4 years. Remarkably, clonotypic distribution was stable not only in late-differentiated but also in less-differentiated T cell subsets. Thus, T cell clonotypes segregate with differentiation, but the clonal composition once established is kept constant for at least several years. These findings reveal novel features of the highly sophisticated control of steady state protective T cell activity in healthy adults.

Purification and biochemical characterization of four iron superoxide dismutases in Trypanosoma cruzi

Four superoxide dismutase (SOD) activities (SOD I, II, III, and IV) have been characterized in the epimastigote form of Trypanosoma cruzi. The total extract was subjected to two successive ammonium sulphate additions between 35 and 85%, and the resulting fraction was purified using two continuous chromatography processes (ion exchange and filtration). Enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing SODs. The molecular masses of the different SODs were 20 kDa (SOD I), 60 kDa (SOD II), 50 kDa (SOD III) and 25 kDa (SOD IV), whereas the isoelectric points were 6.9, 6.8, 5.2 and 3.8, respectively. Subcellular location and digitonin experiments have shown that these SODs are mainly cytosolic, with small amounts in the low-mass organelles (SOD II and SOD I) and the mitochondrion (SOD III), where these enzymes play an important role in minimizing oxidative damage.

Heterologous expression and biochemical characterization of an α1,2-mannosidase encoded by the Candida albicans MNS1 gene

Protein glycosylation pathways, commonly found in fungal pathogens, offer an attractive new area of study for the discovery of antifungal targets. In particular, these post-translational modifications are required for virulence and proper cell wall assembly in Candida albicans, an opportunistic human pathogen. The C. albicans MNS1 gene is predicted to encode a member of the glycosyl hydrolase family 47, with 1,2-mannosidase activity. In order to characterise its activity, we first cloned the C. albicans MNS1 gene into Escherichia coli, then expressed and purified the enzyme. The recombinant Mns1 was capable of converting a Man9GlcNAc2 N-glycan core into Man8GlcNAc2 isomer B, but failed to process a Man5GlcNAc2-Asn N-oligosaccharide. These properties are similar to those displayed by Mns1 purified from C. albicansmembranes and strongly suggest that the enzyme is an ±1,2-mannosidase that is localised to the endoplasmic reticulum and involved in the processing of N-linked mannans. Polyclonal antibodies specifically raised against recombinant Mns1 also immunoreacted with the soluble ±1,2-mannosidases E-I and E-II, indicating that Mns1 could share structural similarities with both soluble enzymes. Due to the high degree of similarity between the members of family 47, it is conceivable that these antibodies may recognise ±1,2-mannosidases in other biological systems as well.

Morphological and biochemical characterization of the aetiological agents of white piedra

The Trichosporon genus is constituted by many species, of which Trichosporon ovoides and Trichosporon inkin are the causative agents of white piedra. They can cause nodules in genital hair or on the scalp. At present, Brazilian laboratory routines generally do not include the identification of the species of Trichosporon genus, which, although morphologically and physiologically distinct, present many similarities, making the identification difficult. The aim of this study was to identify the aetiological agents at the species level of white piedra from clinical specimens. Therefore, both the macro and micro morphology were studied, and physiological tests were performed. Trichosporon spp. was isolated from 10 clinical samples; T. ovoides was predominant, as it was found in seven samples, while T. inkin was identified just in two samples. One isolate could not be identified at the species level. T. inkin was identified for the first time as a white piedra agent in the hair shaft on child under the age of 10.

Molecular and biochemical characterisation of Trypanosoma cruzi phosphofructokinase

The characterisation of the gene encoding Trypanosoma cruzi CL Brener phosphofructokinase (PFK) and the biochemical properties of the expressed enzyme are reported here. In contradiction with previous reports, the PFK genes of CL Brener and YBM strain T. cruzi were found to be similar to their Leishmania mexicana and Trypanosoma brucei homologs in terms of both kinetic properties and size, with open reading frames encoding polypeptides with a deduced molecular mass of 53,483. The predicted amino acid sequence contains the C-terminal glycosome-targeting tripeptide SKL; this localisation was confirmed by immunofluorescence assays. In sequence comparisons with the genes of other eukaryotes, it was found that, despite being an adenosine triphosphate-dependent enzyme, T. cruzi PFK shows significant sequence similarity with inorganic pyrophosphate-dependent PFKs.