Solubilization and electrophoretic studies of cyst wall proteins of a hypotrichous ciliate

A method for induction of synchronous encystment in a hypotrichous ciliate, Paraurostyla sp. is described. Cyst walls, isolated by shaking with glass beads, were analyzed by SDS-polyacrylamide gel electrophoresis. To test optimal conditions of solubilization of cyst wall proteins, different treatments using Triton X-100, EDTA, EGTA, urea, SDS and 2-mercaptoethanol were carried out. At least, 15 different proteins were identified as specific to the cyst wall. Four low molecular weight polypeptides (40, 27–26, 20 and 18 kDa represented aproximately 70% of the cyst wall proteins. The 170, 135 and 40-kDa bands exhibited a PAS-positive reaction. Hydrogen and disulphide bonds were shown to be the most important interactions involving cyst wall proteins. Amino acid composition of cyst wall proteins was also investigated by HPLC. High amounts of glycine, cystine and proline were detected.     

The Diagnosis Of HPRT Deficiency in the 21St Century

We have studied 36 patients with HPRT deficiency, 25 with Lesch-Nyhan syndrome and 11 with partial HPRT deficiency (grades 1 to 3). Patients diagnosed with HPRT deficiency have increased 50% since 2000. The most relevant recent advances have been made in molecular diagnosis. Nevertheless, enzyme determinations are still essential for the diagnosis of HPRT deficiency. Therapy for the neurological manifestations of HPRT deficiency has not advanced. Allopurinol remains the drug of choice to diminish uric acid overproduction, but the optimal allopurinol dose must be established in each patient to prevent xanthine or uric acid urolithiasis, a process aided by sequential determination of urinary oxypurines and uric acid.     

Helminth parasites of the Eurasian otter Lutra lutra in southwest Europe

The helminth fauna in 109 Eurasian otters (Lutra lutra L.) from France, Portugal and Spain was analysed, together with 56 faecal samples collected in Portugal and 23 fresh stools from otters included in a reintroduction programme. Seven helminth species were found in L. lutra in southwest Europe: Phagicola sp. (Trematoda), Aonchotheca putorii, Eucoleus schvalovoj, Strongyloides lutrae, Anisakis (third stage larvae) and Dirofilaria immitis (Nematoda), and Gigantorhynchus sp. (Acanthocephala). Eucoleus schvalovoj was the dominant species throughout southwest Europe. Strongyloides lutrae was significantly more prevalent in the Iberian Peninsula than in France. Apart from these two dominant nematodes and A. putorii, the other helminth species were incidental parasites of L. lutra in southwest Europe. The helminth fauna of L. lutra in southwest Europe is, in general, poorer than that reported in eastern Europe and in all other aquatic mustelids in southwest Europe. Phagicola specimens are reported for the first time in a non-marine wild carnivore in Europe. The prevalences of E. schvalovoj and S. lutrae obtained by necropsy were higher than those observed by coprological analysis using a formalin-ether concentration method (Ritchie). Nevertheless, the culture of fresh faeces appears to be the best method to study infection of L. lutra by Strongyloides.     

Elements of the nitric oxide/cGMP pathway expressed in cerebellar granule cells: biochemical and functional characterisation

It is known that the nitric oxide (NO)/cGMP pathway affects neuronal development and the expression of the different proteins is developmentally dependent in several brain areas. However, so far there are no data on the expression of the proteins involved in this signalling system during the development of the cerebellar granule cell, one of the most widely used models of neuronal development. This study was accordingly designed to analyse the developmental regulation of neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclase subunits (alpha1, alpha2 and beta1) and cGMP-dependent protein kinases (cGK I and cGK II) in cerebellar granule cells through real time-polymerase chain reaction (RT-PCR) and Western blotting. We were able to detect guanylyl cyclase subunits and cGK I and cGK II in cerebellar granule cells at every stage of development examined (cells freshly isolated from 7-day-old rat pups, and cells cultured for 7 days or 14 days). Expression levels, nevertheless, varied significantly at each stage. nNOS, alpha2 and beta1 and cGK II levels increased during granule cell development, while alpha1 and cGK I showed an opposite behaviour pattern; the levels of these latter proteins diminished as the cells matured. The functionality of this pathway was assessed by stimulating cells kept in culture for 7 days with DEA/NO or with N-methyl-D-aspartate (NMDA). Cells responded by increasing intracellular cGMP and activating cGMP-dependent protein kinase activity, which effectively phosphorylated two well-known substrates of this activity, the vasodilator stimulated phosphoprotein (VASP) and the cAMP response element binding protein (CREB). In summary, through both functional and biochemical tests, this is the first demonstration of a complete NO/cGMP signalling transduction pathway in cerebellar granule cells. Our results also indicate the developmental regulation of the proteins in this system.     

Saccharomyces cerevisiae Rrm3p DNA helicase promotes genome integrity by preventing replication fork stalling: viability of rrm3 cells requires the intra-S-phase checkpoint and fork restart activities

Rrm3p is a 5'-to-3' DNA helicase that helps replication forks traverse protein-DNA complexes. Its absence leads to increased fork stalling and breakage at over 1,000 specific sites located throughout the Saccharomyces cerevisiae genome. To understand the mechanisms that respond to and repair rrm3-dependent lesions, we carried out a candidate gene deletion analysis to identify genes whose mutation conferred slow growth or lethality on rrm3 cells. Based on synthetic phenotypes, the intra-S-phase checkpoint, the SRS2 inhibitor of recombination, the SGS1/TOP3 replication fork restart pathway, and the MRE11/RAD50/XRS2 (MRX) complex were critical for viability of rrm3 cells. DNA damage checkpoint and homologous recombination genes were important for normal growth of rrm3 cells. However, the MUS81/MMS4 replication fork restart pathway did not affect growth of rrm3 cells. These data suggest a model in which the stalled and broken forks generated in rrm3 cells activate a checkpoint response that provides time for fork repair and restart. Stalled forks are converted by a Rad51p-mediated process to intermediates that are resolved by Sgs1p/Top3p. The rrm3 system provides a unique opportunity to learn the fate of forks whose progress is impaired by natural impediments rather than by exogenous DNA damage.     

Novel epicatechin derivatives with antioxidant activity modulate interleukin-1beta release in lipopolysaccharide-stimulated human blood

We examine the potential antioxidant activity and the immune function of new epicatechin conjugates obtained by depolymerization of grape polymeric flavanols in the presence of cysteamine or cysteine. When incubated with an erythrocyte suspension, flavanols protected the erythrocyte membrane from hemolysis induced by 2,2'-azo-bis(2-amidinopropane)dihydrochloride (AAPH), an azo free radical initiator. The inhibitory effect was concentration-dependent and the IC50 was 119.8 microM for epicatechin, and 74.9 and 89.4 microM for the cysteine and cysteamine derivatives, respectively. These compounds were tested for their antioxidant activity and their capacity to modulate interleukin-1beta (IL-1beta), which is currently considered to be the major cytokine factor influencing the acute phase of the inflammatory response. At concentrations up to 20 microM, epicatechin and its derivatives inhibited the production of IL-1beta in whole blood incubated in the presence of E. coli lipopolysaccharide (LPS), in a concentration-dependent manner. The most active compound was the cysteamine derivative.     

<Emphasis Type="Italic">Rhizobium etli</Emphasis> maize populations and their competitiveness for root colonization

Rhizobium etli, which normally forms nitrogen-fixing nodules on Phaseolus vulgaris (common bean), is a natural maize endophyte. The genetic diversity of R. etli strains from bulk soil, bean nodules, the maize rhizosphere, the maize root, and inside stem tissue in traditional fields where maize is intercropped with P. vulgaris-beans was analyzed. Based on plasmid profiles and alloenzymes, it was determined that several R. etli types were preferentially encountered as putative maize endophytes. Some of these strains from maize were more competitive maize-root colonizers than other R. etli strains from the rhizosphere or from bean nodules. The dominant and highly competitive strain Ch24-10 was the most tolerant to 6-methoxy-2-benzoxazolinone (MBOA), a maize antimicrobial compound that is inhibitory to some bacteria and fungi. The R. tropici strain CIAT899, successfully used as inoculant of P. vulgaris, was also found to be a competitive maize endophyte in inoculation experiments.