Molecular biology of yeast exoglucanases

Abstract Three exoglucanase (Exg) genes have been reported in Saccharomyces cerevisiae . Gene EXG1 encodes the major isoenzyme (Exgl). Differential glycosylation of the primary translation product throughout the secretory pathway results in the secretion of several glycoforms. The major glycoform (Exglb) contains two short carboxypeptidase Y-like oligosaccharides attached to both potential glycosylation sites present in the molecule. A minor glycoform (Exgla) arises from the former by elongation of the second oligosaccharide. The protein portion is processed in the secretory pathway by the Kex2 protease. Gene EXG2 encodes a 63 kDa polypeptide with 12 potential glycosylation sites. The predicted protein, Exgll, carries a signal peptide at the amino terminus and a glycosyl-phosphatidyl inositol anchoring motif at the carboxyl end. The latter appears responsible for the particulate nature of this isoenzyme, since its elimination results in the secretion of this activity into the culture medium. Gene SSG1 encodes a 52 kDa polypeptide which is specifically synthesized during sporulation of diploids. SSC1 expression is under control of both sexual (a1-α2 element) and nutritional control. Although homozygous ssg1 / ssg1 diploid strains are still able to complete sporulation, they exhibited a delay in the appearance of mature asci. Single or double disruption of EXG1 and EXG2 did not result in any relevant phenotype and the triple mutant behaved as ssg1 /ssg1 . A Exgl-related enzyme is secreted by Candida albicans . All these four enzymes share 8 highly conserved regions in the same relative positions, indicating that they derive from a common ancestor. However, no clear function has so far been demonstrated for them.     

Using Range-Wide Abundance Modeling to Identify Key Conservation Areas for the Micro-Endemic Bolson Tortoise (Gopherus flavomarginatus)

A widespread biogeographic pattern in nature is that population abundance is not uniform across the geographic range of species: most occurrence sites have relatively low numbers, whereas a few places contain orders of magnitude more individuals. The Bolson tortoise Gopherus flavomarginatus is endemic to a small region of the Chihuahuan Desert in Mexico, where habitat deterioration threatens this species with extinction. In this study we combined field burrows counts and the approach for modeling species abundance based on calculating the distance to the niche centroid to obtain range-wide abundance estimates. For the Bolson tortoise, we found a robust, negative relationship between observed burrows abundance and distance to the niche centroid, with a predictive capacity of 71%. Based on these results we identified four priority areas for the conservation of this microendemic and threatened tortoise. We conclude that this approach may be a useful approximation for identifying key areas for sampling and conservation efforts in elusive and rare species.     

Evaluation of Haemophilus influenzae type b carrier status among children 10 years after the introduction of Hib vaccine in Brazil

The aim of the present study was to assess the prevalence of Haemophilus influenzae type b (Hib) nasopharyngeal (NP) colonisation among healthy children where Hib vaccination using a 3p+0 dosing schedule has been routinely administered for 10 years with sustained coverage (> 90%). NP swabs were collected from 2,558 children who had received the Hib vaccine, of whom 1,379 were 12-< 24 months (m) old and 1,179 were 48-< 60 m old. Hi strains were identified by molecular methods. Hi carriage prevalence was 45.1% (1,153/2,558) and the prevalence in the 12-< 24 m and 48-< 60 m age groups were 37.5% (517/1,379) and 53.9% (636/1,179), respectively. Hib was identified in 0.6% (16/2,558) of all children in the study, being 0.8% (11/1,379) and 0.4% (5/1,179) among the 12-< 24 m and 48-< 60 m age groups, respectively. The nonencapsulate Hi colonisation was 43% (n = 1,099) and was significantly more frequent at 48-< 60 m of age (51.6%, n = 608) compared with that at 12-< 24 m of age (35.6%, n = 491). The overall resistance rates to ampicillin and chloramphenicol were 16.5% and 3.7%, respectively; the co-resistance was detected in 2.6%. Our findings showed that the Hib carrier rate in healthy children under five years was very low after 10 years of the introduction of the Hib vaccine.     

Posttranslational acetylation of α-tubulin constrains protofilament number in native microtubules

1. Download : Download high-res image (438KB)
2. Download : Download full-size image

Highlights? α-TAT mutants have short microtubules and variable protofilament number ? α-tubulin K40 acetylation promotes interprotofilament salt bridges ? α-tubulin K40 acetylation is a key constraint on protofilament number in vivo     

High supply of NO3 ? mitigates salinity effects through an enhancement in the efficiency of photosystem II and CO2 assimilation in Jatropha curcas plants

This study was performed to determine if a high supply of N-NO₃ ^? is capable of mitigating negative salinity effects on photosynthesis and growth through the stimulation of nitrate assimilation, which could act as an sink from photosynthetic electron transport chain and restrict the over reduction in thylakoid membrane in Jatropha curcas leaves. The experiment was arranged in a factorial design with two nitrate concentrations (1 and 10?mM) and two NaCl levels (0 and 100?mM). Salt-stressed plants supplied with high NO₃ ^? demonstrated a higher nitrate uptake rate, nitrate reductase activity and soluble-protein content when compared with plants that presented low nitrate uptake. High nitrate assimilation was associated with higher leaf growth, CO₂ assimilation and lower membrane damage in salt-stressed plants. The superior performance of salt-stressed plants grown with high NO₃ ^? was indicated by a higher effective quantum yield of PSII and electron transport rate and lower energy excess at the PSII level and non-photochemical quenching. Interestingly, a high NO₃ ^? level in the absence of NaCl did not alter the leaf growth, photochemical activity and gas exchange parameters when compared with plants supplied with low nitrate. The proline and glycinebetaine contents were similarly increased in both low- and high-NO₃ ^? salt-stressed plants. Our data suggest that the favorable effects induced by high nitrate supply were possibly associated with stimulation in the nitrate assimilatory pathway. This process might have acted as a sink of electrons from the thylakoid membranes minimizing photo-damage and stimulating CO₂ assimilation under salinity in J. curcas.     

Resistance to osmotic stress of horse spermatozoa: the role of ionic pumps and their relationship to cryopreservation success

To study the resistance of horse spermatozoa against hyperosmotic stress, cells were incubated in solutions of 600 to 4000 mOsm(undisturbed media). Then, semen was immediately placed into an iso-osmotic solution (disrupted media). Incubation in undisturbed media decreased sperm viability in an osmolarity- and temperature-dependent manner. Viability was further decreased in disrupted media, with the effect dependent upon the initial osmolarity of the media and on the temperature. Treatment with ouabain or amiloride impaired the resistance of horse spermatozoa to hyperosmotic stress. Very few correlations were strong between viability after hyperosomotic stress and quality parameters of fresh and frozen-thawed horse semen. The results indicate that the usefulness of resistance to hyperosmotic stress in assessing frozen-thawed semen quality is compromised, since other factors are involved in the resistance to freezing-thawing. Both Na (+)K (+) ATP-ase and the Na (+)H (+) antiporter act in the resistance to hyperosmotic stress in horse spermatozoa.     

Subjecting horse spermatozoa to hypoosmotic incubation: effects of ouabain

Although hypoosmotic tests are widely used to assess spermatozoal quality in different species, they have not been used extensively in the stallion. Moreover, the role of the Na (+)K (+), ouabain sensitive-ATP-ase in the response of equine sperm to hypoosmotic shock is not well understood. This study tests two hypotheses: 1) that equine spermatozoa will respond to a hypoosmotic medium by swelling of the tail, and 2) that addition of ouabain will increase the percentage of swollen sperm tails. Ejaculates from 3 stallions were collected with an artificial vagina and diluted in Kenney's medium (Time = 0). Aliquots were randomly selected to be incubated in an isoosmotic (297 mOsm) or different hypoosmotic media that were composed of citrate or of citrate w?th fructose. The osmolarity of the hypoosmotic media with citrate ranged from 18 to 96 mOsm, and the medium composed of citrate plus fructose (HOS medium) was of 153 mOsm. Moreover, aliquots of spermatozoa pretreated with ouabain were added to the isoosmotic medium and also to the HOS and the 96 mOsm citrate medium (ORT medium). Incubation of equine sperm in the hypoosmotic media resulted in a time- and osmolarity-dependent swelling of the sperm tail, reaching maximum values after incubation for 20-30 min in both the HOS and ORT media. Ouabain induced a dose-dependent effect on swollen tails and viability in fresh semen and also affected some parameters related to motility. Ouabain also increased the swelling response in a hypoosmotic medium although viability decreased. The percentage of swollen tails after incubation in ORT and HOS media snowed significant correlations to viability, altered acrosomes and total motility, but not to other parameters of horse semen analysis. Our results suggest that hypoosmotic tests could be used to improve standard horse semen analysis. Additionally, Na (+)K (+)-ATP-ase activity could be related to the response against hypoosmotic shock of horse spermatozoa.     

Human mesenchymal stem cell transformation is associated with a mesenchymal-epithelial transition

Carcinomas are widely thought to derive from epithelial cells with malignant progression often associated with an epithelial-mesenchymal transition (EMT). We have characterized tumors generated by spontaneously transformed human mesenchymal cells (TMC) previously obtained in our laboratory. Immunohistopathological analyses identified these tumors as poorly differentiated carcinomas, suggesting that a mesenchymal-epithelial transition (MET) was involved in the generation of TMC. This was corroborated by microarray and protein expression analysis that showed that almost all mesenchymal-related genes were severely repressed in these TMC. Interestingly, TMC also expressed embryonic antigens and were able to integrate into developing blastocysts with no signs of tumor formation, suggesting a dedifferentiation process was associated with the mesenchymal stem cell (MSC) transformation. These findings support the hypothesis that some carcinomas are derived from mesenchymal rather than from epithelial precursors.