Assembly,plasticity and selective vulnerability to disease of mouse neuromuscular junctions

Although physiological differences among neuromuscular junctions (NMJs) have long been known, NMJs have usually been considered as one type of synapse, restricting their potential value as model systems to investigate mechanisms controlling synapse assembly and plasticity. Here we discuss recent evidence that skeletal muscles in the mouse can be subdivided into two previously unrecognized subtypes, designated FaSyn and DeSyn muscles. These muscles differ in the pattern of neuromuscular synaptogenesis during embryonic development. Differences between classes are intrinsic to the muscles, and manifest in the absence of innervation or agrin. The distinct rates of synaptogenesis in the periphery may influence processes of circuit maturation through retrograde signals. While NMJs on FaSyn and DeSyn muscles exhibit a comparable anatomical organization in postnatal mice, treatments that challenge synaptic stability result in nerve sprouting, NMJ remodeling, and ectopic synaptogenesis selectively on DeSyn muscles. This anatomical plasticity of NMJs diminishes greatly between 2 and 6 months postnatally. NMJs lacking this plasticity are lost selectively and very early on in mouse models of motoneuron disease, suggesting that disease-associated motoneuron dysfunction may fail to initiate maintenance processes at “non-plastic” NMJs. Transgenic mice overexpressing growth-promoting proteins in motoneurons exhibit greatly enhanced stimulus-induced sprouting restricted to DeSyn muscles, supporting the notion that anatomical plasticity at the NMJ is primarily controlled by processes in the postsynaptic muscle. The discovery that entire muscles in the mouse differ substantially in the anatomical plasticity of their synapses establishes NMJs as a uniquely advantageous experimental system to investigate mechanisms controlling synaptic rearrangements at defined synapses in vivo.     

Onychomycosis Due to Neoscytalidium Treated with Oral Terbinafine, Ciclopirox Nail Lacquer and Nail Abrasion: A Pilot Study of 25 Patients

Background

Onychomycosis by Neoscytalidium constitutes chronic infection of the nails, and its frequency has increased in recent decades. Currently, no effective standard treatment exists and literature data remain scarce. This work aimed to conduct a pilot project of combined treatment for this infection.

Methods

Thirty patients were divided into three treatment groups: oral terbinafine plus ciclopirox nail lacquer twice a week; ciclopirox nail lacquer twice a week; and ciclopirox nail lacquer 5 days a week, all associated with nail abrasion when required, for 12 months, with 6 months posttreatment follow-up. Clinical and mycological criteria were used for evaluation.

Results

Twenty-five patients completed the study. Significant clinical lesion reduction in disease occurred in all three treatment groups: 21 patients (84 %) entered the study with more than 50 % of diseased nail plate, at the end of treatment, and at 6-month follow-up, 84 and 96 %, respectively, presented less than 25 % nail lesion. Negative microscopy was observed in 36 % of the patients at the end of treatment and in 24 % of the patients at 6-month follow-up. At treatment completion (12 months), culture was negative in 21 patients (84 %) and in 18 (72 %) at follow-up. It was not possible to establish any clinical or mycological statistical differences between groups (p > 0.05). Global medical evaluation upon treatment completion revealed that one patient (4 %) presented complete cure, 8 (32 %) presented partial cure, 16 (64 %) presented therapeutic failure. At the end of follow-up period, 6 patients (24 %) were considered to have recurrence/reinfection.

Conclusions

The results obtained at the 6-month period of follow-up showed marked improvement (96 % of clinical improvement and 72 % of negative culture) of the patients treated for onychomycosis caused by Neoscytalidium in the three tested groups with no statistical differences between them. Multicentric studies with greater number of patients enrolled are necessary to confirm these results.     

The cell cycle-regulated protein human GTSE-1 controls DNA damage-induced apoptosis by affecting p53 function

GTSE-1 (G2 and S phase-expressed-1) protein is specifically expressed during S and G2 phases of the cell cycle. It is mainly localized to the microtubules and when overexpressed delays the G2 to M transition. Here we report that human GTSE-1 (hGTSE-1) protein can negatively regulate p53 transactivation function, protein levels, and p53-dependent apoptosis. We identified a physical interaction between the C-terminal regulatory domain of p53 and the C-terminal region of hGTSE-1 that is necessary and sufficient to down-regulate p53 activity. Furthermore, we provide evidence that hGTSE-1 is able to control p53 function in a cell cycle-dependent fashion. hGTSE-1 knock-down by small interfering RNA resulted in a S/G2-specific increase of p53 levels as well as cell sensitization to DNA damage-induced apoptosis during these phases of the cell cycle. Altogether, this work suggests a physiological role of hGTSE-1 in apoptosis control after DNA damage during S and G2 phases through regulation of p53 function.     

EARLY TOXIC EFFECTS OF ZINC ON PSII OF SYNECHOCYSTIS AQUATILIS F. AQUATILIS (CYANOPHYCEAE)1

Zinc toxicity on photosynthetic activity in cells of Synechocystis aquatilis f. aquatilis Sauvageau was investigated by monitoring Hill activity and fluorescence. The oxygen‐evolving activity decreased to about 80% of the initial value after exposure to 0.1 mM ZnSO₄ for 1 h. The PSII activity was inhibited by 40% in the presence of zinc concentrations ranging from 0.5 to 5.0 mM, suggesting that the metal effect is limited by zinc uptake. The fluorescence capacity (F_max–F/F_max) decreased from 0.57 to 0.35 and 0.20 in Zn‐treated cells for 15 and 60 min, respectively, thus providing evidence for rapid inactivation of electron transport at PSII. Zinc treatment promoted a rapid increase in PSII fluorescence that was counteracted by addition of 1,4‐benzoquinone, indicating that electron transfer at the reducing side of the PSII reaction center is arrested by zinc. Furthermore, a decline in the fluorescence yield could be observed after 1 h of zinc treatment as well as when Zn‐treated cells were excited in presence of 3‐(3′,4′‐dichlorophenyl)‐1,1‐dimethylurea. Under these conditions, zinc did not affect energy transfer from phycobilisomes to PSII, and the gradual quenching of PSII fluorescence may be due to a decrease in electron flow on the donor side of PSII. However, the 20% increase in the minimal fluorescence intensity (F_o) in parallel to the absence of changes in the maximal fluorescence intensity (F_max), observed in the first hour of zinc treatment, could also suggest a metal‐induced decline in the energy transfer from PSII‐chl a antenna to the PSII reaction center.     

Evolution of rDNA FISH patterns in the Fagaceae

The Fagaceae is one of the most important plant families in European forest ecosystems, and it includes several genera distributed in the Northern hemisphere. In this work we studied the genome organization and evolution within the family, by karyotyping and physically mapping rDNA in ten European and Asian species of the genera Fagus, Quercus, and Castanea. All of the species studied had a chromosome number of 2n=2x=24, except for the first report of a single individual of Quercus suber which proved to be triploid (2n=3x=36). The rDNA physical mapping revealed several patterns: the dominant one is present in European and Asian Quercus subgenus Quercus, and in Castanea sativa and Castanea crenata, consisting of two 18S–25S rDNA loci (one subterminal major and one pericentromeric minor) and one 5S rDNA pericentromeric locus. In Fagus sylvatica and in Quercus sessilifolia, different patterns were observed: four terminal 18S–25S rDNA loci and two 5S rDNA pericentromeric loci in the former, and five 18S–25S rDNA loci (three terminal and two intercalary) and one 5S rDNA pericentromeric locus in the latter. In Castanea mollissima a distinct rDNA distribution pattern with two intercalary 18S–25S rDNA loci and two 5S rDNA was found. These findings suggest rDNA loci restructuring during Castanea evolution, and variability of 18S–25S loci between Quercus and Cyclobalanopsis subgenera.     

B cell response during infection with the MAT a and MAT alpha mating types of Cryptococcus neoformans

In the present study, we compared the B cell response of BALB/c and C57Bl/6 mice during Cryptococcus neoformans infection. This response was investigated using virulent serotype D forms of mating types alpha and a (MAT alpha and MAT a). C57Bl/6 mice showed massive (mainly cerebral) infection by both types, while BALB/c were resistant to infection. Some resistance of C57Bl/6 mice was induced by previous immunization with the capsular polysaccharide from MAT alpha. Passive immunization of C57Bl/6 mice with purified antibody (Ab) obtained from capsular polysaccharide-immunized mice also increased resistance to infection. Both mouse strains showed comparable low IgM response to the capsular polysaccharide from MAT alpha, and only C57Bl/6 mice produced IgM to the polysaccharide of MAT a. Comparable levels of different immunoglobulin (Ig) isotypes against capsular components of MAT alpha and MAT a were detected, and the response of C57Bl/6 mice was higher when compared to that of BALB/c mice. FACS analysis indicated an increase in the percentage of a high-granulosity (side-scatter) splenic subpopulation and in the percentage of splenic Gr-1+ cells in infected C57Bl/6 mice. In addition, the percentage of follicular splenic B cells was decreased after C. neoformans infection of C57Bl/6 mice. This response was more pronounced when we investigated infection induced by the MAT a mating type. Taken together, our results indicate that capsular polysaccharide derived from MAT alpha and MAT a types of C. neoformans have a stimulatory effect upon B cells but that there is no correlation between resistance of BALB/c mice and Ab production. However, the increase in resistance of C57Bl/6 mice parallels the production of Abs and a major change in splenic cell populations.     

Quantitation of drug content in a low dosage formulation by transmission near infrared spectroscopy

A transmission near infrared (NIR) spectroscopic method has been developed for the nondestructive determination of drug content in tablets with less than 1% weight of active ingredient per weight of formulation (m/m) drug content. Tablets were manufactured with drug concentrations of ∼0.5%, 0.7%, and 1.0% (m/m) and ranging in drug content from 0.71 to 2.51 mg per tablet. Transmission NIR spectra were obtained for 110 tablets that constituted the training set for the calibration model developed with partial least squares regression. The reference method for the calibration model was a validated UV spectrophotometric method. Several data preprocessing methods were used to reduce the effect of scattering on the NIR spectra and base the calibration model on spectral changes related to the drug concentration changes. The final calibration model included the spectral range from 11 216 to 8662 cm⁻¹ the standard normal variate (SNV), and first derivative spectral pretreatments. This model was used to predict an independent set of 48 tablets with a root mean standard error of prediction (RMSEP) of 0.14 mg, and a bias of only −0.05 mg per tablet. The study showed that transmission NIR spectroscopy is a viable alternative for nondestructive testing of low drug content tablets, available for the analysis of large numbers of tablets during process development and as a tool to detect drug agglomeration and evaluate process improvement efforts. Published: March 24, 2006     

Calpain-mediated proteolysis of talin regulates adhesion dynamics

Dynamic regulation of adhesion complexes is required for cell migration and has therefore emerged as a key issue in the study of cell motility. Recent progress has been made in defining some of the molecular mechanisms by which adhesion disassembly is regulated, including the contributions of adhesion adaptor proteins and tyrosine kinases. However, little is known about the potential contribution of proteolytic mechanisms to the regulation of adhesion complex dynamics. Here, we show that proteolysis of talin by the intracellular calcium-dependent protease calpain is critical for focal adhesion disassembly. We have generated a single point mutation in talin that renders it resistant to proteolysis by calpain. Quantification of adhesion assembly and disassembly rates demonstrates that calpain-mediated talin proteolysis is a rate-limiting step during adhesion turnover. Furthermore, we demonstrate that disassembly of other adhesion components, including paxillin, vinculin and zyxin, is also dependent on the ability of calpain to cleave talin, suggesting a general role for talin proteolysis in regulating adhesion turnover. Together, these findings identify calpain-mediated proteolysis of talin as a mechanism by which adhesion dynamics are regulated.     

An Outlook on Ovarian Cancer and Borderline Ovarian Tumors: Focus on Genomic and Proteomic Findings

Among the gynaecological malignancies, ovarian cancer is one of the neoplastic forms with the poorest prognosis and with the bad overall and disease-free survival rates than other gynaecological cancers. Ovarian tumors can be classified on the basis of the cells of origin in epithelial, stromal and germ cell tumors. Epithelial ovarian tumors display great histological heterogeneity and can be further subdivided into benign, intermediate or borderline, and invasive tumors. Several studies on ovarian tumors, have focused on the identification of both diagnostic and prognostic markers for applications in clinical practice. High-throughput technologies have accelerated the process of biomolecular study and genomic discovery; unfortunately, validity of these should be still demonstrated by extensive researches on sensibility and sensitivity of ovarian cancer novel biomarkers, determining whether gene profiling and proteomics could help differentiate between patients with metastatic ovarian cancer and primary ovarian carcinomas, and their potential impact on management. Therefore, considerable interest lies in identifying molecular and protein biomarkers and indicators to guide treatment decisions and clinical follow up. In this review, the current state of knowledge about the genoproteomic and potential clinical value of gene expression profiling in ovarian cancer and ovarian borderline tumors is discussed, focusing on three main areas: distinguishing normal ovarian tissue from ovarian cancers and borderline tumors, identifying different genotypes of ovarian tissue and identifying proteins linked to cancer or tumor development. By these targets, authors focus on the use of novel molecules, developed on the proteomics and genomics researches, as potential protein biomarkers in the management of ovarian cancer or borderline tumor, overlooking on current state of the art and on future perspectives of researches.Key Words: Ovarian cancer, borderline ovarian tumors, markers, genomics, proteomics, oncogenes.