Influence of staining and sampling procedures on goat sperm morphometry using the Sperm Class Analyzer

Computer-assisted sperm morphometry analysis (ASMA) has improved the assessment of sperm morphology, but the results depend on the use of adequate sampling and staining procedures of spermatozoa from individual species. In this study, the Sperm Class Analyzer ASMA system was used for the morphometric analysis of goat sperm heads. Semen samples, obtained from four bucks, were used to evaluate the influence of three staining procedures (Diff-Quik, Hemacolor and Harris' Haematoxylin) on the accuracy of image processing and sperm morphometry, the effect of the number of cells analysed and the repeatability of the method. These experiments were performed to obtain objective, accurate and reliable sperm morphometric measurements of goat spermatozoa. Diff-Quik and Harris' Haematoxylin were significantly (p<0.05) more accurate than Hemacolor. However, Diff-Quik obtained the highest proportion of correctly analysed sperm heads (86.06%) and the lowest coefficients of variation on the image processing and morphometric measurements. The staining methods affected significantly the sperm dimensions (p<0.001) with increased values from Diff-Quik than Hemacolor and Harris' Haematoxylin, respectively (Diff-Quik>Hemacolor>Harris' Haematoxylin). No differences in morphometric parameters were found when 100, 150, 175 or 200 spermatozoa were analysed. The repeatability of results obtained was very high since no differences were found when measuring the same sperm on multiple attempts. In conclusion, to obtain objective, accurate and repeatable sperm morphometric measurements by the Sperm Class Analyzer system in goats, the analysis of 100 spermatozoa from slides which have been previously stained with Diff-Quik is recommended.     

Signals from the sympathetic nervous system regulate hematopoietic stem cell egress from bone marrow

Hematopoietic stem and progenitor cells (HSPC), attracted by the chemokine CXCL12, reside in specific niches in the bone marrow (BM). HSPC migration out of the BM is a critical process that underlies modern clinical stem cell transplantation. Here we demonstrate that enforced HSPC egress from BM niches depends critically on the nervous system. UDP-galactose ceramide galactosyltransferase-deficient (Cgt(-/-)) mice exhibit aberrant nerve conduction and display virtually no HSPC egress from BM following granulocyte colony-stimulating factor (G-CSF) or fucoidan administration. Adrenergic tone, osteoblast function, and bone CXCL12 are dysregulated in Cgt(-/-) mice. Pharmacological or genetic ablation of adrenergic neurotransmission indicates that norepinephrine (NE) signaling controls G-CSF-induced osteoblast suppression, bone CXCL12 downregulation, and HSPC mobilization. Further, administration of a beta(2) adrenergic agonist enhances mobilization in both control and NE-deficient mice. Thus, these results indicate that the sympathetic nervous system regulates the attraction of stem cells to their niche.     

Effect of diastolic pressure on MLC2v phosphorylation in the rat left ventricle

The effect of passive muscle stretch on the extent of MLC2v phosphorylation was investigated. We used an isolated rat heart preparation and controlled the passive pressure of the left ventricle (LV) at 0 or 15 mmHg. The hearts were flash frozen and the LV free wall was split into epicardial and the endocardial halves. The samples were solubilized using a novel method that minimizes changes in the phosphate content of MLC2v under non-denaturing conditions. The proteins were separated by urea glycerol PAGE and identified by mass spectrometry and Western blots. At 0 mmHg passive pressure, the extent of MLC2v phosphorylation of the epicardium (34.1+/-1.7%) was the same as that of the endocardium (35.3+/-3.4%). At 15 mmHg passive pressure, we found a significant increase in MLC2v phosphorylation in the epicardium (to 41.5+/-2.0%) and a significant reduction in the endocardium (to 24.2+/-1.2%), giving rise to a gradient in the extent of MLC2v phosphorylation from epicardium (high) to endocardium (low). These changes in MLC2v phosphorylation that take place in response to increased diastolic pressure are likely to impact on the calcium sensitivity of actomyosin interaction (with an increased sensitivity towards the epicardium) and may play a role in the Frank-Starling mechanism of the heart.     

Local wavelet-vaguelette-based functional classification of gene expression data

This paper focuses on the problem of functional statistical classification of gene expression curves. A local-wavelet-vaguelette-based functional logistic regression approach is presented. This approach is specially suitable for the classification of non-stationary singular (non-differentiable) curves. The performance of the methodology proposed is illustrated by implementing it for the classification of yeast cell-cycle temporal gene expression profiles. A simulation study is also carried out for comparison with other functional classification methodologies.     

The residue 179 is involved in product specificity of the <Emphasis Type="Italic">Bacillus circulans</Emphasis> DF 9R cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferases (CGTases) are important enzymes in biotechnology because of their ability to produce cyclodextrin (CD) mixtures from starch whose relative composition depends on enzyme source. A multiple alignment of 46 CGTases and Shannon entropy analysis allowed us to find differences and similarities that could be related to product specificity. Interestingly, position 179 has Gly in all the CGTases except in that from Bacillus circulans DF 9R which possesses Gln. The absence of a side chain at that position has been considered as a strong requirement for substrate binding and cyclization process. Therefore, we constructed two mutants of this enzyme, Q179L and Q179G. The activity and kinetic parameters of Q179G remained unchanged while the Q179L mutant showed a different CDs ratio, a lower catalytic efficiency, and a decreased ability to convert starch into CDs. We show that position 179 is involved in CGTase product specificity and must be occupied by Gly—without a side chain—or by amino acid residues able to interact with the substrate through hydrogen bonds in a way that the cyclization process occurs efficiently. These findings are also explained on the basis of a structural model.     

Differential expression of disialic acids in the cerebellum of senile mice

It is known that disialic acids (diSia) are present in the mammalian brain. However, the precise anatomical distribution and the chronology of its expression along life are not well studied yet. It is accepted that the transfer of diSia in the brain is mediated mainly by the enzyme ST8Sia III (α2,8-sialyltransferase III). We studied the expression of diSia glycoepitopes and of the ST8Sia III gene in different structures of the mouse brain at different postnatal stages by immunohistochemistry and real-time polymerase chain reaction, respectively. C57BL/6 mice of different stages were used. Samples of hippocampus, olfactory bulb, cortex and cerebellum were processed for studies of molecular biology and immunohistochemistry. Histological analysis revealed an important decrease in diSia labeling in the senile cerebellum compared with other structures and stages (P???0.001). In concordance with these results, a significant decrease in ST8Sia III gene expression was found in the cerebellum of senile animals (P?相似文献   

A combination of hydrodynamical and statistical modelling reveals non-stationary climate effects on fish larvae distributions

Biological processes and physical oceanography are often integrated in numerical modelling of marine fish larvae, but rarely in statistical analyses of spatio-temporal observation data. Here, we examine the relative contribution of inter-annual variability in spawner distribution, advection by ocean currents, hydrography and climate in modifying observed distribution patterns of cod larvae in the Lofoten-Barents Sea. By integrating predictions from a particle-tracking model into a spatially explicit statistical analysis, the effects of advection and the timing and locations of spawning are accounted for. The analysis also includes other environmental factors: temperature, salinity, a convergence index and a climate threshold determined by the North Atlantic Oscillation (NAO). We found that the spatial pattern of larvae changed over the two climate periods, being more upstream in low NAO years. We also demonstrate that spawning distribution and ocean circulation are the main factors shaping this distribution, while temperature effects are different between climate periods, probably due to a different spatial overlap of the fish larvae and their prey, and the consequent effect on the spatial pattern of larval survival. Our new methodological approach combines numerical and statistical modelling to draw robust inferences from observed distributions and will be of general interest for studies of many marine fish species.     

Exogenous Schwann Cells Migrate,Remyelinate and Promote Clinical Recovery in Experimental Auto-Immune Encephalomyelitis

Schwann cell (SC) transplantation is currently being discussed as a strategy that may promote functional recovery in patients with multiple sclerosis (MS) and other inflammatory demyelinating diseases of the central nervous system (CNS). However this assumes they will not only survive but also remyelinate demyelinated axons in the chronically inflamed CNS. To address this question we investigated the fate of transplanted SCs in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in the Dark Agouti rat; an animal model that reproduces the complex inflammatory demyelinating immunopathology of MS. We now report that SCs expressing green fluorescent protein (GFP-SCs) allografted after disease onset not only survive but also migrate to remyelinate lesions in the inflamed CNS. GFP-SCs were detected more frequently in the parenchyma after direct injection into the spinal cord, than via intra-thecal delivery into the cerebrospinal fluid. In both cases the transplanted cells intermingled with astrocytes in demyelinated lesions, aligned with axons and by twenty one days post transplantation had formed Pzero protein immunoreactive internodes. Strikingly, GFP-SCs transplantation was associated with marked decrease in clinical disease severity in terms of mortality; all GFP-SCs transplanted animals survived whilst 80% of controls died within 40 days of disease.