Desiccation and osmotic stress increase the abundance of mRNA of the tonoplast aquaporin BobTIP26-1 in cauliflower cells

Changes in vacuolar structure and the expression at the RNA level of a tonoplast aquaporin (BobTIP26-1) were examined in cauliflower (Brassicaoleracea L. var. botrytis) under water-stress conditions. Gradual drying out of slices of cauliflower floret tissue caused its collapse, with a shrinkage in tissue and cell volumes and an apparent vesiculation of the central vacuole, whereas osmotic stress resulted in plasmolysis with a collapse of the cytoplasm and the central vacuole within. Osmotic stress caused a rapid and substantial increase in BobTIP26 mRNA in slices of floret tissue. Exposure of tissue slices to a regime of desiccation showed a slower but equally large rise in BobTIP26 mRNA followed by a rapid decline upon rehydration. In situ hybridization showed that BobTIP26-2 mRNA is expressed most highly in meristematic and expanding cells of the cauliflower florets and that desiccation strongly increased the expression in those cells and in differentiated cells near the xylem vessels. These data indicate that under water-deficit conditions, expression of the tonoplast aquaporin gene in cauliflower is subject to a precise regulation that can be correlated with important cytological changes in the cells. Received: 21 October 1998 / Accepted: 10 February 1999     

Three-dimensional electron microscopy analysis of ndc10-1 mutant reveals an aberrant organization of the mitotic spindle and spindle pole body defects in Saccharomyces cerevisiae

Kinetochore components play a major role in regulating the transmission of genetic information during cell division. Ndc10p, a kinetochore component of the essential CBF3 complex in budding yeast is required for chromosome attachment to the mitotic spindle. ndc10-1 mutant was shown to display chromosome mis-segregation as well as an aberrant mitotic spindle (Goh and Kilmartin, 1993). In addition, Ndc10p localizes along the spindle microtubules (Muller-Reichert et al., 2003). To further understand the role of Ndc10p in the mitotic apparatus, we performed a three-dimensional electron microscopy (EM) reconstruction of mitotic spindles from serial sections of cryo-immobilized ndc10-1 mutant cells. This analysis reveals a dramatic reduction in the number of microtubules present in the half-spindle, which is connected to the newly formed spindle pole body (SPB) in ndc10-1 cells. Moreover, in contrast to wild-type (WT) cells, ndc10-1 cells showed a significantly lower signal intensity of the SPB components Spc42p and Spc110p fused with GFP, in mother cell bodies compared with buds. A subsequent EM analysis also showed clear defects in the newly formed SPB, which remains in the mother cell during anaphase. These results suggest that Ndc10p is required for maturation of the newly formed SPB. Intriguingly, mutations in other kinetochore components, ndc80-1 and spc24-1, showed kinetochore detachment from the spindle, similar to ndc10-1, but did not display defects in SPBs. This suggests that unattached kinetochores are not sufficient to cause SPB defects in ndc10-1 cells. We propose that Ndc10p, alongside its role in kinetochore–microtubule interaction, is also essential for SPB maturation and mitotic spindle integrity.     

Altérations morphologiques des spermatozoïdes en microscopie électronique: indications, phénotypes, fécondance, et pronostic de fertilité

Conventional semen analysis (sperm count) is limited to examination of spermatozoa at a magnification of x1,000, which may be insufficient in rare situations. Electron microscopy sperm examination allows high-power (x 100,000) analysis of sperm organelles and quantification of abnormalities of the constituents involved in sperm mobility and fertility potential. Electron microscopy sperm morphology examination is rarely indicated and is reserved to: 1) severe monomorphic and stable teratospermia (globozoospermia = spermatozoa with a round head and no acrosome, pinheads = decapitated spermatozoa), 2) partial (asthenospermia) or total (akinetospermia) alteration of sperm mobility and/or quality of sperm movement. All of these anomalies are associated with primary infertility. Globozoospermia and pinheads can be detected by light microscopy. Electron microscopy sperm morphology examination precisely identifies and quantifies sperm abnormalities. Pathological phenotypes have a heterogeneous expression. The organelles of spermatozoa other than those primarily involved in the pathological phenotype may also present alterations. Globozoospermia is generally characterized by the absence of elongation of the nucleus, and absence of the acrosome and the post-acrosomal region. The implantation fossa and basal plate are generally missing in decapitated spermatozoa. Asthenospermia may be an indication for electron microscopy sperm examination when it is not associated with necrospermia. Sperm with fibrous sheath dysplasia (FSD) generally present a short flagella and very low overall mobility, less than 5%. The various phenotypes are characterized by abnormal arrangements of the constituents of the fibrous sheath and 20% of patients also present respiratory tract disease. In primary ciliary dyskinesia (PCD), spermatozoa are often immobile and present a normal morphology on light microscopy. Apart from the complete form with absent axoneme, incomplete forms are also observed with absence of the dynein arms, peripheral doublets, microtubules. These phenotypes have a low prevalence in the population of infertile men. A familial incidence, parental consanguinity, and a high incidence in certain geographical regions are frequently reported, suggesting the existence of one or several genetic mechanisms. Despite the limited state of knowledge at the present time, couples must be informed about the possible transmission of the phenotype to their descendants. All men with these phenotypes are spontaneously infertile. The only alternative fertilization technique is intracytoplasmic sperm injection (ICSI). According to the literature and our own experience, the results of ICSI with sperm presenting these phenotypes are poorer than those of ICSI in general. Electron microscopy is not only a diagnostic tool in severe male infertility, but also a prognostic indicator of the success of management by ICSI, which must be evaluated for each case.     

Molecular signals in the trafficking of Toxoplasma gondii protein MIC3 to the micronemes

The protozoan parasite Toxoplasma gondii is equipped with a sophisticated secretory apparatus, including three distinct exocytic organelles, named micronemes, rhoptries, and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of T. gondii infection. We have shown that MIC3 is processed in a post-Golgi compartment and that the MIC3 propeptide and epidermal growth factor (EGF) modules contain microneme-targeting information. The minimal requirement for microneme delivery is defined by the propeptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the propeptide, the dimerization of MIC3, and the chitin binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependent manner.     

Testing competing measures of profitability for mobile resources

Optimal diet theory often fails to predict a forager’s diet choice when prey are mobile. Because they escape or defend themselves, mobile prey are likely to increase the forager’s handling time, thereby decreasing its fitness gain rate. Many animals have been shown to select their prey so as to maximize either their fitness gain or their fitness gain rate. However, no study has yet compared directly these two measures of profitability by generating testable predictions about the choice of the forager. Under laboratory conditions, we compared these two measures of profitability, using the aphid parasitoid Aphidius colemani and its host, Myzus persicae. Fitness gain was calculated for parasitoids developing in each host instar by measuring life-history traits such as developmental time, sex ratio and fecundity. Fitness gain rate was estimated by dividing fitness gain by handling time, the time required to subdue the host. Fourth instar aphids provided the best fitness gain to parasitoids, whereas second instar aphids were the most profitable in terms of fitness gain rate. Host choice tests showed that A. colemani females preferred second instar hosts, suggesting that their decision maximizes fitness gain rate over fitness gain. Our results indicate that fitness gain rate is a reliable predictor of animal’s choice for foragers exploiting resources that impose additional time cost due to their mobility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The effect of MMP inhibitor GM6001 on early fibroblast-mediated collagen matrix contraction is correlated to a decrease in cell protrusive activity

Although fibroblasts play an essential part during the wound healing response, the mechanisms by which they mediate tissue remodelling and contraction are still unclear. Using live cell and matrix imaging within 3D free-floating fibroblast-populated collagen lattices as a model for tissue contraction, we compared the behaviour of a range of fibroblasts with low and high contraction abilities and analysed the effect of the broad spectrum MMP-inhibitor GM6001 on cell behaviour and matrix contraction. We identified two mechanisms underlying matrix contraction, one via direct cell-mediated contractile activity, the second through matrix degradation. These appear to be linked to cell morphology and regulated by the collagen concentration within the matrix. Cells with a rounded morphology proliferated in the matrix but did not remodel it efficiently, resulting in a poor ability to contract matrices. Cells with an elongated morphology showed higher levels of protrusive activity, leading to efficient matrix remodelling and contraction. GM6001 inhibited week-long matrix contraction to various extents with the different cell lines. However, quantitative analysis of the cell protrusive activity showed that GM6001 consistently decreased cell dynamics in 3D by about 20%, and this was correlated with a significant reduction in early matrix contraction. Overall our results suggest that although fibroblast-mediated matrix contraction depends on both cell dynamics and MMP-mediated matrix degradation, the efficiency of GM6001 treatment in preventing contraction might be linked to a direct effect on cell dynamics.     

Simultaneous determination of ivabradine and its metabolites in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective, sensitive and reproducible liquid chromatographic method with tandem mass spectrometric detection has been developed and validated for the analysis of a new specific bradycardic agent, ivabradine (S 16257) and six potentially active metabolites in human plasma. Isolation of these compounds and of the internal standard was performed by an automated solid-phase extraction system using Oasis cartridges. Separation and detection of ivabradine and its metabolites were achieved using a C₁₈ column and a MS–MS detector with a positive electrospray ionization source. Ivabradine and its metabolites gave a linear response ranging from 0.1 or 0.2 to 20 ng/ml and the limits of quantitation ranged from 0.1 to 0.2 ng/ml using a 0.5 ml plasma sample size. A complete validation demonstrated the method to be accurate, precise and specific for the simultaneous quantification of ivabradine and its metabolites in human plasma. The method was subsequently applied to the quantitative determination of ivabradine and its metabolites in human plasma samples from healthy volunteers participating in a clinical study to provide pharmacokinetic data.