Life and death of female gametes during oogenesis and folliculogenesis

The vertebrate ovary is an extremely dynamic organ in which excessive or defective follicles are rapidly and effectively eliminated early in ontogeny and thereafter continuously throughout reproductive life. More than 99% of follicles disappear, primarily due to apoptosis of granulosa cells, and only a minute fraction of the surviving follicles successfully complete the path to ovulation. The balance between signals for cell death and survival determines the destiny of the follicles. An abnormally high rate of cell death followed by atresia can negatively affect fertility and eventually lead irreversibly to premature ovarian failure. In this review we provide a short overview of the role of programmed cell death in prenatal differentiation of the primordial germ cells and in postnatal folliculogenesis. We also discuss the issue of neo-oogenesis. Next, we highlight molecules involved in regulation of granulosa cell apoptosis. We further discuss the potential use of scores for apoptosis in granulosa cells and characteristics of follicular fluid as prognostic markers for predicting the outcome of assisted reproduction. Potential therapeutic strategies for combating premature ovarian failure are also addressed.     

Species relationships in the genus Vasconcellea (Caricaceae) based on molecular and morphological evidence

Validity of the taxa currently recognized in the genus Vasconcellea was analyzed by investigating morphological and molecular data from 105 specimens of this genus and six specimens of the related genus Carica. Taxon identification of these specimens was compared with clustering in two phenetic dendrograms generated with 36 morphological characters and 254 amplified fragment length polymorphic (AFLP) markers. Moreover, cytoplasmic haplotypes were assessed using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) of one mitochondrial and two chloroplast DNA regions. Results show that the morphological data set, containing mainly vegetative characteristics, merely reveals external resemblance between specimens, which is not directly associated with genetic relationships and taxon validity. Phenotypic plasticity and intercompatibility between several species are likely to confuse morphological delimitation of the taxa. Based on the results of our study, several specimens that could not be identified with the currently used identification key (1) could be attributed to a known taxon, which should be extended to include a higher range of morphological variability or (2) could be hypothesized to be of hybrid origin. Because of the high intraspecific variation within V. microcarpa and V. × heilbornii, revision of these taxa is recommended.     

Evolution of GHF5 endoglucanase gene structure in plant-parasitic nematodes: no evidence for an early domain shuffling event

Background  

Endo-1,4-beta-glucanases or cellulases from the glycosyl hydrolase family 5 (GHF5) have been found in numerous bacteria and fungi, and recently also in higher eukaryotes, particularly in plant-parasitic nematodes (PPN). The origin of these genes has been attributed to horizontal gene transfer from bacteria, although there still is a lot of uncertainty about the origin and structure of the ancestral GHF5 PPN endoglucanase. It is not clear whether this ancestral endoglucanase consisted of the whole gene cassette, containing a catalytic domain and a carbohydrate-binding module (CBM, type 2 in PPN and bacteria) or only of the catalytic domain while the CBM2 was retrieved by domain shuffling later in evolution. Previous studies on the evolution of these genes have focused primarily on data of sedentary nematodes, while in this study, extra data from migratory nematodes were included.     

Phosphoinositide-protein interactions of the plasma-membrane Ca2(+)-transport ATPase as revealed by fluorescence energy transfer.

Fluorescence energy transfer has been used to study the interaction of various phospholipids with the erythrocyte (Ca2+ + Mg2+)-ATPase. The fluorescence energy transfer between tryptophan residues of the (Ca2+ + Mg2+)-ATPase purified from erythrocytes and pyrene-labelled analogues of phosphatidylcholine (Pyr-PC), phosphatidylinositol (Pyr-PI), phosphatidylinositol 4-phosphate (Pyr-PIP), phosphatidylinositol 4,5-bisphosphate (Pyr-PIP2), phosphatidylglycerol (Pyr-PG) and phosphatidic acid (Pyr-PA) was measured. A positive correlation was found between the number of negative charges on the phospholipids (PIP2 greater than PIP greater than PA greater than PI = PG greater than PC) and the potency of their pyrene-labelled analogues to act as quantum acceptors in fluorescence energy transfer from the tryptophan residues of the (Ca2+ + Mg2+)-ATPase. This is the first time that a physical interaction between PIP/PIP2 and an intrinsic membrane protein has been demonstrated. The dependence of the energy transfer on the number of negative charges of the phospholipids closely resembles the previously demonstrated charge dependence of the enzymatic activity of the (Ca2+ + Mg2+)-ATPase (Missiaen, L., Raeymaekers, L., Wuytack, F., Vrolix, M., Desmet, H. and Casteels, R. (1989) Biochem. J. 263, 687-694). It is concluded that the stimulation of the (Ca2+ + Mg2+)-ATPase activity by negatively charged phospholipids is based on a binding of these lipids to the (Ca2+ + Mg2+)-ATPase and that the negative charges are a major modulatory factor for this interaction.     

Expression of endoplasmic-reticulum Ca2(+)-pump isoforms and of phospholamban in pig smooth-muscle tissues.

The expression of the gene 2 sarcoplasmic/endoplasmic-reticulum Ca2(+)-pump isoforms (SERCA2a and SERCA2b) and of phospholamban was studied in pig smooth muscle of the stomach, longitudinal ileum, pulmonary artery and aorta. mRNA levels were determined using an RNAase protection assay. The SERCA2 isoforms and phospholamban were tested on Western blots with a panel of antibodies, some of which were isoform-specific. The pig smooth-muscle tissues all contained comparable SERCA2 mRNA levels, but these levels were 10-20-fold lower than SERCA2 mRNA levels in cardiac muscle. Of the SERCA2 mRNAs in smooth muscle, 72-81% encoded the non-muscle isoform (SERCA2b), and Western blot analysis with isoform-specific antibodies confirmed that the SERCA2b isoform is the predominant endoplasmic-reticulum Ca2(+)-pump in smooth muscle. In contrast with SERCA2 mRNA levels, phospholamban mRNA levels varied by 12-fold between the different pig smooth-muscle tissues, with low and very low levels in the pig pulmonary artery and the pig aorta respectively. The differential expression of phospholamban was also confirmed on Western blots. The finding that the phospholamban content varied between the different smooth-muscle tissues whereas the SERCA2 expression remained rather constant indicates that, in pig smooth muscle, the expression of phospholamban is not coupled with that of SERCA2.     

Smooth-muscle endoplasmic reticulum contains a cardiac-like form of calsequestrin

It is proposed that smooth-muscle endoplasmic reticulum contains calsequestrin and that this protein in smooth muscle resembles cardiac calsequestrin more than the skeletal-muscle form. This proposal is based on seven similarities between the smooth-muscle protein and cardiac calsequestrin. Proteins with an Mr of 55,000 can be extracted from the membranes of smooth muscle and of cardiac muscle using 100 mM Na2CO3. The protein from smooth muscle binds to phenyl-Sepharose in the absence of Ca2+ and is released by 10 mM CaCl2, as has been observed for cardiac calsequestrin. The protein from smooth muscle comigrates with the cardiac calsequestrin on Laemmli-type SDS-polyacrylamide gel electrophoresis. The protein of Mr 55,000 from smooth muscle and cardiac calsequestrin both strain blue with the carbocyanine dye Stains-all. Both proteins present similar one-dimensional Cleveland peptide maps although minor differences might exist. From an analysis of subcellular membranes separated by sucrose gradient centrifugation it is concluded that the protein with Mr 55,000 from the smooth muscle is confined to the endoplasmic reticulum, the same subcellular structure from which, in heart muscle, calsequestrin can be isolated. Antibodies raised against canine cardiac calsequestrin bind to a protein of similar Mr in smooth-muscle endoplasmic reticulum. In addition to the calsequestrin, three other extrinsic proteins with an Mr of 130,000, 100,000 and 63,000, stain blue with Stains-all and occur in the endoplasmic reticulum of smooth muscle.     

Antibodies to the calmodulin-binding Ca2+-transport ATPase from smooth muscle

Antibodies were raised against a calmodulin-binding CaMg-ATPase (Ca2+-transport ATPase) from smooth muscle. The binding of these antibodies to a number of related Ca2+-transport ATPases was studied. Antibodies to the calmodulin-binding ATPase from porcine antrum (stomach) smooth muscle do not only bind to this CaMg-ATPase, but also to the corresponding enzyme in porcine erythrocytes. However, they do not bind to the CaMg-ATPase from sarcoplasmic reticulum of porcine skeletal muscle. The binding of these antibodies to the CaMg-ATPase of smooth muscle, does not inhibit the enzyme activity.     

CCS52 and DEL1 genes are key components of the endocycle in nematode‐induced feeding sites

The establishment of galls and syncytia as feeding sites induced by root‐knot and cyst nematodes, respectively, involves a progressive increase in nuclear and cellular size. Here we describe the functional characterization of endocycle activators CCS52A, CCS52B and a repressor of the endocycle, DEL1, during two types of nematode feeding site development in Arabidopsis thaliana. In situ hybridization analysis showed that expression of CCS52A1 and CCS52B was strongly induced in galls and syncytia and DEL1 was stably but weakly expressed throughout feeding site development. Down‐regulation and over‐expression of CCS52 and DEL1 in Arabidopsis drastically affected giant cell and syncytium growth, resulting in restrained nematode development, illustrating the need for mitotic activity and endo‐reduplication for feeding site maturation. Exploiting the mechanism of endo‐reduplication may be envisaged as a strategy to control plant‐parasitic nematodes.     

5-sulfonyl-benzimidazoles as selective CB2 agonists-part 2

In a previous communication, the SAR of a series of potent and selective 5-sulfonyl-benzimidazole CB2-receptor agonists was described. The lack of in vivo activity of compounds from this series was attributed to their poor solubility and metabolic stability. In this Letter, we report on the further optimization of this series, leading to the relatively polar and peripherically acting CB2 agonists 41 and 49. Although both compounds were not active in acute pain models, the less selective compound 41 displayed good, sustained activity in a chronic model of neuropathic pain without the tolerance observed with morphine. In addition, both 41 and 49 delayed the onset of clinical symptoms in an experimental model for Multiple sclerosis.     

Tomato sap flow, stem and fruit growth in relation to water availability in rockwool growing medium

Background and aims

Irrigation strategies for glasshouse tomato are often based on solar radiation sums. However, due to new energy-saving climate control, current strategies might result in inappropriate irrigation. Because of the limited water buffering capacity of soilless growing media like rockwool, this could have adverse effects on fruit production and quality. We present an overview of tomato plant ecophysiological responses to substrate water availability to allow the evaluation of mechanistic hypotheses about internal plant water storage and depletion and reversible stem-fruit water transport.

Methods

The hydraulic properties of the growing medium were determined and plant water uptake, stem and fruit diameter variations were studied.

Results

A low substrate matric suction (?2 to ?3?kPa) had a significant effect on stem and fruit growth dynamics. The substrate water retention curve indicated a sharp decrease in hydraulic conductivity, limiting the water availability for plant roots significantly.

Conclusions

The hydraulic properties of the growing medium are of utmost importance for plant water uptake, and should therefore be incorporated in plant models describing water flow. Internally stored water responds instantaneously to varying water availability and rates of water backflow from tomato fruits can be quite substantial.