Intracellular calcium oscillations in articular chondrocytes induced by basic calcium phosphate crystals lead to cartilage degradation.

OBJECTIVE: Basic calcium phosphate (BCP) crystals, including octacalcium phosphate (OCP), carbonated-apatite (CA) and hydroxyapatite (HA) crystals are associated with destructive forms of osteoarthritis. Mechanisms of BCP-induced cartilage breakdown remain incompletely understood. We assessed the ability of BCP to induce changes in intracellular calcium (iCa(2+)) content and oscillations and the role of iCa(2+) in BCP-induced cartilage degradation. METHODS: Bovine articular chondrocytes (BACs) and bovine cartilage explants (BCEs) were stimulated with BCP or monosodium urate (MSU) crystals. iCa(2+) levels were determined by spectrofluorimetry and oscillations by confocal microscopy. mRNA expression of matrix metalloproteinase 3 (MMP-3), a disintegrin and metalloprotease with thrombospondin-like motifs 4 (ADAMTS-4) and ADAMTS-5 was assessed by quantitative real-time PCR. Glycosaminoglycan (GAG) release was measured in the supernatants of BCE cultures. RESULTS: All three BCP crystals significantly increased iCa(2+) content. OCP also induced iCa(2+) oscillations. Rate of BACs displaying iCa(2+) oscillations increased over time, with a peak after 20 min of stimulation. OCP-induced iCa(2+) oscillations involved both extracellular Ca(2+) (eCa(2+)) influx and iCa(2+) stores. Indeed, OCP-induced iCa(2+) oscillations decreased rapidly in Ca(2+)-free medium. Both voltage- and non-voltage-dependent Ca(2+) channels were involved in eCa(2+) influx. BCP crystal-induced variation in iCa(2+) content was associated with BCP crystal-induced cartilage matrix degradation. However, iCa²(+) was not associated with OCP crystal-induced mRNA expression of MMP-3, ADAMTS-4 or ADAMTS-5. CONCLUSION: BCP crystals can induce variation in iCa(2+) content and oscillations in articular chondrocytes. Furthermore, BCP crystal-induced changes in iCa(2+) content play a pivotal role in BCP catabolic effects on articular cartilage.

Clinical relevance of cagA and vacA gene polymorphisms in Helicobacter pylori isolates from Senegalese patients.

The molecular epidemiology of Helicobacter pylori in Africa is poorly documented. From January 2007 to December 2008, we investigated 187 patients with gastric symptoms in one of the main tertiary hospitals in Dakar, Senegal. One hundred and seventeen patients were culture-positive for H. pylori. Polymorphisms in vacA and cagA status were investigated by PCR; the 3'-region of cagA was sequenced, and EPIYA motifs were identified. Bacterial heterogeneity within individuals was extensively assessed by using an approach based on vacA and cagA heterogeneity. Fourteen per cent of H. pylori-positive patients displayed evidence of mixed infection, which may affect disease outcome. Patients with multiple vacA alleles were excluded from subsequent analyses. Among the final study population of 105 patients, 29 had gastritis only, 61 had ulcerated lesions, and 15 had suspicion of neoplasia based on endoscopic findings. All cases of suspected neoplasia were histologically confirmed as gastric cancer (GC). The cagA gene was present in 73.3% of isolates. CagA proteins contained zero (3.7%), one (93.9%) or two (2.4%) EPIYA-C segments, and all were western CagA. Most of the isolates possessed presumed high-vacuolization isotypes (s1i1m1 (57.1%) or s1i1m2 (21.9%)). Despite the small number of cases, GC was associated with cagA (p 0.03), two EPIYA-C segments in the C-terminal region of CagA (p 0.03), and the s1 vacA allele (p 0.002). Multiple EPIYA-C segments were less frequent than reported in other countries, possibly contributing to the low incidence of GC in Senegal.

Changes in oligosaccharide content and antioxidant enzyme activities in developing bean seeds as related to acquisition of drying tolerance and seed quality.

Seeds of bean (Phaseolus vulgaris cv. Vernel) were collected throughout their development on the plant and dried at 15 degrees C and 75% relative humidity to a final moisture content of about 16% (fresh weight basis) to determine whether the onset of tolerance to this drying condition was related to changes in soluble sugars or the activities of the main antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR). Measurements of soluble sugars and enzyme activities were made after drying the seeds, and drying tolerance was evaluated by the ability of dried seeds to germinate and to produce normal seedlings. Seeds became tolerant to drying at 45 d after anthesis, a time marking physiological maturity. At physiological maturity, the moisture content of seeds was about 50-55% (fresh weight basis) and seed dry matter reached about 190 mg per seed. Seed vigour, evaluated by controlled deterioration and conductivity measurements, continued to increase after seed mass maturity, but decreased when seeds remained thereafter for more than 7 d on the plant. Acquisition of drying tolerance was coincident with an accumulation of raffinose and stachyose. Dried-tolerant seeds were also characterized by a high amount of sucrose, the most abundant sugar, and by a low content of monosaccharides. The (raffinose+stachyose)/sucrose ratio increased during seed filling, reaching a value close to 1 when all the seeds became tolerant to drying, and maintaining this proportion during the final stages of maturation. Acquisition of drying tolerance was also related to a reorientation of the enzymatic antioxidant defence system. Drying-tolerant dried seeds displayed high CAT and GR activities and low SOD and APX activities, while the opposite condition was observed in immature dried seeds. The shift in antioxidant enzymes corresponded to the beginning of the maturation-drying phase. These results suggest that oligosaccharide metabolism and enzymatic antioxidant defences may be involved in acquisition of drying tolerance during bean seed development, but are not related to seed vigour.

Water content, raffinose, and dehydrins in the induction of desiccation tolerance in immature wheat embryos

Desiccation tolerance is initiated in wheat (Triticum aestivum L.) embryos in planta at 22 to 24 d after anthesis, at the time that the embryo water content has decreased from about 73% fresh weight (2.7 g water/g dry weight) to about 65% fresh weight (1.8 g water/g dry weight). To determine if desiccation tolerance is fully induced by the loss of a relatively small amount of water, detached wheat grains were treated to reduce the embryo water content by just a small amount to approximately 69% (2.2 g water/g dry weight). After 24 h of such incipient water loss, subsequently excised embryos were able to withstand severe desiccation, whereas those embryos that had not previously lost water could not. Therefore, a relatively small decrease in water content for only 24 h acts as the signal for the development of desiccation tolerance. Embryos that were induced into tolerance by a 24-h water loss had no detectable raffinose. The oligosaccharide accumulated at later times even in embryos of detached grains that had not become desiccation tolerant, although tolerant embryos (i.e. those that previously had lost some water) contained larger amounts of the carbohydrate. It is concluded that desiccation tolerance and the occurrence of raffinose are not correlated. Immunodetected dehydrins accumulated in embryos in planta as desiccation tolerance developed. Detachment of grains induced the appearance of dehydrins at an earlier age, even in embryos that had not been made desiccation tolerant by incipient drying. It is concluded that a small reduction in water content induces desiccation tolerance by initiating changes in which dehydrins might participate but not by their interaction with raffinose.

Physiological behaviour of encapsulated somatic embryos.

Somatic embryos are characterised by the absence of any protection, a very low reserve level and a high water content. The effects on the respiration and the radicular elongation of somatic embryos of a non toxic and easy to use hydrogel, such as alginate, have been studied. Respiration or germination rates decreased with an increase in alginate concentration. When the encapsulated somatic embryos were placed in a liquid medium, there was very little difference between the germination rates observed at different alginate concentrations, either with or without an additional PEI layer. The effect of capsule concentration on germination rate was significatively different when the artificial seeds were growing on solid media. Besides the anoxic treatment upon encapsulated somatic embryos, storage for one month also decreased the germination rate.

Fructose 2,6-bisphosphate in germinating oat seeds. A biochemical study of seed dormancy.

When dormant oat seeds were imbibed at the non-permissive temperature of 30 degrees C, the concentration of phosphoenolpyruvate and of glycerate 3-phosphate, which are two inhibitors of phosphofructokinase 2, increased almost linearly during 30 h. By contrast, these metabolites increased only after a lag period of about 10 h in non-dormant seeds imbibed at the same temperature. As a consequence of this, the concentration of the C3 derivatives remained always remarkably lower in non-dormant than in dormant seeds. Accordingly, the concentration of fructose 2,6-bisphosphate, which increased similarly in the two types of seeds during the first 8 h after the start of inhibition, then reached a plateau in dormant seeds but continued to increase for another 8 h in non-dormant seeds, reaching a maximal value a few hours before the beginning of radicle protrusion. When the dormant seeds were imbibed at the permissive temperature of 10 degrees C, the evolution of all metabolites was slowed down but behaved like that of non-dormant seeds imbibed at 30 degrees C. Experiments in which the dormant seeds were submitted to a jump from 10 degrees C to 30 degrees C and vice versa, always provoked reverse changes in the concentration of the C3 derivatives and of fructose 2,6-bisphosphate, the latter being increased in all conditions that allowed germination. Dormant seeds were also allowed to germinate at 30 degrees C by imbibition during 24 h in the presence of 3% ethanol. Again, this permissive treatment caused an arrest in the accumulation of C3 derivatives and an increase in fructose 2,6-bisphosphate. Another, apparently unrelated, biochemical difference between dormant and non-dormant oat seeds was their inorganic pyrophosphate content, which was approximately five-fold higher in non-dormant than in dormant seeds. This difference was observed before and persisted during imbibition as long as measurement could be made and was not affected by the temperature jumps or by ethanol. In contrast to the phosphoric esters under investigation, pyrophosphate was not preferentially located in the embryo.

Seed germination and seedling development in the mango (Mangifera indica L.).

Mango (Mangifera indica L., cv Ruby) seeds taken from ripe fruit showed no dormancy. They germinated at temperatures between 5 and 40 degrees C, but germination was most rapid near the upper end of this range (25-40 degrees C). The fresh seeds had a high moisture content (85%, dry weight basis) and quickly died on dehydration. The optimal temperature for growth of the seedlings was close to 30 degrees C. High temperatures (40 degrees C) and temperatures below 15 degrees C were lethal. Growth of the stem occurred in successive flushes separated by rest periods. When the leaves of the preceding flush finished growing, the axis lengthened beneath the apical bud.

Effect of Temperature, Oxygen, and Gibberellic Acid on the Development of Photosensitivity in Oldenlandia corymbosa L. Seeds during Their Incubation in Darkness.

Two successive phases can be distinguished in the development of the responsiveness to light in Oldenlandia corymbosa L. seeds during their incubation in darkness. During phase I, the responsiveness to light increases with time if there is sufficient O(2), and the higher the temperature, the faster the increase. This phase is stimulated by gibberellic acid. During the following phase (II), seeds remain responsive to light at 10 or 20 degrees C, but lose their responsiveness at higher temperature (>/=30 degrees C). This second phase depends on O(2): loss of responsiveness is accelerated at lower O(2) concentration. Phase II is only slightly affected by gibberellic acid. The results are discussed in terms of variation of phytochrome and of a reaction along the transduction chain initiated by phototransformation of this pigment, which is finally expressed in germination.

Effect of the Intensity and Duration of Light at Various Temperatures on the Germination of Oldenlandia corymbosa L. Seeds.

At temperatures below 35 to 40 degrees C, fairly intense continuous white light (13 watts per square meter) inhibits germination of Oldenlandia corymbosa L. seeds, and the lower the temperature, the greater the inhibition. However, such lighting may enable seeds to germinate later in the dark; their degree of germinability depends both on the duration of lighting and on the temperature during lighting and after transfer to the dark.

Ultrastructural changes in the endoplasmic reticulum during dormancy release of apple embryos (Pyrus malus L.).

Apple embryos were treated by cold (0°C) within the fruits, to break their dormancy; the controls were treated at 12°C or at 20°C. Ultrastructural features of meristematic cells in the embryonic axis were compared for each treatment. The organization of the cells of dormant embryos was described: Endoplasmic reticulum consisted in some short rough cisternae; lipid droplets regularly arranged near the plasmalemma constituted a kind of shell; mitochondria had a few cristae; and dictyosomes were rarely observed. All these features are typical of dry seeds. After cold treatments, the only evolution observed was in the endoplasmic reticulum, where highly organized stacks appeared progressively as a function of time at 0°C. An intermediate temperature (12°C) induced similar formations in the reticulum but they were rarely observed and their degree of organization was lower than that obtained at 0°C. At 20°C, endoplasmic reticulum resembled that of the dormant embryo cells. The relation between the appearance of these structures in the reticulum and the disappearance of dormancy induced by cold is discussed.

[Breaking of dormancy of the apple embryo (Pirus malus L.) in the absence of oxygen and cold]. / Levée de dormance de L'embryon de Pommier (Pirus malus L.) en l'absence d'oxygène et de froid.

The dormancy of apple embryos may be entirely broken without cold by keeping them in N2-atmosphere. The breaking of dormancy under these conditions is more rapid than that obtained by cold treatment.