Girdling affects carbohydrate-related gene expression in leaves,bark and roots of alternate-bearing citrus trees

Effects of girdling on carbohydrate status and carbohydrate-related gene expression in citrus trees were investigated. Alternate-bearing 'Murcott' (a Citrus reticulata hybrid of unknown origin) trees were girdled during autumn (25 Sep. 2001) and examined 10 weeks later. Girdling brought about carbohydrate (soluble sugar and starch) accumulation in leaves and shoot bark above the girdle, in trees during their fruitless, 'off' year. Trees during their heavy fruit load, 'on' year did not accumulate carbohydrates above the girdle due to the high demand for carbohydrates by the developing fruit. Girdling caused a strong decline in soluble sugar and starch concentrations in organs below the girdle (roots), in both 'on' and 'off' trees. Expression of STPH-L and STPH-H (two isoforms of starch phosphorylase), Agps (ADP-glucose pyrophosphorylase, small subunit), AATP (plastidic ADP/ATP transporter), PGM-C (phosphoglucomutase) and CitSuS1 (sucrose synthase), all of which are associated with starch accumulation, was studied. It was found that gene expression is related to starch accumulation in all 'off' tree organs. RNA levels of all the genes examined were high in leaves and bark that accumulated high concentrations of starch, and low in roots with declining starch concentrations. It may be hypothesized that changes in specific sugars signal the up- and down-regulation of genes involved in starch synthesis.     

Antiplatelet properties of novel N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone derivatives

This paper describes the design, synthesis and pharmacological evaluation of new N-acylhydrazone (NAH) compounds, belonging to the N-substituted-phenyl-1,2,3-triazole-4-acylhydrazone class (2a-p). Classical heteroaromatic ring bioisosterism strategies were applied to the previously reported N-phenylpyrazolyl-4-acylhydrazone derivative 1, elected as lead-compound due to its important anti-aggregating profile on arachidonic acid induced platelet aggregation (IC(50)=24+/-0.5 micro M), from which emerge this new series 2. These new compounds 2a-p were readily synthesized, characterized and tested on platelet aggregation assays induced by collagen (5 micro g/mL), ADP (5 micro M) and arachidonic acid (100 micro M) in rabbit citrated platelet-rich plasma. Compounds 2b, 2d, and 2h were found to be the most potent, exhibiting a significant antiplatelet activity on arachidonic acid- and collagen-induced platelet aggregation. In addition, these new antiplatelet agents are free of gastric ulcerogenic effect and presented discrete anti-inflammatory and analgesic properties. The N-para-chlorophenyltriazolyl-4-acylhydrazone compound 2h produced the highest inhibitory effect on collagen (IC(50)=21.6+/-0.4 micro M) and arachidonic acid-induced platelet aggregation (IC(50)=2.2+/-0.06 micro M), suggesting that the nature of the substituent on the phenyl ring of the N-heteroaromatic system of NAH moiety may be an important structural requirement for the improvement of antiplatelet activity, in comparison with lead-series 1.     

Chromatin-mediated cortical granule redistribution is responsible for the formation of the cortical granule-free domain in mouse eggs

A cortical granule-free domain (CGFD) overlies the metaphase chromatin in fully mature mouse eggs. Although a chromatin-induced localized release of cortical granules (CG) during maturation is thought to be a major contributing factor to its formation, there are indications that CG redistribution may also be involved in generating the CGFD. We performed experiments to determine the relative contributions of CG exocytosis and redistribution in generating the CGFD. We found that the CGFD-inducing activity was not specific to female germ cell chromatin and was heat stable but sensitive to DNase and protease treatment. Surprisingly, chelation of egg intracellular Ca(2+) levels did not prevent CGFD formation in response to microinjection of exogenous chromatin, suggesting that development of the CGFD was not a result of CG exocytosis. This finding was confirmed by the lack of CG exudate on the plasma membrane surface of the injected eggs and the absence of conversion of ZP2 to ZP2(f) during formation of the new CGFD. Moreover, clamping intracellular Ca(2+) did not prevent the formation of the CGFD during oocyte maturation, but did inhibit the maturation-associated release of CGs between metaphase I and II. Results of these experiments suggest that CG redistribution is the dominant factor in formation of the CGFD.     

A high-throughput alphavirus-based expression cloning system for mammalian cells

We have developed a widely applicable functional genomics strategy based on alphavirus expression vectors. The technology allows for rapid identification of genes encoding a functional activity such as binding of a defined ligand. Complementary DNA (cDNA) libraries were expressed in mammalian cells following infection with recombinant Sindbis virus (SIN replicon particles), a member of the Alphavirus genus. Virus-infected cells that specifically bound a ligand of choice were isolated using fluorescence-activated cell sorting (FACS). Replication-competent, infective SIN replicon particles harboring the corresponding cDNA were amplified in a next step. Within one round of selection, viral clones encoding proteins recognized by monoclonal antibodies or Fc-fusion molecules could be isolated and sequenced. Moreover, using the same viral libraries, a plaque-lift assay was established that allowed the identification of secreted, intracellular, and membrane proteins.     

Egg-to-embryo transition is driven by differential responses to Ca(2+) oscillation number

Ca(2+) oscillations and signaling represent a basic mechanism for controlling many cellular events. Activation of mouse eggs entrains a temporal series of Ca(2+)-dependent events that include cortical granule exocytosis, cell cycle resumption with concomitant decreases in MPF and MAP kinase activities, and recruitment of maternal mRNAs. The outcome is a switch in cellular differentiation, i.e., the conversion of the egg into the zygote. By activating mouse eggs with experimentally controlled and precisely defined Ca(2+) transients, we demonstrate that each of these events is initiated by a different number of Ca(2+) transients, while their completion requires a greater number of Ca(2+) transients than for their initiation. This combination of differential responses to the number of Ca(2+) transients provides strong evidence that a single Ca(2+) transient-driven signaling system can initiate and drive a cell into a new developmental pathway, as well as can account for the temporal sequence of cellular changes associated with early development.     

Involvement of endothelin-1 and its receptors in PGF2alpha-induced luteolysis in the rat

The possible mediatory role of endothelin-1 (ET-1) in prostaglandin F(2alpha) (PGF(2alpha))-induced luteolysis in the rat was examined. The effect of PGF(2alpha) was tested on day 9 of pregnancy either in vivo, by injecting cloprostenol, an analog of PGF(2alpha) or in vitro, in isolated intact corpora lutea incubated with PGF(2alpha). Luteolysis was confirmed by progesterone determination in the peripheral blood serum or in the culture medium, respectively. Administration of cloprostenol (.0025 mg/rat) induced within 1 hr, a significant fall (from 56.8 to 27.6 ng/ml, P < 0.0001) in serum progesterone concentrations that was associated with an increased expression of the mRNA to ET-1 and its protein product in rat luteal tissue. Elevated level of ET-1 were also determined at the spontaneous regression of the CL, upon parturition. Expression of the ET receptors, ETA and ETB was not affected by cloprostenol. On the other hand, this PGF(2alpha) analog induced expression of luteal VEGF mRNA. In vitro experiments demonstrate that the LH (100 ng/ml)-induced increase in luteal progesterone secretion was reduced by PGF(2alpha) (1 microg/ml). The inhibitory effect of PGF(2alpha) was reversed by BQ123 (10(- 7) M), that is a selective ETA receptor antagonist. We conclude that the PGF(2alpha)-induced elevation in luteal expression of ET-1 combined with the reversal of its luteolytic effect by an ETA receptor antagonist suggest that ET-1 may take part in the PGF(2alpha)-induced luteolysis in the rat.     

Genomewide search for type 2 diabetes susceptibility genes in four American populations

Type 2 diabetes is a serious, genetically influenced disease for which no fully effective treatments are available. Identification of biochemical or regulatory pathways involved in the disease syndrome could lead to innovative therapeutic interventions. One way to identify such pathways is the genetic analysis of families with multiple affected members where disease predisposing genes are likely to be segregating. We undertook a genomewide screen (389-395 microsatellite markers) in samples of 835 white, 591 Mexican American, 229 black, and 128 Japanese American individuals collected as part of the American Diabetes Association's GENNID study. Multipoint nonparametric linkage analyses were performed with diabetes, and diabetes or impaired glucose homeostasis (IH). Linkage to diabetes or IH was detected near markers D5S1404 (map position 77 cM, LOD = 2.80), D12S853 (map position 82 cM, LOD = 2.81) and GATA172D05 (X-chromosome map position 130 cM, LOD = 2.99) in whites, near marker D3S2432 (map position 51 cM, LOD = 3.91) in Mexican Americans, and near marker D10S1412 (map position 14 cM, LOD = 2.39) in African Americans mainly collected in phase 1 of the study. Further analyses showed evidence for interactions between the chromosome 5 locus and region on chromosome 12 containing the MODY 3 gene (map position 132 cM) and between the X-chromosome locus and region near D12S853 (map position 82 cM) in whites. Although these results were not replicated in samples collected in phase 2 of the GENNID study, the region on chromosome 12 was replicated in samples from whites described by Bektas et al. (1999).