Expression of expansin genes in strawberry varieties with contrasting fruit firmness.

Fruit softening is associated with cell wall disassembly mediated by the action of a complex set of enzymes and proteins. Expansins, a group of proteins with unknown enzymatic activity, are proposed to be involved in this process. In order to study the involvement of expansins in strawberry fruit softening we have analyzed the expression level of five expansin mRNAs (FaEXP1, FaEXP2, FaEXP4, FaEXP5 and FaEXP6) in the cultivars "Selva", "Camarosa" and "Toyonaka", which differ in fruit firmness during ripening. We have found a correlation between mRNA expression levels and fruit firmness for FaEXP1, FaEXP2 and FaEXP5. For these three mRNAs we have observed higher expression levels in the softest cultivar (Toyonaka) than in the other two firmer cultivars (Selva and Camarosa) at the beginning of ripening. This correlation was not found in the case of FaEXP4 and FaEXP6, although both genes displayed a different expression pattern in the three cultivars analyzed. Western-blot analysis revealed that the accumulation of expansin proteins begins earlier in the softest cultivar during ripening.     

Interleukin-15 and interferon-gamma participate in the cross-talk between natural killer and monocytic cells required for tumour necrosis factor production

We have characterized the lymphocyte subset and the receptor molecules involved in inducing the secretion of TNF by monocytic cells in vitro. The TNF secreted by monocytic cells was measured when they were co-cultured with either resting or IL-15-stimulated lymphocytes, T cells, B cells or natural killer (NK) cells isolated from the peripheral blood of healthy subjects and from the synovial fluid from patients with inflammatory arthropathies. Co-culture with IL-15-activated peripheral blood or synovial fluid lymphocytes induced TNF production by monocytic cells within 24 hours, an effect that was mainly mediated by NK cells. In turn, monocytic cells induced CD69 expression and IFN-gamma production in NK cells, an effect that was mediated mainly by beta2 integrins and membrane-bound IL-15. Furthermore, IFN-gamma increased the production of membrane-bound IL-15 in monocytic cells. Blockade of beta2 integrins and membrane-bound IL-15 inhibited TNF production, whereas TNF synthesis increased in the presence of anti-CD48 and anti-CD244 (2B4) monoclonal antibodies. All these findings suggest that the cross-talk between NK cells and monocytes results in the sustained stimulation of TNF production. This phenomenon might be important in the pathogenesis of conditions such as rheumatoid arthritis in which the synthesis of TNF is enhanced.     

Self-assembly of monoglycerides in beta-lactoglobulin adsorbed films at the air-water interface. Structural, topographical, and rheological consequences

In this work, we have analyzed the structural, topographical, and surface dilatational characteristics of pure beta-lactoglobulin adsorbed films and the effect of the self-assembly of monoglycerides (monopalmitin or monoolein) in beta-lactoglobulin films at the air-water interface. Measurements were performed in a single device that incorporates a Wilhelmy-type film balance, Brewster angle microscopy, and interfacial dilatational rheology. The structural and topographical characteristics of beta-lactoglobulin adsorbed and spread films are similar. However, the surface dilatational modulus of beta-lactoglobulin films shows a complex behavior depending on film formation. The self-assembly of monoglyceride in a beta-lactoglobulin adsorbed film has an effect on the structural, topographical, and dilatational properties of the mixed films, depending on the interfacial composition and the surface pressure (pi). At low pi, a mixed film of monoglyceride and beta-lactoglobulin may exist. At high pi (after the collapse of beta-lactoglobulin), the mixed films are dominated by monoglyceride molecules. However, the small amounts of collapsed beta-lactoglobulin have a significant effect on the surface dilatational properties of the mixed films. Protein displacement by monoglyceride is higher for monopalmitin than for monoolein. However, some degree of interaction exists between proteins and monoglycerides, and these interactions are more evident in adsorbed films than in spread films.     

Nerve growth factor increases in pancreatic beta cells after streptozotocin-induced damage in rats

We investigated short-term in vivo and in vitro effects of streptozotocin (STZ) on pancreatic beta cells. Male Wistar rats were treated with 75 mg/kg STZ, and, after 4 hrs blood glucose and insulin were measured and islet cells were isolated, cultured for 16 hrs, and challenged with 5.6 and 15.6 mM glucose. Treated rats showed hyperglycemia (approximately 14 mM) and a 70% decrease in serum insulin levels as compared with controls. Although insulin secretion by isolated beta cells from STZ-treated rats was reduced by more than 80%, in both glucose concentrations, nerve growth factor (NGF) secretion by the same cells increased 10-fold. Moreover, NGF messenger RNA (mRNA) expression increased by 30% as compared with controls. Similar results were obtained in an in vitro model of islet cells, in which cells were exposed directly to STZ for 1, 2, and 4 hrs and then challenged for 3 hrs with the same glucose concentrations. Our data strongly suggest that an early increase in NGF production and secretion by beta cells could be an endogenous protective response to maintain cell survival and that diabetes mellitus may occur when this mechanism is surpassed.     

Further evidence of association between mutations in FGFR2 and syndromic craniosynostosis with sacrococcygeal eversion

BACKGROUND: Pfeiffer syndrome (PS; OMIM #101600) is an autosomal dominant disorder characterized by craniosynostosis, midface hypoplasia, broad thumbs, brachydactyly, broad great toes, and variable syndactyly. CASE: We report a case of PS (type 3) with tracheal and visceral involvement and sacrococcygeal eversion. The patient shows facial dysmorphism with macrocephaly, dolichocephaly, and trigonocephaly, and an asymmetric skull, bilateral and severe exophthalmia with shallow orbits and ocular hypertelorism, downslanting palpebral fissures, constant strabismus, short anterior cranial base, and midface hypoplasia. CONCLUSIONS: Molecular analysis of the FGFR2 gene in this patient revealed a point mutation (c.890G>C NM_000141). This mutation leads to the substitution of the residue tryptophan at position 290 to cysteine in the protein (p.Try290Cys). These data reinforce the hypothesis that the p.Trp290Cys mutation is more often associated with a severe and poor prognosis of PS. Furthermore they suggest that the presence of sacrococcygeal defects is not associated with any specific FGFR2 mutation.     

Immunolocalization of chitin synthases in the phytopathogenic dimorphic fungus Ustilago maydis

Conserved polypeptides of the chitin synthase genes UmCHS3 and UmCHS6 from the phytopathogenic fungus Ustilago maydis were utilized as immunogens to obtain polyclonal antibodies that were purified by affinity procedures. Because of their similarities at the regions encoded by either polypeptide, it was concluded that anti-Chs3 antibodies recognized both Chs3 and Chs4 chitin synthases, whereas anti-Chs6 antibodies recognized Chs6 and Chs8 polypeptides. These antibodies were used to analyze the localization of the corresponding chitin synthases in U. maydis cells, using both indirect immunofluorescence microscopy and immunoelectron microscopy with colloidal-gold-labeled secondary antibodies. It was observed that chitin synthase proteins were accumulated both in the surface and in the cytoplasm of the fungal cells. Electron microscopy images revealed the accumulation of clusters of gold particles in vesicles, providing evidence for the possible origin and destination of chitin synthases in the fungal cells.     

Capillary electromigration methods for the study of collagen

This review paper gives an overview of capillary electromigration methods used in the analysis of collagen. Analyses of the parent chains as well as of the bromcyane and collagenase fragments of collagens are presented. Methods include capillary zone electrophoresis, capillary gel electrophoresis, micellar electrokinetic chromatography as well as combinations of HPLC and capillary electrophoresis, and capillary electrophoresis with mass spectrometry.     

Compensatory effect of the minor Streptomyces lividans type I signal peptidases on the SipY major signal peptidase deficiency as determined by extracellular proteome analysis

The developmentally complex bacterium Streptomyces lividans has the ability to produce and secrete a significant amount of protein and possesses four different type I signal peptidase genes (sipW, sipX, sipY and sipZ) that are unusually clustered in its chromosome. 2-DE and subsequent MS of extracellular proteins showed that proteins with typical export signals for type I and type II signal peptidases are the main components of the S. lividans secretome. Secretion of extracellular proteins is severely reduced in a strain deficient in the major type I signal peptidase (SipY). This deficiency was efficiently compensated by complementation with any of the other three signal peptidases as deduced from a comparison of the corresponding 2-D PAGE patterns with that of the wild-type strain.