Proteomic and functional analysis of the mitotic Drosophila centrosome

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.     

Asterless is a centriolar protein required for centrosome function and embryo development in Drosophila

BACKGROUND: Centrosomes, the major organizers of the microtubule network in most animal cells, are composed of centrioles embedded in a web of pericentriolar material (PCM). Recruitment and stabilization of PCM on the centrosome is a centriole-dependent function. Compared to the considerable number of PCM proteins known, the molecular characterization of centrioles is still very limited. Only a few centriolar proteins have been identified so far in Drosophila, most related to centriole duplication. RESULTS: We have cloned asterless (asl) and found that it encodes a 120 kD highly coiled-coil protein that is a constitutive pancentriolar and basal body component. Loss of asl function impedes the stabilization/maintenance of PCM at the centrosome. In embryos deficient for Asl, development is arrested right after fertilization. Asl shares significant homology with Cep 152, a protein described as a component of the human centrosome for which no functional data is yet available. CONCLUSIONS: The cloning of asl offers new insight into the molecular composition of Drosophila centrioles and a possible model for the role of its human homolog. In addition, the phenotype of asl-deficient flies reveals that a functional centrosome is required for Drosophila embryo development.     

Deconstructing common variable immunodeficiency by genetic analysis

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency. Patients have recurrent bacterial infections and an increased risk of developing autoimmune diseases, lung damage, and selected cancers. Since 2003, four genes have been shown to be mutated in CVID patients: ICOS, TNFRSF13B (encoding TACI), TNFRSF13C (encoding BAFF-R) and CD19. Heterozygous mutations in TNFRSF13B are also associated with CVID, whereas the other three genes are purely recessive. Recent genetic linkage studies have also identified possible loci for dominant CVID genes on chromosomes 4q, 5p and 16q. These findings markedly improved the genetic diagnosis of CVID and point towards new strategies for future genetic studies. In addition, some CVID genes might be relevant to more common diseases such as asthma and stroke.     

Extracellular matrix and growth factors in the pathogenesis of some craniofacial malformations

The normal development of cranial primordia and orofacial structures involves fundamental processes in which growth, morphogenesis, and cell differentiation take place and interactions between extracellular matrix (ECM) components, growth factors and embryonic tissues are involved. Biochemical and molecular aspects of craniofacial development, such as the biological regulation of normal or premature cranial suture fusion, has just begun to be understood, thanks mainly to studies performed in the last decade. Several mutations has been identified in both syndromic and non-syndromic craniosynostosis patients throwing new light onto the etiology, classification and developmental pathology of these diseases. In the more common craniosynostosis syndromes and other skeletal growth disorders, the mutations were identified in the genes encoding fibroblast growth factor receptor types 1-3 (FGFR1, 2 and 3) where they are dominantly acting and affect specific and important protein binding domain. The unregulated FGF signaling during intramembranous ossification is associated to the Apert and Crouzon syndrome. The non syndromic cleft of the lip and/or palate (CLP) has a more complex genetic background if compared to craniosynostosis syndrome because of the number of involved genes and type of inheritance. Moreover, the influence of environmental factor makes difficult to clarify the primary causes of this malformation. ECM represents cell environment and results mainly composed by collagens, fibronectin, proteoglycans (PG) and hyaluronate (HA). Cooperative effects of ECM and growth factors regulate regional matrix production during the morphogenetic events, connective tissue remodelling and pathological states. In the present review we summarize the studies we performed in the last years to better clarify the role of ECM and growth factors in the etiology and pathogenesis of craniosynostosis and CLP diseases.     

Isoflavonoid derivatives from Lophira alata stem heartwood

Six isoflavonoid derivatives among which three are new have been isolated from the stem heartwood of Lophira alata. The structures were elucidated from spectroscopic and chemical evidences. Two have unusual carbon skeletons, possibly resulting from a variant of isoflavonoid biogenesis. The two compounds form the first members of a new subclass of flavonoid compounds which we call "isobiflavonoids". The presence of these isoflavonoid compounds in this plant of the Ochnaceae family has important chemotaxonomic implications since it modifies the botanic distribution of isoflavonoid compounds in non-leguminous plants.     

Estradiol protects cultured articular chondrocytes from oxygen-radical-induced damage

Osteoarthritis (OA) is aggravated in menopausal women possibly because of changed serum estrogen levels. Estradiol has been postulated to affect oxidative stress induced by reactive oxygen species (ROS) in articular chondrocytes. We generated ROS in cultured bovine articular chondrocytes by incubating them with combined Fe₂SO₄, vitamin C, and hydrogen peroxide. The release of thiobarbituric-acid-reactive substances (TBARS, lipid peroxidation) and lactate dehydrogenase (LDH, membrane damage) was measured photometrically. Various estradiol doses and vitamin E, serving as control with an established anti-oxidative capacity, were applied either upon each exchange of medium and during radical production (strategy 1) or only during radical production (strategy 2). In chondrocytes incubated according to strategy 1, the production of TBARS and LDH release were significantly suppressed by 10^–10–10^–4 M estradiol or by vitamin E. Under strategy 2, the production of TBARS was significantly suppressed at estradiol concentrations higher than 10^–6 M, whereas LDH release was inhibited at concentrations of 10^–6–10^–4 M. Vitamin E showed no significant effects. As repeated application of estradiol and vitamin E produced the best results, estradiol, like vitamin E, was speculated to accumulate in the plasma membrane and to decrease membrane fluidity resulting in protection against lipid peroxidation (non-genomic effect). Thus, in contrast to the neuroprotective effect of 17-estradiol in supraphysiological doses reported recently, the anti-oxidative potential of estradiol appears to protect articular chondrocytes from ROS-induced damage when the hormone is given repeatedly in a physiological range. Decreased estradiol levels may therefore contribute to menopausal OA in the long term.     

Two cyclopeptides from the seeds of Annona cherimola

Bioassay-guided fractionation of cytotoxic of methanol extract of the seeds of Annona cherimola provided two novel cyclic peptides, cherimolacyclopeptide E (1) and cherimolacyclopeptide F (2), which exhibited significant cytotoxic activity against the KB (human nasopharyngeal carcinoma) cell culture system. The peptide 1 and 2 were elucidated by MS/MS fragmentation experiments using a Q-TOF mass spectrometer equipped with an ESI source, extensive 2D NMR analyses and chemical degradation.