Wolf–Hirschhorn syndrome-associated chromosome changes are not mediated by olfactory receptor gene clusters nor by inversion polymorphism on 4p16

The basic genomic defect in Wolf–Hirschhorn syndrome (WHS), including isolated 4p deletions and various unbalanced de novo 4p;autosomal translocations and above all t(4p;8p), is heterogeneous. Olfactory receptor gene clusters (ORs) on 4p were demonstrated to mediate a group of WHS-associated t(4p;8p)dn translocations. The breakpoint of a 4-Mb isolated deletion was also recently reported to fall within the most distal OR. However, it is still unknown whether ORs mediate all 4p-autosomal translocations, or whether they are involved in the origin of isolated 4p deletions. Another unanswered question is whether a parental inversion polymorphism on 4p16 can act as predisposing factor in the origin of WHS-associated rearrangements. We investigated the involvement of the ORs in the origin of 73 WHS-associated rearrangements. No hotspots for rearrangements were detected. Breakpoints on 4p occurred within the proximal or the distal olfactory receptor gene cluster in 8 of 73 rearrangements (11%). These were five t(4p;8p) translocations, one t(4p;7p) translocation and two isolated terminal deletions. ORs were not involved in one additional t(4p;8p) translocation, in a total of nine different 4p;autosomal translocations and in the majority of isolated deletions. The presence of a parental inversion polymorphism on 4p was investigated in 30 families in which the 4p rearrangements, all de novo, were tested for parental origin (7 were maternal and 23 paternal). It was detected only in the mothers of 3 t(4p;8p) cases. We conclude that WHS-associated chromosome changes are not usually mediated by low copy repeats. The 4p16.3 inversion polymorphism is not a risk factor for their origin. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Web Resources: Electronic Database Information: Online Mendelian Inheritance in Man (OMIM), (for WHS [MIM 194190]; Ensembl Human Map, ; UCSC, . An erratum to this article can be found at     

Differential expression of cysteine proteases in developmental stages of the parasitic ciliate Ichthyophthirius multifiliis

An expressed sequence tag database of the freshwater fish parasite, Ichthyophthirius multifiliis (Ciliophora) was analyzed to seek for proteases potentially involved in the invasion and degradation of host tissues during infection. The translation of the database revealed two cathepsin L cysteine proteases (Icp1 and Icp2) of the C1A peptidase subfamily. The analysis of Icp1 and Icp2 sequences suggested that both proteases would be synthesized as preproproteins, with a mature domain of 27.9 and 22.8 kDa, respectively. Their expression level was determined in the trophont parasitic stage, in the tomont reproductive stage, and in the theront infective stage by real-time RT-PCR. ICP1 and ICP2 were significantly upregulated in trophont and theront stages in comparison with the tomont stage. Mature peptides of Icp1 and Icp2 were identified in crude extracts of I. multifiliis trophonts by LC-MS/MS. Zymograms showed three to seven activity bands at the optimum pH of cathepsin L cysteine proteases. Two bands displaying cysteine protease activity were identified by inhibition with E-64. They represented the major proteolytic activity of the trophont stage at pH 5-7, suggesting that cysteine proteases play an important role in the infection process.     

Modification of sialidase levels and sialoglycoconjugate pattern during erythroid and erytroleukemic cell differentiation

Glycosphingolipids and glycoproteins play pivotal roles in the complex series of events governing cell adhesion and signal transduction. Aberrant glycosilation, typical of tumor cells, represents a key event in the induction of invasion and metastasis. Sialidases remove sialic acid residues from sialoconjugates and, in mammals, these enzymes have been proved to be involved in several cellular phenomena, including cell proliferation and differentiation, membrane function, and malignant transformation. Herein we show that only the lysosomal sialidase Neu1 and the plasma membrane-associated sialidase Neu3 are expressed in CFU-E erythroid precursors and K562 erythroleukemic cells. Tumour cells show much higher expression levels than CFU-E cells and, during differentiation, the content of the two enzymes progressively decreases. The sialoglycoconjugate pattern is different in the two cell types. In fact, the differentiating erythroid precursors show an increase of the typical erythrocyte sphingolipids, whereas K562 cells treated with butyrate show a marked increase of GD1a, GM2, PE, and ceramide. Finally, during differentiation the sialoglycoprotein content of erythroid cells shows a marked increase, and in K562 cells the process induces the synthesis of some sialoglycoprotein typical of the erythroid membrane. Overall, these results point out the great differences in sialoglycoconjugate and sialidase patterns exhibited by normal and tumour cells. The ganglioside nomenclature proposed by Svennerholm L. (1980) Adv. Exp. Mod. Biol. 125, 11.     

Evidence for outer hair cell driven oscillatory fluid flow in the tunnel of corti

Outer hair cell (OHC) somatic motility plays a key role in mammalian cochlear frequency selectivity and hearing sensitivity, but the mechanism of cochlear amplification is not well understood and remains a matter of controversy. We have visualized and quantified the effects of electrically evoked OHC somatic motility within the gerbil organ of Corti using an excised cochlear preparation. We found that OHC motility induces oscillatory motion of the medial olivocochlear fibers where they cross the tunnel of Corti (ToC) in their course to innervate the OHCs. We show that this motion is present at physiologically relevant frequencies and remains at locations distal to the OHC excitation point. We interpret this fiber motion to be the result of oscillatory fluid flow in the ToC. We show, using a simple one-dimensional hydromechanical model of the ToC, that a fluid wave within the tunnel can travel without significant attenuation for distances larger than the wavelength of the cochlear traveling wave at its peak. This ToC fluid wave could interact with the cochlear traveling wave to amplify the motion of the basilar membrane. The ToC wave could also provide longitudinal coupling between adjacent sections of the basilar membrane, and such coupling may be critical for cochlear amplification.     

The structure of the Shiga toxin 2a A‐subunit dictates the interactions of the toxin with blood components

Hemolytic uremic syndrome (eHUS) is a severe complication of human infections with Shiga toxins (Stxs)‐producing Escherichia coli. A key step in the pathogenesis of eHUS is the interaction of Stxs with blood components before the targeting of renal endothelial cells. Here, we show that a single proteolytic cleavage in the Stx2a A‐subunit, resulting into two fragments (A1 and A2) linked by a disulfide bridge (cleaved Stx2a), dictates different binding abilities. Uncleaved Stx2a was confirmed to bind to human neutrophils and to trigger leukocyte/platelet aggregate formation, whereas cleaved Stx2a was ineffective. Conversely, binding of complement factor H was confirmed for cleaved Stx2a and not for uncleaved Stx2a. It is worth noting that uncleaved and cleaved Stx2a showed no differences in cytotoxicity for Vero cells or Raji cells, structural conformation, and contaminating endotoxin. These results have been obtained by comparing two Stx2a batches, purified in different laboratories by using different protocols, termed Stx2a(cl; cleaved toxin, Innsbruck) and Stx2a(uncl; uncleaved toxin, Bologna). Stx2a(uncl) behaved as Stx2a(cl) after mild trypsin treatment. In this light, previous controversial results obtained with purified Stx2a has to be critically re‐evaluated; furthermore, characterisation of the structure of circulating Stx2a is mandatory to understand eHUS‐pathogenesis and to develop therapeutic approaches.     

MgcRacGAP interacts with cingulin and paracingulin to regulate Rac1 activation and development of the tight junction barrier during epithelial junction assembly

The regulation of Rho-family GTPases is crucial to direct the formation of cell–cell junctions and tissue barriers. Cingulin (CGN) and paracingulin (CGNL1) control RhoA activation in epithelial cells by interacting with RhoA guanidine exchange factors. CGNL1 depletion also inhibits Rac1 activation during junction assembly. Here we show that, unexpectedly, Madin–Darby canine kidney epithelial cells depleted of both CGN and CGNL1 (double-KD cells) display normal Rac1 activation and tight junction (TJ) formation, despite decreased junctional recruitment of the Rac1 activator Tiam1. The expression of the Rac1 inhibitor MgcRacGAP is decreased in double-KD cells, and the barrier development and Rac1 activation phenotypes are rescued by exogenous expression of MgcRacGAP. MgcRacGAP colocalizes with CGN and CGNL1 at TJs and forms a complex and interacts directly in vitro with CGN and CGNL1. Depletion of either CGN or CGNL1 in epithelial cells results in decreased junctional localization of MgcRacGAP but not of ECT2, a centralspindlin-interacting Rho GEF. These results provide new insight into coordination of Rho-family GTPase activities at junctions, since apical accumulation of CGN and CGNL1 at TJs during junction maturation provides a mechanism to spatially restrict down-regulation of Rac1 activation through the recruitment of MgcRacGAP.     

RGDechi-hCit: αvβ3 Selective Pro-Apoptotic Peptide as Potential Carrier for Drug Delivery into Melanoma Metastatic Cells

αvβ3 integrin is an important tumor marker widely expressed on the surface of cancer cells. Recently, we reported some biological features of RGDechi-hCit, an αvβ3 selective peptide antagonist. In the present work, we mainly investigated the pro-apoptotic activity of the molecule and its ability to penetrate the membrane of WM266 cells, human malignant melanoma cells expressing high levels of αvβ3 integrin. For the first time we demonstrated the pro-apoptotic effect and the ability of RGDechi-hCit to enter into cell overexpressing αvβ3 integrin mainly by clathrin- and caveolin-mediated endocytosis. Furthermore, we deepened and confirmed the selectivity, anti-adhesion, and anti-proliferative features of the peptide. Altogether these experiments give insight into the biological behavior of RGDechi-hCit and have important implications for the employment of the peptide as a new selective carrier to deliver drugs into the cell and as a therapeutic and diagnostic tool for metastatic melanoma. Moreover, since the peptide shows a pro-apoptotic effect, a great perspective could be the development of a new class of selective systems containing RGDechi-hCit and pro-apoptotic molecules or other therapeutic agents to attain a synergic action.     

Accelerated evolution and coevolution drove the evolutionary history of AGPase sub-units during angiosperm radiation

Background and Aims

ADP-glucose pyrophosphorylase (AGPase) is a key enzyme of starch biosynthesis. In the green plant lineage, it is composed of two large (LSU) and two small (SSU) sub-units encoded by paralogous genes, as a consequence of several rounds of duplication. First, our aim was to detect specific patterns of molecular evolution following duplication events and the divergence between monocotyledons and dicotyledons. Secondly, we investigated coevolution between amino acids both within and between sub-units.

Methods

A phylogeny of each AGPase sub-unit was built using all gymnosperm and angiosperm sequences available in databases. Accelerated evolution along specific branches was tested using the ratio of the non-synonymous to the synonymous substitution rate. Coevolution between amino acids was investigated taking into account compensatory changes between co-substitutions.

Key Results

We showed that SSU paralogues evolved under high functional constraints during angiosperm radiation, with a significant level of coevolution between amino acids that participate in SSU major functions. In contrast, in the LSU paralogues, we identified residues under positive selection (1) following the first LSU duplication that gave rise to two paralogues mainly expressed in angiosperm source and sink tissues, respectively; and (2) following the emergence of grass-specific paralogues expressed in the endosperm. Finally, we found coevolution between residues that belong to the interaction domains of both sub-units.

Conclusions

Our results support the view that coevolution among amino acid residues, especially those lying in the interaction domain of each sub-unit, played an important role in AGPase evolution. First, within SSU, coevolution allowed compensating mutations in a highly constrained context. Secondly, the LSU paralogues probably acquired tissue-specific expression and regulatory properties via the coevolution between sub-unit interacting domains. Finally, the pattern we observed during LSU evolution is consistent with repeated sub-functionalization under ‘Escape from Adaptive Conflict’, a model rarely illustrated in the literature.     

The ESR2 AluI 1730G>A (rs4986938) gene polymorphism is associated with fibrinogen plasma levels in postmenopausal women

The incidence of cardiovascular disease (CVD) and resultant morbidity and mortality are highly increased in postmenopausal women. Recent observations indicate the involvement of estrogen receptor beta in the pathogenesis of CVD, and the potential role of ESR2 gene polymorphisms as independent risk factors for CVD. We aimed to investigate the possible association between the ESR2 AluI 1730G>A gene polymorphism (rs4986938) with different CVD risk markers, such as body mass index (BMI), blood fibrinogen, glucose and insulin, homeostasis model assessment of insulin resistance and urinary F2-isoprostanes, in 89 postmenopausal women. Genotyping for ESR2 1730G>A polymorphism showed the higher prevalence of heterozygous GA1730 genotype than either wild-type GG1730 or homozygously mutated AA1730 genotype (50.6 vs 34.8 or 14.6%, respectively). Statistical analysis of between-group variability revealed that mean levels of the examined CVD risk markers, except BMI and fibrinogen, were within the normal range in all subjects grouped to different ESR2 1730G>A genotypes. Interestingly, only fibrinogen levels were statistically different in AA1730 carriers compared with other genotypes. The analysis of genotype relative risk showed a significant elevation of plasma fibrinogen in AA1730 carriers compared with GG + GA ones. The present data strongly indicate that genotyping for the ESR2 AluI 1730G>A gene variant should be included in a screening panel for assessment of cardiovascular risk in menopausal women.