Spatiotemporal organization and mechanosensory function of podosomes

Podosomes are small, circular adhesions formed by cells such as osteoclasts, macrophages, dendritic cells, and endothelial cells. They comprise a protrusive actin core module and an adhesive ring module composed of integrins and cytoskeletal adaptor proteins such as vinculin and talin. Furthermore, podosomes are associated with an actin network and often organize into large clusters. Recent results from our laboratory and others have shed new light on podosome structure and dynamics, suggesting a revision of the classical “core-ring” model. Also, these studies demonstrate that the adhesive and protrusive module are functionally linked by the actin network likely facilitating mechanotransduction as well as providing feedback between these two modules. In this commentary, we briefly summarize these recent advances with respect to the knowledge on podosome structure and discuss force distribution mechanisms within podosomes and their emerging role in mechanotransduction.     

Isolated Bovine Spinal Motoneurons Have Specific Ganglioside Antigens Recognized by Sera from Patients with Motor Neuron Disease and Motor Neuropathy

The gangliosides GM1 and GD1b have recently been reported to be potential target antigens in human motor neuron disease (MND) or motor neuropathy. The mechanism for selective motoneuron and motor nerve impairment by the antibodies directed against these gangliosides, however, is not fully understood. We recently investigated the ganglioside composition of isolated bovine spinal motoneurons and found that the ganglioside pattern of the isolated motoneurons was extremely complex. GM1, GD1a, GD1b, and GT1b, which are major ganglioside components of CNS tissues, were only minor species in motoneurons. Among the various ganglioside species in motoneurons, several were immunoreactive to sera from patients with MND and motor neuropathy. One of these gangliosides was purified from bovine spinal cord and characterized as N-glycolylneuraminic acid-containing GM1 [GM1(NeuGc)] by compositional analysis, fast atom bombardment mass spectra, and the use of specific antibodies. Among seven sera with anti-GM1 antibody activities, five sera reacted with GM1(NeuGc) and two did not. Two other gangliosides, which were recognized by another patient's serum, appeared to be specific for motoneurons. We conclude that motoneurons contained, in addition to the known ganglioside antigens GM1 and GD1b, other specific ganglioside antigens that could be recognized by sera from patients with MND and motor neuropathy.     

Plasma Membrane-Associated Glycohydrolases Along Differentiation of Murine Neural Stem Cells

The activities of plasma membrane associated sialidase Neu3, total β-glucosidase, CBE-sensitive β-glucosidase, non-lysosomal β-glucosyl ceramidase GBA2, β-galactosidase, β-hexosaminidase and sphingomyelinase were determined at three different stages of differentiation of murine neural stem cell cultures, corresponding to precursors, commited progenitors, and differentiated cells. Cell immunostaining for specific markers of the differentiation process, performed after 7 days in culture in presence of differentiating agents, clearly showed the presence of oligodendrocytes, astrocytes and neurons. Glial cells were the most abundant. Sialidase Neu3 after a decrease from progenitors to precursors, showed an increase parallel to the differentiation process. All the other glycosidases increased their activity along differentiation. The activity of CBE-sensitive β-glucosidase and GBA2 were very similar at the precursor stage, but CBE-sensitive β-glucosidase increased 7 times while GBA2 only two in the differentiated cells. In addition, we analysed also sphingomyelinase as enzyme specifically associated to sphingolipids. The activity of this enzyme increased from precursors to differentiated cells.     

Enzymes from Sulfolobus shibatae for the production of trehalose and glucose from starch

Enzymes that convert starch and dextrins to α,α-trehalose and glucose were found in cell homogenates of the hyperthermophilic acidophilic archaeon Sulfolobus shibatae DMS 5389. Three enzymes were purified and characterized. The first, the S. shibatae trehalosyl dextrin-forming enzyme (SsTDFE), transformed starch and dextrins to the corresponding trehalosyl derivatives with an intramolecular transglycosylation process that converted the glucosidic linkage at the reducing end from α-1,4 to α-1,1. The second, the S. shibatae trehalose-forming enzyme (SsTFE), hydrolyzed the α-1,4 linkage adjacent to the α-1,1 bond of trehalosyl dextrins, forming trehalose and lower molecular weight dextrins. These two enzymes had molecular masses of 80 kDa and 65 kDa, respectively, and showed the highest activities at pH 4.5. The apparent optimal temperature for activity was 70°C for SsTDFE and 85°C for SsTFE. The third enzyme identified was an α-glycosidase (SsαGly), which catalyzed the hydrolysis of the α-1,4 glucosidic linkages in starch and dextrins, releasing glucose in a stepwise manner from the nonreducing end of the polysaccharide chain. The enzyme had a molecular mass of 313 kDa and showed the highest activity at pH 5.5 and at 85°C. Received: October 29, 1997 / Accepted: April 29, 1998     

Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H(2)O(2) induced oxidative damage

Hydroxytyrosol (2-(3′,4′-dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuronide metabolites of HT, 3′-O-β-d-glucuronide and 4′-O-β-d-glucuronide derivatives and 2-(3′,4′-dihydroxyphenyl)ethanol-1-O-β-d-glucuronide, in comparison with the parent compound, to inhibit H₂O₂ induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H₂O₂ treatment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellular-signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the highest concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited.     

Curcumin and cyclopalladated complexes: a recipe for bifunctional biomaterials

The first examples of binuclear and mononuclear ortho-palladated complexes based on a functionalized 2-phenylquinoline ligand have been synthesized and fully characterized. Conjugating cyclopalladated fragments to curcumin family biologically active beta-diketones gives in one single molecule two different functionalities. The structural variations based on the curcuminoid structure have been tested for their in vitro cytotoxic activity. The activity of complexes comprised of a cyclopalladated fragment conjugated to functionalized bioactive ligands, represents the potential of organometallic systems in generating new bifunctional biomaterials.     

Low-level lead exposure increases systolic arterial pressure and endothelium-derived vasodilator factors in rat aortas

Chronic lead exposure induces hypertension and alters endothelial function. However, treatment with low lead concentrations was not yet explored. We analyzed the effects of 7 day exposure to low lead concentrations on endothelium-dependent responses. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent dose 0.05 μg/100 g, i.m. to cover daily loss) or vehicle; blood levels attained at the end of treatment were 9.98 μg/dL. Lead treatment had the following effects: increase in systolic blood pressure (SBP); reduction of contractile response to phenylephrine (1 nM-100 μM) of aortic rings; unaffected relaxation induced by acetylcholine (0.1 nM-300 μM) or sodium nitroprusside (0.01 nM-0.3 μM). Endothelium removal, N(G)-nitro-L-arginine methyl ester (100 μM) and tetraethylammonium (2 mM) increased the response to phenylephrine in treated rats more than in untreated rats. Aminoguanidine (50 μM) increased but losartan (10 μM) and enalapril (10 μM) reduced the response to phenylephrine in treated rats. Lead treatment also increased aortic Na(+)/K(+)-ATPase functional activity, plasma angiotensin-converting enzyme (ACE) activity, protein expression of the Na(+)/K(+)-ATPase alpha-1 subunit, phosphorylated endothelial nitric oxide synthase (p-eNOS), and inducible nitric oxide synthase (iNOS). Our results suggest that on initial stages of lead exposure, increased SBP is caused by the increase in plasma ACE activity. This effect is accompanied by increased p-eNOS, iNOS protein expression and Na(+)/K(+)-ATPase functional activity. These factors might be a compensatory mechanism to the increase in SBP.     

Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD.