The involvement of CD44 and its novel ligand galectin-8 in apoptotic regulation of autoimmune inflammation

The synovial fluid (SF) cells of rheumatoid arthritis (RA) patients express a specific CD44 variant designated CD44vRA. Using a cellular model of this autoimmune disease, we show in this study that the mammalian lectin, galectin-8 (gal-8), is a novel high-affinity ligand of CD44vRA. By affinity chromatography, flow cytometry, and surface plasmon resonance, we demonstrate that gal-8 interacts with a high affinity (K(d), 6 x 10(-9) M) with CD44vRA. We further demonstrate that SF cells from RA patients express and secrete gal-8, to a concentration of 25-65 nM, well within the concentration of gal-8 required to induce apoptosis of SF cells. We further show that not all gal-8 remains freely soluble in the SF and at least part forms triple complexes with CD44 and fibrinogen that can be detected, after fibrinogen immunoprecipitation, with Abs against fibrinogen, gal-8 and CD44. These triple complexes may therefore increase the inflammatory reaction by sequestering the soluble gal-8, thereby reducing its ability to induce apoptosis in the inflammatory cells. Our findings not only shed light on the receptor-ligand relationships between CD44 and gal-8, but also underline the biological significance of these interactions, which may affect the extent of the autoimmune inflammatory response in the SF of RA patients.     

Synthesis and structure-activity relationship of N-acyl-Gly-, N-acyl-Sar- and N-blocked-boroPro inhibitors of FAP, DPP4, and POP

The structure-activity relationship of various N-acyl-Gly-, N-acyl-Sar-, and N-blocked-boroPro derivatives against three prolyl peptidases was explored. Several N-acyl-Gly- and N-blocked-boroPro compounds showed low nanomolar inhibitory activity against fibroblast activation protein (FAP) and prolyl oligopeptidase (POP) and selectivity against dipeptidyl peptidase-4 (DPP4). N-Acyl-Sar-boroPro analogs retained selectivity against DPP4 and potent POP inhibitory activity but displayed decreased FAP inhibitory activity.     

The ingestion of a Nectandra membranacea extract changes the bioavailability of technetium-99m radiobiocomplex in rat organs

The radiobiocomplexes labeled with technetium-99m (Tc-99m) have been widely used in nuclear medicine in single photon emission computed tomography and in basic research. The aim of this study was to assess the influence of a Nectandra membranacea extract on the bioavailability of the sodium pertechnetate (Na(99m)TcO(4)) radiobiocomplex in rat organs. The animals were treated with a N. membranacea extract (30 mg/ ml), for 6 days. Na(99m)TcO(4) was injected, the organs were isolated and weighed, and the radioactivity was determined in each organ (%ATI/organ). The %ATI/organ was divided by the mass of each organ to calculate the %ATI/g. A significant increase of the %ATI/organ of Na(99m)TcO(4) was observed in muscle and thyroid as well as in the %ATI/g in the heart, kidney and thyroid. These findings could result from the interaction between components of the plant extract and the radiobiocomplex which may influence the uptake Na(99m)TcO(4) in rat organs. Therefore, precaution is suggested in the interpretation of nuclear medicine results in patients using this herb.     

Autophagy facilitates glycolysis during Ras-mediated oncogenic transformation

The protumorigenic functions for autophagy are largely attributed to its ability to promote cancer cell survival in response to diverse stresses. Here we demonstrate an unexpected connection between autophagy and glucose metabolism that facilitates adhesion-independent transformation driven by a strong oncogenic insult-mutationally active Ras. In cells ectopically expressing oncogenic H-Ras as well as human cancer cell lines harboring endogenous K-Ras mutations, autophagy is induced following extracellular matrix detachment. Inhibiting autophagy due to the genetic deletion or RNA interference-mediated depletion of multiple autophagy regulators attenuates Ras-mediated adhesion-independent transformation and proliferation as well as reduces glycolytic capacity. Furthermore, in contrast to autophagy-competent cells, both proliferation and transformation in autophagy-deficient cells expressing oncogenic Ras are insensitive to reductions in glucose availability. Overall, increased glycolysis in autophagy-competent cells facilitates Ras-mediated adhesion-independent transformation, suggesting a unique mechanism by which autophagy may promote Ras-driven tumor growth in specific metabolic contexts.     

Advanced procedures for separation and analysis of low molecular weight inhibitor (NCXIF) of the cardiac sodium-calcium exchanger

A low molecular weight inhibitor (NCX(IF)) of the cardiac Na/Ca exchanger, isolated from the calf ventricle tissue, is capable of regulating the muscle strip's contractility and relaxation without involving the beta-activation pathway. The structural analysis of NCX(IF) requires highly purified preparations that fulfill the demanding requirements for mass spectra and NMR analyses. No such preparation is yet available. To this end, new HPLC procedures were developed by a combination of the reverse phase, normal phase, and HILIC (hydrophilic liquid chromatography) techniques. The specific activity of NCX(IF) is 10(5) times higher in the purified preparations (as compared to the crude extract) showing a 2-5% yield of total inhibitory activity and 20-100 microg content of final material. The purification yield reveals that 1 kg ventricle muscle contains 0.1-0.2 mg NCX(IF), meaning that the tissue concentrations of NCX(IF) may reach 10(-7)-10(-6) M. The diode-array scanning of purified preparations of NCX(IF) shows a homogeneous 3D peak with a maximal absorption at 202 nm. These spectral properties may represent a five-membered ring (e.g., proline, histidine) and/or simple chemical groups (like amine, carbonyl, ester, etc.), but not an aromatic ring or complex conjugates (alkyne, alkene, aldehyde, etc.). NCX(IF) does not respond to phenol/sulfur reagent, suggesting that it lacks reducing (aldo) sugar. NCX(IF) shows a faint response to fluorescamine, meaning that it may contain an amino group (or its derivative). It is believed that a combination of presently developed procedures with LC/MS and LC/MS/MS may provide a useful tool for structural analysis of NCX(IF).     

Chemoattractant signals and beta 2 integrin occupancy at apical endothelial contacts combine with shear stress signals to promote transendothelial neutrophil migration

Lymphocyte transendothelial migration (TEM) is promoted by fluid shear signals and apical endothelial chemokines. Studying the role of these signals in neutrophil migration across differently activated HUVEC in a flow chamber apparatus, we gained new insights into how neutrophils integrate multiple endothelial signals to promote TEM. Neutrophils crossed highly activated HUVEC in a beta(2) integrin-dependent manner but independently of shear. In contrast, neutrophil migration across resting or moderately activated endothelium with low-level beta(2) integrin ligand activity was dramatically augmented by endothelial-presented chemoattractants, conditional to application of physiological shear stresses and intact beta(2) integrins. Shear stress signals were found to stimulate extensive neutrophil invaginations into the apical endothelial interface both before and during TEM. A subset of invaginating neutrophils completed transcellular diapedesis through individual endothelial cells within <1 min. Our results suggest that low-level occupancy of beta(2) integrins by adherent neutrophils can mediate TEM only if properly coupled to stimulatory shear stress and chemoattractant signals transduced at the apical neutrophil-endothelial interface.     

The F-BAR domain of SRGP-1 facilitates cell-cell adhesion during C. elegans morphogenesis

Robust cell-cell adhesion is critical for tissue integrity and morphogenesis, yet little is known about the molecular mechanisms controlling cell-cell junction architecture and strength. We discovered that SRGP-1 is a novel component of cell-cell junctions in Caenorhabditis elegans, localizing via its F-BAR (Bin1, Amphiphysin, and RVS167) domain and a flanking 200-amino acid sequence. SRGP-1 activity promotes an increase in membrane dynamics at nascent cell-cell contacts and the rapid formation of new junctions; in addition, srgp-1 loss of function is lethal in embryos with compromised cadherin-catenin complexes. Conversely, excess SRGP-1 activity leads to outward bending and projections of junctions. The C-terminal half of SRGP-1 interacts with the N-terminal F-BAR domain and negatively regulates its activity. Significantly, in vivo structure-function analysis establishes a role for the F-BAR domain in promoting rapid and robust cell adhesion during embryonic closure events, independent of the Rho guanosine triphosphatase-activating protein domain. These studies establish a new role for this conserved protein family in modulating cell-cell adhesion.     

Infantile cerebellar-retinal degeneration associated with a mutation in mitochondrial aconitase, ACO2

Degeneration of the cerebrum, cerebellum, and retina in infancy is part of the clinical spectrum of lysosomal storage disorders, mitochondrial respiratory chain defects, carbohydrate glycosylation defects, and infantile neuroaxonal dystrophy. We studied eight individuals from two unrelated families who presented at 2-6 months of age with truncal hypotonia and athetosis, seizure disorder, and ophthalmologic abnormalities. Their course was characterized by failure to acquire developmental milestones and culminated in profound psychomotor retardation and progressive visual loss, including optic nerve and retinal atrophy. Despite their debilitating state, the disease was compatible with survival of up to 18 years. Laboratory investigations were normal, but the oxidation of glutamate by muscle mitochondria was slightly reduced. Serial brain MRI displayed progressive, prominent cerebellar atrophy accompanied by thinning of the corpus callosum, dysmyelination, and frontal and temporal cortical atrophy. Homozygosity mapping followed by whole-exome sequencing disclosed a Ser112Arg mutation in ACO2, encoding mitochondrial aconitase, a component of the Krebs cycle. Specific aconitase activity in the individuals' lymphoblasts was severely reduced. Under restrictive conditions, the mutant human ACO2 failed to complement a yeast ACO1 deletion strain, whereas the wild-type human ACO2 succeeded, indicating that this mutation is pathogenic. Thus, a defect in mitochondrial aconitase is associated with an infantile neurodegenerative disorder affecting mainly the cerebellum and retina. In the absence of noninvasive biomarkers, determination of the ACO2 sequence or of aconitase activity in lymphoblasts are warranted in similarly affected individuals, based on clinical and neuroradiologic grounds.