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- Topographic Distribution of Cortical Projection Cells in the Rat Subiculum. [JOURNAL ARTICLE]
- Neurosci Res 2014 Dec 13.
The topographic distribution of subicular pyramidal cells, which give rise to projections to the entorhinal cortex, presubiculum, parasubiculum, and the retrosplenial granular cortex, was investigated in the rat using retrograde labeling with wheat germ agglutinin-horseradish peroxidase. Using two-dimensional unfolded maps of the entire hippocampal and parahippocampal fields, we found that the cells originating the projections to the above cortical areas were consistently observed throughout the entire septotemporal extent of the subiculum. In the transverse plane, most of the cortical projection cells were vertically located in the middle region of the subicular pyramidal cell layer. The cells giving rise to the projections to the lateral entorhinal cortex were predominantly located in the most proximal (near CA1), superficial region. Few cortical projection cells were located in the deepest (adjacent to the angular bundle) region. The distribution of cortical projection cells showed an oblique tri-laminar pattern, which was similar to the previously reported laminal pattern of subcortical projection cells in the subiculum. These results suggest that cortical projection cells in middle and superficial regions of the subiculum may correspond to layer V of the isocortex and cells in the deepest region corresponding to layer VI.
- Cortical fast-spiking parvalbumin interneurons enwrapped in the perineuronal net express the metallopeptidases Adamts8, Adamts15 and Neprilysin. [JOURNAL ARTICLE]
- Mol Psychiatry 2014 Dec 16.
The in situ hybridization Allen Mouse Brain Atlas was mined for proteases expressed in the somatosensory cerebral cortex. Among the 480 genes coding for protease/peptidases, only four were found enriched in cortical interneurons: Reln coding for reelin; Adamts8 and Adamts15 belonging to the class of metzincin proteases involved in reshaping the perineuronal net (PNN) and Mme encoding for Neprilysin, the enzyme degrading amyloid β-peptides. The pattern of expression of metalloproteases (MPs) was analyzed by single-cell reverse transcriptase multiplex PCR after patch clamp and was compared with the expression of 10 canonical interneurons markers and 12 additional genes from the Allen Atlas. Clustering of these genes by K-means algorithm displays five distinct clusters. Among these five clusters, two fast-spiking interneuron clusters expressing the calcium-binding protein Pvalb were identified, one co-expressing Pvalb with Sst (PV-Sst) and another co-expressing Pvalb with three metallopeptidases Adamts8, Adamts15 and Mme (PV-MP). By using Wisteria floribunda agglutinin, a specific marker for PNN, PV-MP interneurons were found surrounded by PNN, whereas the ones expressing Sst, PV-Sst, were not.Molecular Psychiatry advance online publication, 16 December 2014; doi:10.1038/mp.2014.162.
- Efficacy and safety of rituximab in auto-immune hemolytic anemia: A meta-analysis of 21 studies. [REVIEW]
- Autoimmun Rev 2014 Dec 9.
This study aims to evaluate the response to rituximab (RTX) treatment in auto-immune hemolytic anemia (AIHA) patients.Studies were selected from MEDLINE up to March 2014. Two investigators independently extracted data on study design, patient characteristics, clinical features (AIHA type, disease duration, previous treatments), dose-schedule of rituximab, duration of treatment follow-up, and toxicities. Pooled overall response rate (ORR) and complete response (CR) rates were evaluated to determine RTX efficacy and toxicity by calculating the weighted mean proportion with fixed or random-effects models in case of heterogeneity (p<0.1 or I(2)>50%).Twenty-one studies encompassing 409 patients were included in the meta-analysis. The characteristics of the entire analyzed cohort reported were as follows: mean male proportion: 43%, mean age: 50years, splenectomized patients range: 0-50%. Warm AIHA, primary AIHA and adults were mostly represented. With the random-effect model, the overall response rate (ORR) was 73% (95% CI 64-81%, 20 studies encompassing 402 patients). CR rate was 37% (95% CI 26-49%, 20 studies including 397 patients). The ORRs were close to 70% for warm AIHA (79%, 95% CI 60-90%, 11 studies, 154 patients), primary AIHA (67%, 95% CI 49-81%, 10 studies, 161 patients), and secondary AIHA (72%, 95% CI 60-82%, 8 studies, 66 patients). The ORR was 57% (95% CI 47-66%, 6 studies, 109 patients) for cold agglutinin disease (CAD). The CR rate was 42% (95% CI 27-58%, 11 studies, 154 patients) for warm AHAI, 32% (95% CI 17-51%, 11 studies, 176 patients) for primary AIHA, 46% (95% CI 30-62%, 9 studies, 87 patients) for secondary AIHA and only 21% (95% CI 6-51%, 7 studies, 118 patients) for CAD. Definitive response rates were evaluated during follow-up. CR rate was the highest within 2 to 4months after RTX (13 studies, 203 patients, CR=70% [57-80%]). As for toxicities, 38 adverse events in 364 patients were noted (42% (95% CI 35-48%)). Sixteen events were infusion-linked side effects, mostly chills and fever, whereas twenty-two were severe. Only one opportunistic Pneumocystis jiroveci pneumonia was reported. Seventeen patients out of 364 (4.6%) died during follow-up. In univariate mixed-effect meta-regressions, ORR and CR were significantly associated with warm AIHA (p=0.002) and mean age (p<0.001), and marginally associated with disease type (p=0.06 and 0.005, respectively).Rituximab seems to be a safe and effective therapy for AIHA in this meta-analysis of observational studies. The authors suggest that it could be used at an earlier point in therapy, before more toxic immunosuppressive drugs, or in place of splenectomy in some cases.
- How does fluorescent labeling affect the binding kinetics of proteins with intact cells? [JOURNAL ARTICLE]
- Biosens Bioelectron 2014 Nov 22.:412-416.
Fluorescent labeling is a mainstream technology for detecting molecular binding. Despite the importance, few studies have been devoted to quantitatively examine the effect of labeling on the molecular binding processes. Here we present a quantitative study on the binding kinetics of fluorescent-labeled and un-labeled molecules (lectin proteins) with glycoproteins on the membrane of cells using surface plasmon resonance imaging (SPRi) technique. The study shows that fluorescent labeling has a significant influence on the binding behaviors of proteins, especially the association processes, and the influence depends sensitively on the charge of fluorescent labels. It further shows that the labels also affect the local distribution of probe proteins, due to the inhomogeneous surface charge distribution of the cell membrane. Our work indicates that fluorescent labeling in general affects the binding behaviors, but proper design of the label will help to minimize its effect.
- Regulation of vesicle transport and cell motility by Golgi-localized Dbs. [Journal Article]
- Small GTPases 2014 Oct 2; 5(4):1-12.
DBS/MCF2L has been recently identified as a risk locus for osteoarthritis. It encodes a guanine nucleotide exchange factor (Dbs) that has been shown to regulate both normal and tumor cell motility. In the current study, we have determined that endogenous Dbs is predominantly expressed as 2 isoforms, a 130 kDa form (Dbs-130) that is localized to the Golgi complex, and an 80 kDa form (Dbs-80) that is localized to the endoplasmic reticulum (ER). We have previously described an inhibitor that binds to the RhoGEF domain of Dbs and blocks its transforming activity. Here we show that the inhibitor localizes to the Golgi, where it specifically interacts with Dbs-130. Inhibition of endogenous Dbs-130 activity is associated with reduced levels of activated Cdc42, enlarged Golgi, and resistance to Brefeldin A-mediated Golgi dispersal, suggesting a role for Dbs in vesicle transport. Cells treated with the inhibitor exhibit normal protein transport from the ER to the Golgi, but are defective in transport from the Golgi to the plasma membrane. Inhibition of Dbs-130 in MDA-MB-231 human breast tumor cells limits motility in both transwell and wound healing assays, but appears to have no effect on the organization of the microtubule cytoskeleton. The reduced motility is associated with a failure to reorient the Golgi toward the leading edge. This is consistent with the Golgi localization, and suggests that the Dbs-130 regulates aspects of the secretory pathway that are required to support cell polarization during directed migration.
- N-Glycans in Xenopus laevis testis characterised by lectin histochemistry. [JOURNAL ARTICLE]
- Reprod Fertil Dev 2014 Jul 22.
Analysis of glycan chains of glycoconjugates is difficult because of their considerable variety. Despite this, several functional roles for these glycans have been reported. N-Glycans are oligosaccharides linked to asparagine residues of proteins. They are synthesised in the endoplasmic reticulum (ER) in a unique way, and later modified in both the ER and Golgi apparatus, developing different oligosaccharide chains. An essential role for complex N-glycans in mammalian spermatogenesis has been reported. The aim of the present study was to analyse the N-glycans of the Xenopus laevis testis by means of lectin histochemistry. Five lectins were used that specifically recognise mannose-containing and complex glycans, namely Galanthus nivalis agglutinin (GNA) from snowdrops, concanavalin A (Con A) from the Jack bean, Lens culinaris agglutinin (LCA) from lentils and Phaseolus vulgaris erythroagglutinin (PHA-E) and P. vulgaris leukoagglutinin (PHA-L) from the common bean. GNA and Con A labelled the interstitium and most of the germ cell types, whereas LCA and PHA-E showed affinity only for the interstitium. A granular cytoplasmic region was labelled in spermatogonia and spermatocytes by GNA and PHA-L, whereas GNA and LCA labelled a spermatid region that is probably associated with the centriolar basal body of the nascent flagellum. There was no specific labelling in the acrosome. Some unexpected results were found when deglycosylative pretreatments were used: pre-incubation of tissue sections with peptide N glycosidase F, which removes N-linked glycans, reduced or removed labelling with most lectins, as expected. However, after this pretreatment, the intensity of labelling remained or increased for Con A in the follicle (Sertoli) and post-meiotic germ cells. The ?-elimination procedure, which removes O-linked glycans, revealed new labelling patterns with GNA, LCA and PHA-L, suggesting that some N-glycans were masked by O-glycans, and thus they became accessible to these lectins only after removal of the O-linked oligosaccharides. The functional role of the glycan chains identified could be related to the role of N-glycans involved in mammalian spermatogenesis reported previously.
- Exposure of Paracentrotus lividus male gametes to engineered nanoparticles affects skeletal bio-mineralization processes and larval plasticity. [JOURNAL ARTICLE]
- Aquat Toxicol 2014 Nov 25.:181-191.
The aim of this study is to contribute to the understanding of the mechanisms underlying nanoparticle (NP)-induced embryotoxicity in aquatic organisms. We previously demonstrated that exposure of male gametes to NPs causes non-dose-dependent skeletal damage in sea urchin (Paracentrotus lividus) larvae. In the present study, the molecular mechanisms responsible for these anomalies in sea urchin development from male gametes exposed to cobalt (Co), titanium dioxide (TiO2) and silver (Ag) NPs were investigated by histochemical, immunohistochemical and Western blot analyses. P. lividus sperm were exposed to different NP concentrations (from 0.0001 to 1mg/L). The distribution of molecules related to skeletogenic cell identification, including ID5 immunoreactivity (IR), wheat germ agglutinin (WGA) affinity and fibronectin (FN) IR, were investigated by confocal laser scanning microscopy at the gastrula (24h) and pluteus (72h) stages. Our results identified a spatial correspondence among PMCs, ID5 IR and WGA affinity sites. The altered FN pattern suggests that it is responsible for the altered skeletogenic cell migration, while the Golgi apparatus of the skeletogenic cells, denoted by their WGA affinity, shows different aspects according to the degree of anomalies caused by NP concentrations. The ID5 IR, a specific marker of skeletogenic cells in sea urchin embryos (in particular of the msp130 protein responsible for Ca(2+) and Mg(2+) mineralization), localized in the cellular strands prefiguring the skeletal rods in the gastrula stage and, in the pluteus stage, was visible according to the degree of mineralization of the skeleton. In conclusion, the present study suggests that the investigated NPs suspended in seawater interfere with the bio-mineralization processes in marine organisms, and the results of this study offer a new series of specific endpoints for the mechanistic understanding of NP toxicity.
- Histological and lectin histochemical studies on the olfactory mucosae of the Korean roe deer, Capreolus pygargus. [JOURNAL ARTICLE]
- Tissue Cell 2014 Nov 18.
The morphological features of the olfactory mucosae of Korean roe deer, Capreolus pygargus, were histologically studied using the ethmoid turbinates containing the olfactory mucosae from six roe deer (male, 2-3 years old). The ethmoid turbinates were embedded in paraffin, and histochemically evaluated in terms of the mucosal characteristics. Lectin histochemistry was performed to investigate the carbohydrate-binding specificity on the olfactory mucosa. Lectins, including Triticum vulgaris wheat germ agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), and soybean agglutinin (SBA) were used for the N-acetylglucosamine, fucose and N-acetylgalactosamine carbohydrate groups, respectively. Histologically, the olfactory mucosa, positioned mainly in the caudal roof of the nasal cavity, consisted of the olfactory epithelium and the lamina propria. The olfactory epithelium consisted of protein gene product (PGP) 9.5-positive olfactory receptor cells, galectin-3-positive supporting cells and basal cells. Bowman's glands in the lamina propria were stained by both the periodic acid Schiff reagent and alcian blue (pH 2.5). Two types of lectin, WGA and SBA, were labeled in free border, receptor cells, supporting cells and Bowman's glands, with the exception of basal cells, while UEA-I was labeled in free border, supporting cells and Bowman's glands, but not in receptor cells and basal cells, suggesting that carbohydrate terminals on the olfactory mucosae of roe deer vary depending on cell type. This is the first morphological study of the olfactory mucosa of the Korean roe deer to evaluate carbohydrate terminals in the olfactory mucosae.
- Inhibition of autophagy recovers cardiac dysfunction and atrophy in response to tail-suspension. [JOURNAL ARTICLE]
- Life Sci 2014 Dec 2.
Physical inactivity during space flight or prolonged bed rest may cause cardiac dysfunction and atrophy, but the exact mechanism that governs the regulation of myocardial dysfunction and cardiac atrophy remains poorly understood. Autophagy, a protein degradation pathway, has recently been shown to be involved in the regulation of cardiac dysfunction and atrophy. In this study, we investigated the relationships between dysfunction and inactivity-induced atrophy and autophagy in rat cardiac tissue.Physical inactivity was simulated by a tail suspension model, and cardiac function was examined by echocardiography. Cardiac atrophy was measured by wheat germ agglutinin staining and autophagic activity was detected by Western blot analysis and immunofluorescence staining.We demonstrated that cardiac function, especially contractility, declined and the area of cardiac atrophy increased in the tail-suspended cardiac tissue. Additionally, the cross-sectional area of myocardial cells decreased; however, apoptosis did not increase with tail suspension. Similarly, the expression of autophagy-related proteins and the number of autophagosomes were elevated in the tail-suspended cardiac tissue. Moreover, the administration of chloroquine, an autophagy inhibitor, reversed cardiac dysfunction and atrophy via the suppression of autophagic activity during suspension. Our results indicate that autophagy facilitates the development and progression of cardiac dysfunction and atrophy induced by tail suspension.Our studies hint that the components of the autophagy-related signaling pathway are potential therapeutic targets for the treatment of cardiac diseases induced by physical activity.
- Characterization of a Ricin-resistant Mutant of Leishmania donovani that Expresses Lipophosphoglycan. [JOURNAL ARTICLE]
- Glycobiology 2014 Dec 3.
The abundant cell-surface lipophosphoglycan (LPG) of Leishmania parasites plays a central role throughout the eukaryote's life cycle. A number of LPG-defective mutants and their complementing genes have been isolated and have proven invaluable in assessing the importance of LPG and related glycoconjugates in parasite virulence. While ricin agglutination selection protocols frequently result in lpg- mutants, one L. donovani variant we isolated, named JABBA, was found to be lpg+. Procyclic (logarithmic) JABBA expresses significant amounts of a large-sized LPG, larger than observed from procyclic wild-type but similar in size to LPG from wild-type from metacyclic (stationary) phase. Structural analysis of the LPG from logarithmically-grown JABBA by capillary electrophoresis protocols revealed that it averaged 30 repeat units composed of the unsubstituted Gal(β1,4)Man(α1)-PO4 typical of wild-type L. donovani. Analysis of JABBA LPG caps indicated that 20% are branched trisaccharide Gal(β1,4)[Glc(β1,2)]Man and tetrasaccharide Gal(β1,4)[Glc(β1,2)Man(α1,2)]Man instead of the usual Gal(β1,4) Man and Man (α1,2) Man terminating caps. Consistent with these structural observations, analyses of the relevant glycosyltransferases in JABBA microsomes involved in LPG biosynthesis showed a two-fold increase in elongating mannosylphosphoryltransferase activity and up-regulation of a β-glucosyltransferase activity. Furthermore, the caps of JABBA LPG are cryptic in presentation as shown by the loss of binding by the lectins, ricin, peanut agglutinin and concanavalin A and reduced accessibility of the terminal galactose residues to oxidation by galactose oxidase. These results indicate that LPG from JABBA is intriguingly similar to the larger LPG in wild-type parasites that arises following the differentiation of the non-infectious procyclic promastigotes to infectious, metacyclic forms.