Characterization of a novel C-type lectin-like gene, LSECtin: demonstration of carbohydrate binding and expression in sinusoidal endothelial cells of liver and lymph node

A new C-type lectin-like gene encodes 293 amino acids and maps to chromosome 19p13.3 adjacent to the previously described C-type lectin genes, CD23, dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), and DC-SIGN-related protein (DC-SIGNR). The four genes form a tight cluster in an insert size of 105 kb and have analogous genomic structures. The new C-type lectin-like molecule, designated liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin), is a type II integral membrane protein of approximately 40 kDa in size with a single C-type lectin-like domain at the COOH terminus, closest in homology to DC-SIGNR, DC-SIGN, and CD23. LSECtin mRNA was only expressed in liver and lymph node among 15 human tissues tested, intriguingly neither expressed on hematopoietic cell lines nor on monocyte-derived dendritic cells (DCs). Moreover, LSECtin is expressed predominantly by sinusoidal endothelial cells of human liver and lymph node and co-expressed with DC-SIGNR. LSECtin binds to mannose, GlcNAc, and fucose in a Ca(2+)-dependent manner but not to galactose. Our results indicate that LSECtin is a novel member of a family of proteins comprising CD23, DC-SIGN, and DC-SIGNR and might function in vivo as a lectin receptor.     

Inhibition of histone deacetylases 1 and 6 enhances cytarabine-induced apoptosis in pediatric acute myeloid leukemia cells

Background

Pediatric acute myeloid leukemia (AML) remains a challenging disease to treat even with intensified cytarabine-based chemotherapy. Histone deacetylases (HDACs) have been reported to be promising therapeutic targets for treating AML. However, HDAC family members that are involved in chemotherapy sensitivities remain unknown. In this study, we sought to identify members of the HDAC family that are involved in cytarabine sensitivities, and to select the optimal HDACI that is most efficacious when combined with cytarabine for treating children with AML.

Methodology

Expression profiles of classes I, II, and IV HDACs in 4 pediatric AML cell lines were determined by Western blotting. Inhibition of class I HDACs by different HDACIs was measured post immnunoprecipitation. Individual down-regulation of HDACs in pediatric AML cells was performed with lentiviral shRNA. The effects of cytarabine and HDACIs on apoptosis were determined by flow cytometry analysis.

Results

Treatments with structurally diverse HDACIs and HDAC shRNA knockdown experiments revealed that down-regulation of both HDACs 1 and 6 is critical in enhancing cytarabine-induced apoptosis in pediatric AML, at least partly mediated by Bim. However, down-regulation of HDAC2 may negatively impact cytarabine sensitivities in the disease. At clinically achievable concentrations, HDACIs that simultaneously inhibited both HDACs 1 and 6 showed the best anti-leukemic activities and significantly enhanced cytarabine-induced apoptosis.

Conclusion

Our results further confirm that HDACs are bona fide therapeutic targets for treating pediatric AML and suggest that pan-HDACIs may be more beneficial than isoform-specific drugs.     

Fluorescent metal nanoshell and CK19 detection on single cell image

In this article, we report the synthesis strategy and optical properties of a novel type of fluorescence metal nanoshell when it was used as imaging agent for fluorescence cell imaging. The metal nanoshells were made with 40 nm silica cores and 10 nm silver shells. Unlike typical fluorescence metal nanoshells which contain the organic dyes in the cores, novel metal nanoshells were composed of Cy5-labelled monoclonal anti-CK19 antibodies (mAbs) on the external surfaces of shells. Optical measurements to the single nanoparticles showed that in comparison with the metal free labelled mAbs, the mAb-Ag complexes displayed significantly enhanced emission intensity and dramatically shortened lifetime due to near-field interactions of fluorophores with metal. These metal nanoshells were found to be able to immunoreact with target cytokeratin 19 (CK19) molecules on the surfaces of LNCAP and HeLa cells. Fluorescence cell images were recorded on a time-resolved confocal microscope. The emissions from the metal nanoprobes could be clearly isolated from the cellular autofluorescence backgrounds on the cell images as either individuals or small clusters due to their stronger emission intensities and shorter lifetimes. These emission signals could also be precisely counted on single cell images. The count number may provide an approach for quantifying the target molecules in the cells.     

"Schizophrenic" micellization associated with coil-to-helix transitions based on polypeptide hybrid double hydrophilic rod-coil diblock copolymer

A polypeptide hybrid double hydrophilic diblock copolymer (DHBC), poly( N-isopropylacrylamide)- b-poly( l-glutamic acid) (PNIPAM- b-PLGA), was synthesized via the ring-opening polymerization of gamma-benzyl- l-glutamate N-carboxyanhydride (BLG-NCA) using monoamino-terminated PNIPAM as the macroinitiator, followed by deprotection of benzyl groups under alkaline conditions. Containing a thermoresponsive PNIPAM block and a pH-responsive PLGA block, the obtained polypeptide hybrid diblock copolymer molecularly dissolves in aqueous solution at alkaline pH and room temperature but supramolecularly self-assembles into PNIPAM-core micelles at alkaline pH and elevated temperatures and PLGA-core micelles at acidic pH and room temperature accompanied with coil-to-helix transition of the PLGA sequence. The pH- and thermoresponsive "schizophrenic" micellization behavior of PNIPAM- b-PLGA diblock copolymer has been investigated by (1)H NMR, optical transmittance, fluorescence probe measurement, transmission electron microscopy (TEM), dynamic and static laser light scattering (LLS), and circular dichroism (CD) spectroscopy. Moreover, the micellization process was investigated employing stopped-flow light scattering technique. The pH-induced micelle growth of PNIPAM- b-PLGA in aqueous solution exhibits drastically different kinetics compared to that of conventional pH-responsive DHBCs, probably due to the stabilization effects exerted by the formed alpha-helix secondary structures within the PLGA core at low pH. Exhibiting "schizophrenic" micellization, the polypeptide sequence of PNIPAM- b-PLGA can either locate within micelle cores or stabilizing coronas. The incorporation of polypeptide block into DHBCs can endow them with structural versatility, tunable spatial arrangement of chain segments within self-assembled nanostructures, and broader applications in the field of biomedicines.     

The morphology and haemodynamics of the rabbit renal artery: evaluation by conventional and contrast-enhanced ultrasonography

The purpose of this study was to test the morphology and haemodynamics of the renal artery in the rabbit as evaluated by conventional and contrast-enhanced ultrasonography (CEUS). The morphology and haemodynamics of the rabbit renal artery, including the diameter, which were measured using B-mode ultrasonography (US), colour Doppler US and CEUS, and systolic velocity, diastolic velocity and resistive index (RI) were measured using pulsed wave Doppler US. CEUS was used to measure the renal artery diameter: 0.21 ± 0.04 cm (right) and 0.21 ± 0.03 cm (left). Values of the main renal artery diameter obtained from CEUS significantly correlated with those of digital subtraction angiography. The blood flow velocity of the right main renal artery was 44.20 ± 8.71/18.92 ± 6.26 cm/s (systolic/diastolic) and 36.30 ± 6.89/17.64 ± 5.58 cm/s (systolic/diastolic), at its origin from the aorta and at the renal hilus, respectively. The blood flow velocity of the left main renal artery was 45.10 ± 8.49/19.00 ± 6.80 cm/s (systolic/diastolic) and 41.70 ± 10.25/19.55 ± 7.90 cm/s (systolic/diastolic), at its origin from the aorta and at the renal hilus, respectively. Conventional US provides a more feasible modality for measuring the morphology and haemodynamics of the rabbit renal artery. CEUS is a more accurate method for measuring diameter. This information on the morphology and haemodynamics of the rabbit renal artery might be helpful for researchers.     

Time Course of Current of Injury Is Related to Acute Stability of Active-Fixation Pacing Leads in Rabbits

Background

Magnitude of current of injury (COI) consequent to pacemaker lead fixation is recognized as a predictor of acute lead stability. It is unclear whether dynamic monitoring of COI after lead fixation provides additional information beyond a single assessment performed at the time of fixation.

Objectives

This study was aimed to test the hypothesis that the time course of COI is related to acute lead stability.

Methods and Results

Active fixation leads with fixed screw were anchored to either Langendorff-perfused rabbit hearts endocardially or in vivo hearts epicardially in manners of contact the helix with no rotation, half rotation and full rotation, respectively. Intracardiac electrogram (EGM) was monitored dynamically from onset to resolution of COI, and magnitudes of intrinsic R wave and COI, including ST-segment elevation, ST/R and intracardiac EGM duration (IED), were measured. A digital force gauge was applied to assess lead stability. In vitro, COI in contacted leads was significantly smaller than those in half rotated (p<0.05) and fully rotated leads (p<0.05), and presented most precipitous recovery to baseline (1.5±1.1 min, p<0.05). Half-rotated and fully rotated leads manifested the same magnitude of COI right after placement. However, the time course of COI was significantly longer in fully rotated leads than that in half rotated leads (26.5±2.8 min vs. 5.6±2.0 min, p<0.05). Similar findings were observed in vivo. The time course of COI was significantly correlated with the force needed to detach the lead from myocardium (r = 0. 72, n = 48, p<0.001).

Conclusions

Time course of COI is related to acute lead stability in rabbits. One might be misled by a single assessment of COI magnitude right after lead placement, whereas persistence of COI is likely to be a useful indicator of adequate lead stability.     

Scorpion Venom Heat-Resistant Peptide (SVHRP) Enhances Neurogenesis and Neurite Outgrowth of Immature Neurons in Adult Mice by Up-Regulating Brain-Derived Neurotrophic Factor (BDNF)

Scorpion venom heat-resistant peptide (SVHRP) is a component purified from Buthus martensii Karsch scorpion venom. Although scorpions and their venom have been used in Traditional Chinese Medicine (TCM) to treat chronic neurological disorders, the underlying mechanisms of these treatments remain unknown. We applied SVHRP in vitro and in vivo to understand its effects on the neurogenesis and maturation of adult immature neurons and explore associated molecular mechanisms. SVHRP administration increased the number of 5-bromo-2’-dexoxyuridine (BrdU)-positive cells, BrdU- positive/neuron-specific nuclear protein (NeuN)-positive neurons, and polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive immature neurons in the subventricular zone (SVZ) and subgranular zone (SGZ) of hippocampus. Furthermore immature neurons incubated with SVHRP-pretreated astrocyte-conditioned medium exhibited significantly increased neurite length compared with those incubated with normal astrocyte-conditioned medium. This neurotrophic effect was further confirmed in vivo by detecting an increased average single area and whole area of immature neurons in the SGZ, SVZ and olfactory bulb (OB) in the adult mouse brain. In contrast to normal astrocyte-conditioned medium, higher concentrations of brain-derived neurotrophic factor (BDNF) but not nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) was detected in the conditioned medium of SVHRP-pretreated astrocytes, and blocking BDNF using anti-BDNF antibodies eliminated these SVHRP-dependent neurotrophic effects. In SVHRP treated mouse brain, more glial fibrillary acidic protein (GFAP)-positive cells were detected. Furthermore, immunohistochemistry revealed increased numbers of GFAP/BDNF double-positive cells, which agrees with the observed changes in the culture system. This paper describes novel effects of scorpion venom-originated peptide on the stem cells and suggests the potential therapeutic values of SVHRP.     

Oligomerization state-dependent hyperlipidemic effect of angiopoietin-like protein 4

Angiopoietin-like protein 4 (Angptl4) is the second member of the angiopoietin-like family of proteins previously shown to increase plasma triglyceride (TG) levels in vivo. We recently reported that Angptl4 is a variable-sized oligomer formed by intermolecular disulfide bonds and undergoes regulated proteolytic processing upon secretion. We now show that adenoviral overexpression of Angptl4 potently increases plasma TG levels by a mechanism independent of food intake or hepatic VLDL secretion. We determined that cysteine residues at positions 76 and 80 of Angptl4, conserved among mouse, rat, and human, are required to form higher order structures. By generating adenoviral expression vectors of Angptl4 containing different epitope tags at both N and C termini, we show that loss of oligomerization results in decreased stability of the N-terminal coiled-coil domain of Angptl4 as well as decreased ability to increase plasma TG levels, suggesting that intermolecular disulfide bond formation plays important roles in determining the magnitude of the hyperlipidemic effect of Angptl4. Because Angptl4 is more potent than Angptl3 in increasing plasma TG levels in mice, inappropriate oligomerization of Angptl4 could be associated with disorders of lipid metabolism in vivo.