RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain

Amyloid-beta peptide (Abeta) interacts with the vasculature to influence Abeta levels in the brain and cerebral blood flow, providing a means of amplifying the Abeta-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Abeta infusion and studies in genetically manipulated mice show that Abeta interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Abeta across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Abeta-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Abeta in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Abeta-vascular interactions, including development of cerebral amyloidosis.     

Trends in the Use of Nephron-Sparing Surgery over 7 Years: An Analysis Using the R.E.N.A.L. Nephrometry Scoring System

Objective

To analyze trends in the use of partial nephrectomy, we evaluated which individual factors of renal nephrometry score (RNS) influenced the operative approach bi-annually from 2008 to 2014.

Materials and Methods

We performed a retrospective review of renal cell carcinoma treated by surgery in 2008, 2010, 2012, and 2014. The complexity of renal masses was measured using the R.E.N.A.L. nephrometry scoring system with CT or MRI. Group comparison in terms of operation year and surgical type (partial nephrectomy versus radical nephrectomy) was performed. We developed a nomogram to quantitate the likelihood of selecting partial nephrectomy over radical nephrectomy.

Results

A total of 1106 cases (237 in 2008, 225 in 2010, 292 in 2012, and 352 in 2014) were available for the study. Over the study period, the proportion of partial nephrectomies performed increased steadily from 21.5% in 2008 to 66.5% in 2014 (p < 0.05). Furthermore, use of partial nephrectomy increased steadily in all RNS complexity groups (low, moderate, and high) (p < 0.05). In the analysis of individual components of RNS, values of the R and N components increased statistically by year in the partial nephrectomy group (p < 0.05). Average AUC was 0.920.

Conclusions

The proportion of partial nephrectomies performed sharply increased over the study period. Additionally, over the study period, more partial nephrectomies were performed for renal masses of larger size and closer to the collecting system and main renal vessels. A nomogram developed based on this recent data set provides significant predictive value for surgical decision making.     

The neuronal EGF-related gene Nell2 interacts with Macf1 and supports survival of retinal ganglion cells after optic nerve injury

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01).     

Affinity chromatography of the D1 dopamine receptor from rat corpus striatum

The D1 dopamine receptor from rat corpus striatum has been purified 200-250-fold by using a newly developed biospecific affinity chromatography matrix based on a derivative of the D1 selective antagonist SCH 23390. This compound, (RS)-5-(4-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-1H-3-benz azepin-7-o l (SCH 39111), possesses high affinity for the D1 receptor and, when immobilized on Sepharose 6B through an extended spacer arm, was able to adsorb digitonin-solubilized D1 receptors. The interaction between the solubilized receptor and the affinity matrix was biospecific. Adsorption of receptor activity could be blocked in a stereoselective fashion [SCH 23390 greater than SCH 23388; (+)-butaclamol greater than (-)-butaclamol]. The elution of [3H]SCH 23390 activity from the gel demonstrated similar stereoselectivity for antagonist ligands. Agonists eluted receptor activity with a rank order of potency consistent with that of a D1 receptor [apomorphine greater than dopamine greater than (-)-epinephrine much greater than LY 171555 greater than serotonin]. SCH 39111-Sepharose absorbed 75-85% of the soluble receptor activity, and after the gel was washed extensively, 35-55% of the absorbed receptor activity could be eluted with 100 microM (+)-butaclamol with specific activities ranging from 250 to 450 pmol/mg of protein. The affinity-purified receptor retains the ligand binding characteristics of a D1 dopamine receptor. This affinity chromatography procedure should prove valuable in the isolation and molecular characterization of the D1 dopamine receptor.     

Chemical cross-linking and mass spectrometric identification of sites of interaction for UreD, UreF, and urease

Synthesis of active Klebsiella aerogenes urease requires four accessory proteins to generate, in a GTP-dependent process, a dinuclear nickel active site with the metal ions bridged by a carbamylated lysine residue. The UreD and UreF accessory proteins form stable complexes with urease apoprotein, comprised of UreA, UreB, and UreC. The sites of protein-protein interactions were explored by using homobifunctional amino group-specific chemical cross-linkers with reactive residues being identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) of tryptic peptides. On the basis of studies of the UreABCD complex, UreD is capable of cross-linking with UreB Lys(9), UreB Lys(76), and UreC Lys(401). Furthermore UreD appears to be positioned over UreC Lys(515) according to decreased reactivity of this residue compared with its reactivity in UreD-free apoprotein. Several UreB-UreC and UreC-UreC cross-links also were observed within this complex; e.g. UreB Lys(76) with the UreC amino terminus, UreB Lys(9) with UreC Lys(20), and UreC Lys(515) with UreC Lys(89). These interactions are consistent with the proximate surface locations of these residues observed in the UreABC crystal structure. MALDI-TOF MS analyses of UreABCDF are consistent with a cross-link between the UreF amino terminus and UreB Lys(76). On the basis of an unexpected cross-link between UreB Lys(76) and UreC Lys(382) (distant from each other in the UreABC structure) along with increased side chain reactivities for UreC Lys(515) and Lys(522), UreF is proposed to induce a conformational change within urease that repositions UreB and potentially could increase the accessibility of nickel ions and CO(2) to residues that form the active site.     

High yield expression in a recombinant <Emphasis Type="Italic">E. coli</Emphasis> of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

Background

Chicken anemia virus (CAV), the causative agent chicken anemia, is the only member of the genus Gyrovirus of the Circoviridae family. CAV is an immune suppressive virus and causes anemia, lymph organ atrophy and immunodeficiency. The production and biochemical characterization of VP1 protein and its use in a subunit vaccine or as part of a diagnostic kit would be useful to CAV infection prevention.

Results

Significantly increased expression of the recombinant full-length VP1 capsid protein from chicken anemia virus was demonstrated using an E. coli expression system. The VP1 gene was cloned into various different expression vectors and then these were expressed in a number of different E. coli strains. The expression of CAV VP1 in E. coli was significantly increased when VP1 was fused with GST protein rather than a His-tag. By optimizing the various rare amino acid codons within the N-terminus of the VP1 protein, the expression level of the VP1 protein in E. coli BL21(DE3)-pLysS was further increased significantly. The highest protein expression level obtained was 17.5 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 2 h. After purification by GST affinity chromatography, the purified full-length VP1 protein produced in this way was demonstrated to have good antigenicity and was able to be recognized by CAV-positive chicken serum in an ELISA assay.

Conclusions

Purified recombinant VP1 protein with the gene's codons optimized in the N-terminal region has potential as chimeric protein that, when expressed in E. coli, may be useful in the future for the development of subunit vaccines and diagnostic tests.