Purification and partial characterization of ceruloplasmin receptors from rat liver endothelium

Ceruloplasmin (CP), a circulating glycoprotein, is known for its copper transport. Recently the spectrum of its activity has been increased to include numerous enzymatic functions. CP binds to the liver endothelium and is transported across the cell via a mechanism involving receptor-mediated endocytosis. To isolate CP receptors, we obtained purified preparations of liver endothelium in rats. The membrane was then isolated by ultracentrifugation and solubilized in Triton X-100. Membrane proteins were labeled with 125I and passed through an affinity column in which CP was covalently linked to Sepharose 4B. Most of the radioactivity was eluted with buffer during the first 5 days. When no more radioactivity was eluted with buffer, elution was done either competitively with cold excess CP or 1 M NaCl. By this technique, a sharp single peak of radioactivity was obtained and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. Under both conditions receptors appeared as a single band with Mr of 35,000 containing 3% carbohydrate and an isoelectric point of 5.2.     

Pyridine-Functionalized Fe3O4 Nanoparticles as a Novel Sorbent for the Preconcentration of Lead and Cadmium Ions in Tree Leaf as a Bioindicator of Urban Traffic Pollution

We have developed a facile and highly sensitive sorbent for cadmium and lead ions. It is based on Fe₃O₄ nanoparticles functionalized with a derivative of picoline and was characterized by scanning electron microscopy, differential thermographic analysis, and elemental analysis. The material can be applied to the preconcentration of lead and cadmium ions. Factors such as the type, concentration and volume of eluent, the pH of the sample solution, the time for extraction, and the volume of the sample were studied. The effects of a variety of ions on preconcentration and recovery of these ions were also investigated. The ions were determined by FAAS, and the limits of detection are <0.8 and <0.061???g?L^?1 for lead and cadmium, respectively. Recoveries and precisions are >98.0?% and <1.3?%, respectively. The method was validated by analyzing several certified leaf reference materials.     

Talent in autism: hyper-systemizing,hyper-attention to detail and sensory hypersensitivity

We argue that hyper-systemizing predisposes individuals to show talent, and review evidence that hyper-systemizing is part of the cognitive style of people with autism spectrum conditions (ASC). We then clarify the hyper-systemizing theory, contrasting it to the weak central coherence (WCC) and executive dysfunction (ED) theories. The ED theory has difficulty explaining the existence of talent in ASC. While both hyper-systemizing and WCC theories postulate excellent attention to detail, by itself excellent attention to detail will not produce talent. By contrast, the hyper-systemizing theory argues that the excellent attention to detail is directed towards detecting ‘if p, then q’ rules (or [input–operation–output] reasoning). Such law-based pattern recognition systems can produce talent in systemizable domains. Finally, we argue that the excellent attention to detail in ASC is itself a consequence of sensory hypersensitivity. We review an experiment from our laboratory demonstrating sensory hypersensitivity detection thresholds in vision. We conclude that the origins of the association between autism and talent begin at the sensory level, include excellent attention to detail and end with hyper-systemizing.     

Localization of cytochrome b5 in rat organs and tissues by immunohistochemistry

We employed an immunohistochemical technique for a study of the localization of cytochrome b₅ in paraffin-embedded tissues of the rat. The presence of cytochrome b₅ was limited to the centrilobular areas of the liver, the distal tubules and the collecting ducts of the kidney and the absorptive cells of the villi in the small intestine.     

Double labeling of transferrin: tritium labeling of sialic acid and 125I or 59Fe labeling of the protein moiety

A method is described in which the glycoprotein transferrin was double labeled. Its sialic acid residues were labeled with 3H through a consecutive oxidation-reduction technique utilizing tritiated NaBH4. Its protein moiety was labeled with either 125I or 59Fe. Incubation of this double-labeled molecule at 4 degrees C with K562 cells gave overlapping curves, indicating identical patterns of binding for all labels. At 37 degrees C, 3H and 125I demonstrated identical patterns while 59Fe was cummulatively retained. This method can be used to follow the fate of other glycoproteins and their possible desialation in vivo.     

Proteoglycan synthesis by cultured liver endothelium: the role of membrane-associated heparan sulfate in transferrin binding

Liver endothelium has been reported to possess membrane receptors for the iron-binding protein transferrin (Tf). Similarly, the core protein of proteoglycans (PG) associated with cell membrane in many cell systems can bind Tf. To find out if membrane-associated proteoglycans can explain Tf-binding ability of liver endothelium, we investigated the synthesis and distribution of proteoglycans by isolated, cultured liver capillary endothelium. Cells were isolated and cultured for 48 h in sulfate-free medium and pulse-labeled with 35SO4. The relative distribution of 35SO4-labeled macromolecules, determined in the extracellular (EC), membrane-associated (MA), and intracellular (IC) pools, was respectively 74, 15, and 10%. Membrane-associated proteoglycan (MA-PG) was further purified by ion exchange and gel chromatography. Glycosaminoglycan (GAG) chain characterization indicated about 78% chondroitin sulfate, 7% dermatan sulfate, and about 14% heparan sulfate (HS). Similar GAG chain characterization was made for PG in the EC and IC pools. Transferrin-binding ability of MA-PG was studied by affinity column chromatography, using CNBr-activated sepharose bound to transferrin. About 15% of the labeled MA-PG was specifically bound to Tf-affinity column and could be eluted by excess soluble Tf. This proportion was similar to the proportion of HS in the total membrane-associated pool. Moreover, the eluted labeled material was susceptible to pretreatment with heparitinase, confirming its HS nature. We conclude that the transport capillary endothelium of the liver can synthesize HS proteoglycans which are membrane-associated and this MA-HS pool can bind transferrin. The finding may provide a molecular basis for transferrin binding to liver endothelium and may explain the subsequent transendothelial transport of iron-transferrin complexes into the liver.     

Apoptin induces apoptosis by changing the equilibrium between the stability of TAp73 and ΔNp73 isoforms through ubiquitin ligase PIR2

Apoptin, a protein derived from the chicken anaemia virus, induces cell death in various cancer cells but shows little or no cytotoxicity in normal cells. The mechanism of apoptin-induced cell death is currently unknown but it appears to induce apoptosis independent of p53 status. Here we show that p73, a p53 family member, is important in apoptin-induced apoptosis. In p53 deficient and/or mutated cells, apoptin induced the expression of TAp73 leading to the induction of apoptosis. Knockdown of p73 using siRNA resulted in a significant reduction in apoptin-induced cytotoxicity. The p53 and p73 pro-apoptotic target PUMA plays an important role in apoptin-induced cell death as knockdown of PUMA significantly reduced cell sensitivity to apoptin. Importantly, apoptin expression resulted in a marked increase in TAp73 protein stability. Investigation into the mechanisms of TAp73 stability showed that apoptin induced the expression of the ring finger domain ubiquitin ligase PIR2 which is involved in the degradation of the anti-apoptotic ?Np73 isoform. Collectively, our results suggest a novel mechanism of apoptin-induced apoptosis through increased TAp73 stability and induction of PIR2 resulting in the degradation of ?Np73 and activation of pro-apoptotic targets such as PUMA causing cancer cell death.