Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.     

Structurally inherent antigenic sites. Localization of the antigenic sites of the alpha-chain of human haemoglobin in three host species by a comprehensive synthetic approach.

The antigenic structure of the alpha-chain of human haemoglobin was studied by a synthetic approach consisting of the synthesis of a series of consecutive overlapping peptides that together systematically represent the entire primary structure of the protein. This approach enabled the identification of a full profile of immunochemically active alpha-chain peptides and the localization of its major 'continuous' antigenic sites. Antibodies to haemoglobin raised in each of three different species (goat, rabbit and mouse) recognize similar sites on the alpha-chain. Further, the molecular locations of these sites coincide with alpha-chain regions extrapolated from antigenic sites of the conformationally similar myoglobin molecule. These findings support our earlier proposed concept of 'structurally inherent antigenic sites', namely that antigenicity is conferred on certain surface regions of proteins by virtue of their three-dimensional locations. Thus the antigenic sites of conformationally related proteins are likely to have similar molecular locations.     

Interaction of exercise and adenosine receptor agonist and antagonist on rat heart antioxidant defense system

This study investigated the interactive effects of acute exercise and adenosine receptor agonist and antagonist on antioxidant enzyme activities, glutathione and lipid peroxidation in the heart of the rat. Male Fisher-344 rats were divided into six groups and treated as follows: (1) saline control; (2) acute exercise (100% VO_2max); (3) R-Phenyl isopropyl adenosine (R-PIA) (3.46 mol/kg, i.p.); (4) theophylline (1.70 mol/kg, i.p.) plus acute exercise; (5) theophylline plus R-PIA; and (6) theophylline. Animals were sacrificed 1 h after treatments; hearts were isolated and analyzed. The results show that acute exercise as well as adenosine receptor agonist R-PIA significantly enhanced cardiac superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GR) activity by 36–135% and 16–51%, respectively. Adenosine receptor agonist R-PIA significantly decreased cardiac GSSG concentration and enhanced GSH/GSSG ratio by 22 and 30%, respectively. Whereas theophylline treatment blocked the activation of antioxidant enzyme activities enhanced by acute exercise and R-PIA. Theophylline treatment significantly increased lipid peroxidation by 43% in the heart of exercised rats. The study concluded that the adenosine receptors are involved in the upregulation of cardiac antioxidant defense system and attenuation of lipid peroxidation due to acute exercise in rats. (Mol Cell Biochem 270: 209–214, 2005)     

Hip joint replacement surgery for idiopathic osteoarthritis aggregates in families

In order to determine whether there is a genetic component to hip or knee joint failure due to idiopathic osteoarthritis (OA), we invited patients (probands) undergoing hip or knee arthroplasty for management of idiopathic OA to provide detailed family histories regarding the prevalence of idiopathic OA requiring joint replacement in their siblings. We also invited their spouses to provide detailed family histories about their siblings to serve as a control group. In the probands, we confirmed the diagnosis of idiopathic OA using American College of Rheumatology criteria. The cohorts included the siblings of 635 probands undergoing total hip replacement, the siblings of 486 probands undergoing total knee replacement, and the siblings of 787 spouses. We compared the prevalence of arthroplasty for idiopathic OA among the siblings of the probands with that among the siblings of the spouses, and we used logistic regression to identify independent risk factors for hip and knee arthroplasty in the siblings. Familial aggregation for hip arthroplasty, but not for knee arthroplasty, was observed after controlling for age and sex, suggesting a genetic contribution to end-stage hip OA but not to end-stage knee OA. We conclude that attempts to identify genes that predispose to idiopathic OA resulting in joint failure are more likely to be successful in patients with hip OA than in those with knee OA.     

Effects of dietary combination of chromium and biotin on growth performance, carcass characteristics, and oxidative stress markers in heat-distressed Japanese quail

Environmental stress causes adverse effects in performance and antioxidant status of poultry. Dietary chromium supplementation promotes the growth rate and feed efficiency of growing poultry and these beneficial effects of chromium appear to be greater under stress. Biotin, a member of the vitamin B complex, is involved in the metabolism of carbohydrates, fats, and proteins. In a previous experiment, we examined the effects of chromium picolinate (CrPic) as a chromium source in birds subjected to high environmental temperature and the data showed that supplementation with CrPic ameliorated the deletorious effect of stress. The study was conducted to determine the effects of a supplementation of combination of CrPic and biotin (Diachrome^TM) on performance, carcass characteristics, levels of oxidative stress markers, serum cholesterol, and glucose concentrations in Japanese quail (Coturnix coturnix japonica) exposed to high ambient temperature of 34°C. Two hundred forty Japanese quail (10 d old) were randomly assigned to 8 treatment groups consisting of 10 replicates of 3 birds. The birds were kept in a temperature-controlled room at 22°C (thermo-neutral [TN] groups) or 34°C (for 8h/d; 09.00 am to 05.00 pm; heat-stress [HS] groups). Birds were fed either a basal (control) diet (TN and HS) or the basal diet supplemented with either 1, 2 or 4 mg of Diachrome/kg of diet. Heat exposure decreased performance when the basal diet was fed (p=0.001). Diachrome supplementation increased feed intake (p=0.001), body weight (p=0.05), feed efficiency (p=0.01), and carcass traits (p≤0.05) variables linearly in birds reared under HS conditions. Serum vitamin C (p=0.05) and vitamin E (p=0.03) concentrations increased, whereas malondialdehyde (MDA) levels in serum and the liver (p=0.01), thigh muscle (p=0.05), and serum cholesterol and glucose concentrations (p=0.05) decreased in supplemented birds reared at a high temperature. It should be noted that when birds were kept at the thermo-neutral temperature, Diachrome supplementation did not affect (p>0.05) the variables measured, with the exception of a reduction in serum cholesterol and glucose. Results of the present study suggest that Diachrome can be considered a protective dietary supplement by reducing the negative effects of high environment temperature on performance and oxidative stress in quail.     

Interaction of regular exercise and chronic nitroglycerin treatment on blood pressure and rat aortic antioxidants

Many cardiac patients undergo exercise conditioning with or without medication. Therefore, we investigated the interaction of exercise training and chronic nitroglycerin treatment on blood pressure (BP), aortic nitric oxide (NO), oxidants and antioxidants in rats. Fisher 344 rats were divided into four groups and treated as follows: (1) sedentary control, (2) exercise training (ET) for 8 weeks, (3) nitroglycerin (15 mg/kg, s.c. for 8 weeks) and (4) ET+nitroglycerin. BP was monitored with tail-cuff method. The animals were sacrificed 24 h after the last treatments and thoracic aorta was isolated and analyzed. Exercise training on treadmill for 8 weeks significantly increased respiratory exchange ratio (RER), aortic NO levels, and endothelial nitric oxide synthase (eNOS) protein expression. Training significantly enhanced aortic glutathione (GSH), reduced to oxidized glutathione (GSH/GSSG) ratio, copper/zinc-superoxide dismutase (CuZn-SOD), Mn-SOD, catalase (CAT), glutathione peroxidase (GSH-Px) glutathione disulfide reductase (GR) activities and protein expressions. Training significantly depleted aortic malondialdehyde (MDA) and protein carbonyls without change in BP. Nitroglycerin administration for 8 weeks significantly increased aortic NO levels and eNOS protein expression. Nitroglycerin significantly enhanced aortic Mn-SOD, CAT, GR and glutathione-S-transferase (GST) activities and protein expressions with decreased MDA levels, protein carbonyls and BP. Interaction of training and nitroglycerin treatment significantly increased aortic NO levels, eNOS protein expression, GSH/GSSG ratio, antioxidant enzymes and normalized BP. The data suggest that the interaction of training and nitroglycerin maintained BP by up-regulating the aortic NO and antioxidants and reducing the oxidative stress in rats.     

Rapid evolution of immunoglobulin superfamily C2 domains expressed in immune system cells

To test the hypothesis that proteins expressed in cells of the vertebrate immune system evolve unusually rapidly, 107 orthologous immunoglobulin C2 domains were compared between human and murine rodent. The analysis showed that the rate of nonsynonymous (amino-acid- altering) nucleotide substitution in these domains was correlated with factors associated with protein structure and with breadth of tissue expression, as well as with the rate of synonymous substitution. However, when such factors were controlled for statistically, there remained a strong positive association between expression in the immune system and nonsynonymous rate, with the highest rates being seen in genes expressed in the immune system only. Certain immune system genes are known to be subject to positive selection favoring diversity at the amino acid level; most of these genes encode receptors that interact directly with foreign antigens. The observed acceleration of the rate of nonsynonymous evolution in C2 domains of immune system proteins may be explained by either (1) reduced constraint at the amino acid level on molecules interacting with immune system receptors that are themselves evolving rapidly due to positive diversifying selection or (2) positive selection favoring amino acid changes correlated with changes in the immune system receptors.