Spherical-supported membranes as platforms for screening against membrane protein targets.

Screening assays performed against membrane protein targets (e.g. phage display) are hampered by issues arising from protein expression and purification, protein stability in detergent solutions and epitope concealment by detergent micelles. Here, we have studied a fast and simple method to improve screening against membrane proteins: spherical-supported bilayer lipid membranes ("SSBLM"). SSBLMs can be quickly isolated via low-speed centrifugation and redispersed in liquid solutions while presenting the target protein in a native-like lipid environment. To provide proof-of-concept, SSBLMs embedding the polytopic bacterial nucleoside transporter NupC were assembled on 100- and 200 nm silica particles. To test specific binding of antibodies, NupC was tagged with a poly-histidine epitope in one of its central loops between two transmembrane helices. Fluorescent labelling, small angle X-ray scattering (SAXS) and cryo-electron microscopy (cryo-EM) were used to monitor formation of the SSBLMs. Specific binding of an anti-his antibody and a gold-nitrilotriacetic acid (NTA) conjugate probe was confirmed with ELISAs and cryo-EM. SSBLMs for screening could be made with purified and lipid reconstituted NupC, as well as crude bacterial membrane extracts. We conclude that SSBLMs are a promising new means of presenting membrane protein targets for (biomimetic) antibody screening in a native-like lipid environment.

Epitope mapping and key amino acid identification of anti-CD22 immunotoxin CAT-8015 using hybrid ß-lactamase display.

Monoclonal antibodies are a commercially successful class of drug molecules and there are now a growing number of antibodies coupled to toxic payloads, which demonstrate clinical efficacy. Determining the precise epitope of therapeutic antibodies is beneficial in understanding the structure-activity relationship of the drug, but in many cases is not done due to the structural complexity of, in particular, conformational protein epitopes. Using the immunotoxin CAT-8015 as a test case, this study demonstrates that a new methodology, hybrid ß-lactamase display, can be employed to elucidate a complex epitope on CD22. Following insertion of random CD22 gene fragments into a permissive site within ß-lactamase, proteins expressed in Escherichia coli were first screened for correct folding by resistance to ampicillin and then selected by phage display for affinity to CAT-8015. The optimal protein region recognised by CAT-8015 could then be used as a tool for fine epitope mapping, using alanine-scanning analysis, demonstrating that this technology is well suited to the rapid characterisation of antibody epitopes.

Cow milk allergy within the spectrum of atopic disorders.

In order to examine the relationship between cow milk allergy (CMA) and atopic disorders in childhood, a consecutive group of 42 infants with IgE mediated CMA was followed for at least 2 years. The incidence of sensitization to common food and inhalant antigens and the development of eczema, asthma, and food allergies was examined for the cohort and compared between patients whose CMA remitted and those with persistent disease. In this cohort the prevalence of eczema was 57%, asthma 69%, egg allergy 67%, peanut allergy 55%, and 83% of infants demonstrated positive skin-prick tests to three or more allergens. At the end of the study CMA had remitted in 13 patients (median age 44 months) whereas in 29 patients it persisted (median age 44 months). Although there was no significant difference in the incidence of eczema or asthma during the study between these two patient groups, the incidence of allergy to egg and peanut butter was significantly greater for children with persistent CMA. Consistent with our hypothesis that children with persistent CMA have a more severe dysregulation of IgE synthesis than those whose disease remits, patients with persistent CMA had a significantly higher incidence of and level of skin sensitivity to inhalant and other dietary allergens. Sensitization to the inhalant allergens Dermatophagoides pteronyssinus, cat dander and rye grass was frequently seen in early infancy and increased during the study period. Thus, children with IgE mediated CMA frequently generate IgE responses to multiple dietary and inhalant allergens in infancy and early childhood and develop immediate hypersensitivity to other foods as well as clinical eczema, and asthma.

Minor burns lead to major changes in synthesis rates of plasma proteins in the liver.

The effect of minor burns on the rates of synthesis of plasma proteins in the liver was studied in white Buffalo rats. Burns of second to third degree, covering 0.8% of total body surface, were produced by short application of a hot piece of metal to the skin under ether anesthesia. Levels of mRNA in extracts from liver removed after 24 hr were measured by hybridization to radioactively labeled specific cDNA probes. The level of mRNA for major acute phase alpha 1-protein (also called cysteine proteinase inhibitor or T1-kininogen) increased 20-fold, that of fibrinogen mRNA 8-fold, and that of alpha 1-acid glycoprotein mRNA about 9-fold. The levels of albumin mRNA and transthyretin mRNA (also called prealbumin) decreased to about 80% of normal and the level of transferrin mRNA did not change significantly. Thus, although the percentage of burnt skin was only very small, a typical acute phase response of plasma protein synthesis in liver was observed.

Nutritional effects of peritonitis in continuous ambulatory peritoneal dialysis (CAPD) patients.

Peritonitis, a major complication of end-stage renal disease patients treated with continuous ambulatory peritoneal dialysis (CAPD), enhances peritoneal protein losses by increasing protein and energy requirements while simultaneously decreasing appetite, usually causing a negative nitrogen balance. The influence of peritonitis on the nutritional status of CAPD patients was evaluated. Fourteen end-stage renal disease patients being treated with CAPD and presenting with peritonitis were randomized to one group with and one without a nutritional supplement. Four CAPD patients without peritonitis served as controls. Anthropometric measurements, laboratory determinations, dietary protein intake, and protein catabolic rate were obtained. The control group lost an average of 9.6 gm protein per 24 hours in the peritoneal fluid vs. an average of 15.1 gm protein per 24 hours lost by patients with peritonitis (p less than .01). Serum albumin did not decrease except in two diabetic patients in whom it dropped an average of 42% and remained low. Nitrogen balance remained positive in all patients except one with diabetes who had very low daily protein intake and caloric intake. The catabolism produced by short uncomplicated peritonitis did not create a negative nitrogen balance in patients eating at least 1 gm protein per kilogram ideal body weight (IBW) and 25 kcal/kg IBW.

Rat choroid plexus specializes in the synthesis and the secretion of transthyretin (prealbumin). Regulation of transthyretin synthesis in choroid plexus is independent from that in liver.

Synthesis of total protein and of transthyretin in rat choroid plexus was studied by measuring the incorporation of radioactive leucine into proteins in choroid plexus tissue incubated in vitro. About 20% of the protein newly synthesized in choroid plexus and about 50% of the newly synthesized protein secreted into the medium was transthyretin. Evidently, the choroid plexus is very active in the biosynthesis of this carrier protein for thyroid hormones and could be an important link in the chemical communication between the body and the central nervous system. Acute inflammation, which leads to a profound rearrangement of the pattern of plasma protein synthesis rates in the liver, produced distinct changes in the levels for plasma protein mRNAs in the liver. The levels of the mRNAs for alpha 1-acid glycoprotein and major acute phase alpha 1-protein increased more than 30-fold, those for transthyretin and albumin decreased to 27 and 57% of normal, respectively. The pattern of the observed changes in the levels of mRNAs for plasma proteins in the liver was independent of whether the acute inflammation was produced by subcutaneous injection of turpentine or intraperitoneal injection of a suspension of talcum. However, levels of transthyretin mRNA in choroid plexus were affected only very slightly, or not at all. Apparently, transthyretin synthesis in liver and choroid plexus is regulated independently during the acute phase response. No mRNA was detected in choroid plexus for albumin, alpha 1-acid glycoprotein, and major acute phase alpha 1-protein under any conditions.

Gluconeogenic and lipogenic properties of isolated kidney tubules from the domestic fowl (Gallus domesticus).

Isolated kidney tubules synthesize glucose actively from fructose, lactate, glycerol and pyruvate and, to a lesser extent, from a variety of amino acids. Ethanol stimulated gluconeogenesis from pyruvate and inhibited it from lactate. The aminotransferase inhibitor, aminooxyacetate, greatly reduced synthesis from lactate but not from pyruvate. Quinolinate inhibited gluconeogenesis from both precursors, indicating an active role for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in the gluconeogenic pathway. Incorporation of lactate or glucose into triglycerides was relatively low, and since no fatty acid synthase (FAS) activity could be detected, probably represented chain elongation or reesterification.

Biotin deficiency and susceptibility to fatty liver and kidney syndrome in broiler chicks: reduced 6-phosphofructokinase (EC 2.7.1.11) activity but normal fructose 2,6-bisphosphate content in birds with hepatomegaly.

In two separate experiments, using different strains, broiler chicks were reared on either a commercial-type chick mash (control) or a fatty liver and kidney syndrome (FLKS)-inducing diet. In Expt a, chicks were killed on day 29 and in Expt b, on day 32. Body-weights and liver weights were measured, and values from those given the control ration used to construct a hepatomegaly index by employing a variant of linear discriminant analysis. Application of the index to FLKS birds revealed a statistically significant bimodal distribution of liver size. The birds with enlarged livers (high index) also possessed metabolic abnormalities in that 6-phosphofructokinase (EC 2.7.1.11; PFK-1) activity (measured at low substrate concentration) was depressed despite the presence of normal, or even slightly elevated fructose 2,6-bisphosphate concentration. This indicates the presence of an uncharacterized regulatory mechanism for PFK-1 in FLKS-susceptible birds.

Activation of phosphofructokinase and pyruvate kinase by 6 phosphogluconate in chicken liver (Gallus domesticus): no evidence for a regulatory role.

Phosphofructokinase and pyruvate kinase in crude extracts of chick liver were activated by 6 phosphogluconate at subsaturating concentrations of their respective substrates. The apparent Ka for phosphofructokinase was 0.79 mM and for pyruvate kinase 0.44 mM. Total pentose phosphate dehydrogenase activity was low in the livers of both fed and starved chicks. Hepatic 6 phosphogluconate concentration was too low to be measured accurately by a conventional spectrophotometric method. It is concluded that the concentration of 6 phosphogluconate is too low for it to have a role in the regulation of phosphofructokinase and pyruvate kinase activities in chicken liver.

Lipogenic enzyme activity and fructose 2,6-bisphosphate concentration in livers of two lines of domestic fowl (Gallus domesticus) selected for different body fat content.

Livers were obtained from two lines of domestic broiler which had been selected for low (lean) and high (fat) plasma very-low-density lipoprotein concentration over three generations. The fat line possessed significantly higher hepatic specific activities of malate dehydrogenase (NADP), ATP citrate lyase and fatty acid synthase, and lower glucose bisphosphatase than the lean line. The glycolytic enzymes, pyruvate kinase and phosphofructokinase, were similar and so was the concentration of fructose 2,6-bisphosphate. This recently discovered metabolic regulator was present at somewhat higher concentrations than previously reported in rats or mice. It exhibited a positive correlation with phosphofructokinase activity (only data for the fat line are shown), and stimulated enzyme activity when added to crude preparations.

The effects of oxalate and glucose on lipogenesis by isolated hepatocytes from normal and biotin-deficient chicks (Gallus domesticus).

Isolated hepatocytes synthesize fatty acids and cholesterol from lactate and acetate with lactate being the more effective substrate. Biotin deficiency decreased fatty acid synthesis from both substrates but stimulated cholesterogenesis. Exposure of intact hepatocytes to oxalate inhibited fatty acid and cholesterol synthesis from lactate, this effect was enhanced in biotin-deficient chicks. A similar effect was not observed when acetate was the substrate. Synthesis of fatty acids from lactate and acetate was stimulated by glucose, biotin deficiency increased this response. Cholesterogenesis was reduced in control but not biotin-deficient chicks.

The effect of biotin deficiency and dietary protein content on lipogenesis, gluconeogenesis and related enzyme activities in chick liver.

Chicks were given biotin-deficient diets containing either suboptimal (low) or supraoptimal (high) concentrations of protein from 1-d-old until they were used during their fourth week of life. The low-protein diet predisposed chicks to develop fatty liver and kidney syndrome and the high-protein diet to develop classical biotin deficiency signs. Two other groups, as controls, received biotin-supplemented rations. Low dietary protein increased lipogenesis by isolated hepatocytes but had little effect on gluconeogenesis compared to high dietary protein. Low dietary protein decreased activities of hepatic isocitrate dehydrogenase (EC 1.1.1.42), fructose-1,6-bisphosphatase (EC 3.1.3.11) and glucose-6-phosphatase (EC 3.1.3.9; GP) and increased activities of fatty acid synthase (FAS), citrate cleavage enzyme (EC 4.1.3.8; CCE) and malate dehydrogenase (decarboxylating) (EC 1.1.1.39). When biotin deficiency was superimposed, the rate of lipogenesis by isolated hepatocytes (from fed birds) was decreased. Gluconeogenesis from lactate and glycerol was also depressed. Activity of GP was further decreased by biotin deficiency on the low-protein regimen and FAS and CCE were further increased. PK activity was increased by biotin deficiency.

The effect of oxalate on gluconeogenesis by isolated chicken hepatocytes. Increased sensitivity to inhibition as a result of biotin deficiency.

Addition of varying concentrations of oxalate to isolated chicken hepatocytes reduced gluconeogenesis from lactate in a manner indicating that pyruvate carboxylase was not the rate-limiting step. With hepatocytes from biotin-deficient chicks, sensitivity to inhibition was increased, and was consistent with pyruvate carboxylase being rate-limiting. Administration of biotin to deficient chicks overnight restores sensitivity to oxalate to normal.

Application and evaluation of an NADH-linked spectrophotometric assay for pyruvate dehydrogenase complex using chicken liver homogenates.

1. A method for measuring the activity of the pyruvate dehydrogenase complex [EC 1.2.4.1] (Hinman and Blass, 1981) has been adapted for use with crude chicken liver homogenates. 2. It was found necessary to remove cytoplasmic lactate dehydrogenase by centrifugation before releasing the complex from the mitochondria by treatment with sodium deoxycholate. 3. Lipoamide dehydrogenase was preferable to phenazine methosulphate as the electron carrier between the complex and NADH. 4. The assay was not suitable for use at 40 degrees C (approximate body temperature) but was satisfactory at 25 degrees C. 5. The assay exhibited the properties expected of pyruvate dehydrogenase complex i.e. a complete dependence on the presence of NAD, coenzyme A and pyruvate and a partial dependence on MgCl2, dithiothreitol and thiamine pyrophosphate. The pH optimum was between 7.8 and 8.0. 6. The mean activity for a group of normally fed young chickens was 654 mumol/g dry weight h and 88 for birds starved overnight.

The post mortem diagnosis of fatal hypoglycaemia using the fatty liver and kidney syndrome of chicks as a model.

1. The concentrations of vitreous humour and plasma glucose were closely correlated in both healthy and fatty liver and kidney syndrome-affected chicks at time of death. 2. The values of vitreous humour glucose and lactate decreased rapidly after death, such that they were not reliable indicators of the presence of hypoglycaemia immediately ante mortem. 3. Hepatic glycogen was extremely low in fatty liver and kidney syndrome-affected birds, whereas significant quantities remained in healthy birds up to at least 24 h post mortem.

Aspects of metabolism related to the occurrence of skin lesions in biotin-deficient chicks.

1. Chicks were fed on biotin-deficient low- and high-protein diets supplemented with increasing concentrations of biotin. 2. Biotin deficiency decreased hepatic activity of pyruvate carboxylase [EC 6.4.1.1] but activity of acetyl-CoA carboxylase [EC 6.4.1.2] was comparatively unaffected. 3. Increasing dietary protein increased the severity of biotin deficiency as assessed by skin lesions and decreased plasma biotin concentrations. 4. The severity of the skin lesions over all the treatments was most closely related to plasma biotin concentration.

Blood pyruvate carboxylase activity as a criterion of biotin status in young pigs.

A basal diet containing isolated soybean protein was supplemented with 100 and 500 microgram biotin/kg and another basal diet containing hen-egg albumen was supplemented with 1300 microgram biotin/kg. The five diets were fed to groups of young pigs from four to 12 weeks old. Growth depression or skin or foot lesions characteristic of biotin deficiency were not seen but the undersurfaces of the hooves of pigs given the basal albumen diet were unusually soft. Blood pyruvate carboxylase activity showed a maximum response to dietary biotin at six weeks but by 12 weeks there was no response to supplemental levels greater than 100 microgram/kg. Plasma biotin concentration gave a good indication of biotin intake at all ages.

Biotin status, blood pyruvate carboxylase (EC 6.4.1.1) activity and performance in broilers under different conditions of bird husbandry and diet processing.

1. Three experiments were conducted to investigate the relationships between blood pyruvate carboxylase (pyruvate: carbon dioxide ligase (ADP-forming), EC 6.4.1.1; PC) activity, growth performance and dietary biotin level in broilers under different husbandry conditions. Blood PC activity was also used to gain information on the ingestion of biotin faecal origin by birds housed on wood shavings and the effect of steam-pelleting on the biotin content of diets. 2. Blood PC activity and relationship to dietary biotin level were similar in birds kept under different husbandry conditions. 3. Biotin requirement for growth was higher in birds kept under conditions that allowed them to reach their growth potential more fully. 4. The contribution of biotin of faecal origin to the biotin intake of birds housed on wood shavings was negligible at 3 weeks of age but by 7 weeks was equivalent to approximately 0.01 mg/kg diet. 5. Steam-pelleting did not affect the stability or availability of biotin of natural or synthetic origin in diets. 6. It is concluded that blood PC activity is a good criterion of biotin status and requirement in fast-growing broilers.