Biomedical subjects
Publications and source records attributed to T M Cox.
Heart disease is a recognised complication of influenza. We report a unique case in which myopericarditis and collapse due to acute influenza A infection was associated with pericardial effusion and tamponade. In addition, the patient had myositis and pleurisy. Emergency pericardiocentesis and inotropic drugs were needed but recovery was complete.
Addition of hemin to reticulocytes inhibits incorporation of iron from transferrin [Ponka, P. & Neuwirt, J. (1969) Blood 33, 609-707]. Heme also regulates protein synthesis in immature erythroid cells through its effects on phosphorylation of the initiation factor eIF-2. We have therefore examined its effects on endocytosis of iron-transferrin and phosphorylation of the transferrin receptor. Hemin (10-50 microM) reduced iron transport but increased cell-associated transferrin. When intracellular iron delivery was inhibited by NH4Cl, no such increase in cell-associated transferrin was seen. During uptake of 125I-labeled transferrin in the steady state, the use of a washing technique to dissociate bound transferrin on the cell membrane showed that radioligand accumulated on the surface of hemin-treated cells. Hemin reduced the initial influx of transferrin, thereby diminishing incorporation of iron. Receptor phosphorylation was investigated by immunoprecipitation of reticulocyte extracts after metabolic labeling with [32P]Pi. In the absence of ligand, phosphorylated receptor was chiefly localized on cell stroma. Exposure to transferrin increased cytosolic phosphorylated receptor from 15-30% to approximately 50% of the total, an effect overcome by hemin treatment. Addition of hemin in the presence of transferrin enhanced net phosphorylated receptor in the reticulocyte in association with a redistribution of phosphorylated receptor to stromal membranes. The findings suggest a possible relationship of phosphorylation to endocytosis of the transferrin receptor in reticulocytes.
These studies were performed to determine whether the reticulocyte can synthesize its own transferrin receptor and, if so, whether synthesis is subject to translational control by intracellular heme. Reticulocytosis (20-35%) was produced by bleeding rabbits and the washed cells were incubated for 1-4 h at 37 degrees C in buffered nutritional medium containing L-[35S]methionine. After washing and detergent lysis in the presence of protease inhibitors, supernatant reticulocyte extracts were analyzed for transferrin receptors by immunoprecipitation with specific ovine receptor antibody raised against denatured rabbit transferrin receptor. Immunoprecipitates were analyzed by SDS-gel electrophoresis and fluorography. Antibody, but not preimmune sheep immunoglobin, consistently precipitated a 35S-labeled protein with an Mr of 90,000 (reduced), coincident with bona fide receptor subunits purified by ligand-affinity chromatography. Incorporation of radioactive methionine was exclusively associated with receptor in reticulocyte stroma, and nascent receptor was not detected on free polyribosomes. Incorporation of radioactivity in the receptor moiety accounted for 0.1-0.2% of total incorporation into TCA insoluble cell protein. Treatment of the cells with 40 micrograms/ml cycloheximide markedly inhibited amino acid incorporation into the receptor, thus indicating de novo synthesis of receptor protein. On treatment of reticulocytes with 4,6 dioxoheptanoate to induce heme deficiency by diminishing the formation of intracellular heme, synthesis of the receptor was inhibited by greater than 50%; synthesis was restored to control rates on addition of 50 microM exogenous hemin. These findings indicate that the reticulocyte retains receptor mRNA and that synthesis of the receptor in erythroid cells is subject to translational regulation by intracellular heme.
To test the discriminatory potential of certain indices of pancreatic function we performed duodenal perfusion studies and measured trypsin, bicarbonate, and lactoferrin outputs, and plasma concentrations of pancreatic polypeptide and motilin in the basal state and during continuous intravenous stimulation with 100 ng kg-1h-1 Ceruletide and 1 CU kg-1h-1 secretin. The following groups were studied: 12 normal volunteers (NV), seven patients with chronic pancreatitis with steatorrhea (CPS), and seven without steatorrhea (CP). Stimulated trypsin outputs, after 45 min of stimulation, were the best discriminant among the groups (NV versus CPS, p less than 0.0005; NV versus CP, p less than 0.005; CP versus CPS, p less than 0.05). Basal trypsin outputs showed similar patterns but failed to discriminate between NV and CP. Bicarbonate outputs were less discriminatory than trypsin outputs. Lactoferrin outputs failed to discriminate, but transient high peak outputs occurred in the initial stimulation period in all four patients with calcific chronic pancreatitis, suggesting a washout phenomenon. Basal motilin levels were elevated in both groups of pancreatitis (p less than 0.05). Stimulated pancreatic polypeptide levels were lower in CPS (NV versus CPS, p less than 0.05) but higher in CP (NV versus CP, p less than 0.005). These differences were also apparent in the basal state. We conclude that the best discrimination among the three groups was achieved by measurement of trypsin outputs, after 45 min of stimulation. In addition, the pancreatic polypeptide response may be used as a marker of residual pancreatic function in chronic pancreatitis.
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Hereditary fructose intolerance (HFI) is a metabolic disorder caused by enzymic deficiency of aldolase B, a genetically distinct cytosolic isoenzyme expressed exclusively in liver, kidney, and intestine. The molecular basis of this enzyme defect has been investigated in three affected individuals from a nonconsanguineous kindred, in whom fructose-l-phosphate aldolase activities in liver or intestinal biopsy samples were reduced to 2-6% of mean control values. To identify a putative enzyme mutant in tissue extracts, aldolase B was purified from human liver by affinity chromatography and monospecific antibodies were prepared from antiserum raised in sheep. Immunodiffusion gels showed a single precipitin line common to pure enzyme and extracts of normal liver and intestine, but no reaction with extracts of brain, muscle, or HFI liver. However, weak positive staining for aldolase in hepatocyte and enterocyte cytosol was demonstrated by indirect immunofluorescence of HFI tissues. This was abolished by pretreatment with pure enzyme protein. Accordingly, a specific radioimmunoassay (detection limit 7.5 ng) was established to quantify immunoreactive aldolase B in human biopsy specimens. Extracts of tissue from affected patients gave 10-25% immunoreactive enzyme in control samples; immunoreactive aldolase in intestinal extracts from four heterozygotes was reduced (to 55%) when compared with seven samples from normal control subjects (P < 0.05). In extracts of HFI tissues, there was a sevenfold reduction in apparent absolute specific activity (1.02 vs. 8.82 U/mg) of immunoreactive fructose-l-phosphate aldolase B, but the apparent specific activity in heterozygotes (7.71 U/mg) was only slightly impaired. Displacement radioimmunotitration of aldolase B in liver supernatants showed a significant (P < 0.005) decrease in antibody avidity for immunoreactive protein in HFI tissue when compared with the pure enzyme or extract of normal control liver. Immunoaffinity chromatography on antialdolase B-Sepharose facilitated isolation and purification of enzyme from liver biopsy specimens. Active aldolase in normal liver, with substrate activity ratios and Michaelis constants identical to biochemically purified human enzyme, could be recovered from antibody columns. Chromatography on monospecific Fab' antialdolase B enabled pure enzyme protein to be retrieved quantitatively from normal control and HFI liver: direct chemical assay showed 1.88 and 1.15 mg aldolase protein/g of tissue, respectively. This confirmed that the catalytic properties of the HFI aldolase were profoundly impaired with specific activities of fructose-l-phosphate cleavage of 7.21 and 0.07 U/mg, respectively. Radioimmunoassay gave estimates of 7.66 and 1.18 U/mg, respectively. Sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that immunopurified aldolase from HFI liver possessed a single subunit size similar to material from control liver extracts: M(r) 39,100 vs. 37,900+/-700 (SD) D, respectively. Electrofocusing under denaturing conditions of aldolase isolated in parallel from control and HFI liver revealed the same complement of subunits and, despite qualitative differences in distribution of bands during degradation, no additional charged species. Fructose phosphate aldolase deficiency in hereditary fructose intolerance is attended by the synthesis of an immunoreactive, but functionally and structurally modified enzyme variant that results from a restricted genetic mutation.
Hereditary fructose intolerance (HFI) is a disorder of visceral carbohydrate metabolism which is transmitted as a recessive character of moderate to high gene prevalence. The condition is caused by enzymic deficiency of aldolase B and is associated with the synthesis of inactive enzyme protein. The molecular structure of aldolase B was examined in tissue samples from four adult patients who were the offspring of non-consanguineous unions. Titration of aldolase protein, by radioimmunoassay, showed that antibody recognition of the inactive enzyme was attenuated differently in two unrelated HFI patients. The existence of separate structural lesions was confirmed by protein blotting and immunodetection of enzyme subunits after sodium dodecyl sulphate/polyacrylamide electrophoresis. In one patient the subunit size was identical to wild type (Mr 38,000) and in the other, a single faint band (Mr 39,000) was identified. Radioimmunotitration studies, in two affected offspring of this latter patient by a proven HFI carrier, also revealed differences in antibody recognition. Segregation of different mutant alleles within this kindred demonstrates heterogeneity in HFI occurring at the same genetic locus. Variations in apparent immunoreactivity of aldolase B in HFI are thus related to overt modification of enzyme subunits and indicate that the disorder results principally from structural rather than regulatory mutations in the aldolase B gene.
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Rabbit intestinal microvillus membranes possess high-affinity receptors for iron whose activity reflects homeostatic changes in mucosal iron transport. To isolate and characterize these membrane components, purified microvilli were radiolabelled with 59Fe(II) and solubilized in Triton X-100. 59Fe in 105000g supernatants co-eluted with a major broad protein peak (Mr approx. 100000) on gel-permeation chromatography and was rendered diffusible by Pronase digestion but not mild periodate degradation. Fluorescence studies with castor-bean lectin conjugates showed specific binding of this affinity probe exclusively to brush-border membranes in the intestinal epithelium. Affinity chromatography of solubilized membrane proteins showed binding to columns of immobilized lectin. Elution with D(+)-galactose released glycoprotein-bound 59Fe purified up to sevenfold over initial membrane extracts. The lectin bound up to 82% of protein-bound 59Fe. In contrast polyspecific antisera raised against rabbit microvilli in guinea-pigs precipitated less than 10% of solubilized radioactivity. Significantly more protein-bound 59Fe in detergent extracts of microvilli purified from bled animals interacted specifically with the lectin, suggesting that membrane glycoprotein receptors are involved in the homeostatic control of intestinal iron transport.
1. Whole-body retention in vivo and uptake of 59Fe-labelled ascorbate and nitrilotriacetate chelates by intestinal slices in vitro were determined in groups of normal control rabbits and rabbits with experimentally-induced Fe deficiency. 2. Over-all absorption as measured by retention of doses of either chelate was greatly increased in conditions of Fe deficiency. 3. Intestinal Fe uptake in vitro was inhibited up to 77% in the presence of 2,4-dinitrophenol and sodium fluoride. Initial rates showed saturation within the concentration range 18-450 mumol/l, suggesting that uptake was brought about by an active transport process. 4. When studied at chelate concentrations of 450 mumol/l, significant regional differences in uptake rates were observed. Uptake in duodenal slices was increased when compared with slices from jejunum and ileum. 5. Fe uptake from ferric and ferrous chelates was greatly enhanced in Fe deficiency. This was chiefly due to increases in uptake by slices from the duodenum, but uptake into slices of distal intestine was also stimulated. 6. Kinetic analysis of Fe uptake by duodenal slices from animals rendered Fe deficient by diet or repeated bleeding indicated in both groups an increased apparent maximum velocity (Vmax) for influx of Fe without significant changes in apparent affinity for Fe. 7. The experiments provide further insight into the nature and regional distribution of transport of Fe into the intestine and suggest, in the rabbit, that important control of Fe absorption may be exerted by an active process operating at this initial entry step.
1. 59Fe binding by microvillus membranes purified from rabbit intestine was studied by means of a microfiltration procedure. 2. Binding activity from ferrous ascorbate chelates was 100-fold greater than from ferric chelates of citrate and nitrilotriacetate. Dual-label experiments indicated dissociation of iron complexes before binding to the membranes. 3. Binding was inhibited at low incubation temperatures and was optimal at neutral pH. 4. Binding activity was reduced in ileal preparations when compared with membranes prepared from proximal intestine. 5. Initial binding velocity followed saturation kinetics over the range 45-450 microM-iron: it was weakly inhibited in the presence of excess Co2+ and V3+. 6. The data provide additional evidence for high-affinity iron-binding sites on the intestinal microvillus membrane and indicate properties that may reflect the functional significance of the binding step in the absorption pathway for iron.
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An in vitro method for measuring initial rates of iron uptake by mucosal biopsies of human duodenum was used to study control mechanisms for iron absorption. Within the physiological range of intraluminal concentrations (18--450 mumol/l) iron influx has many features of active, carrier-mediated transport. In biopsies form six patients with iron deficiency anaemia, uptake rates were increased 2--3-fold at the higher concentrations, when compared with normal controls (P less than 0.01) and overall were related inversely to serum transferrin saturation. Uptake was examined in four anaemic patients before and after therapy: the enhanced uptake fell to normal after repletion with iron, but was not reduced in two patients treated initially by red cell transfusion alone. Total mucosal iron in the anaemic patients was significantly lower at 58+/-7 nmol/mg protein, compared with 129+/-25 nmol/mg in normal subjects (P less than 0.05). In the serial studies, iron therapy for 6 weeks corrected the anaemia but did not restore mucosal iron levels to normal, even though uptake had fallen to control values. The experiments indicate that iron deficiency reversibly induces brush border iron carriers, and suggest that in man initial entry into the enterocyte rather than cellular retention of iron is a major regulatory step in the control of iron absorption.
Initial rates of iron uptake into fresh specimens of human duodenal mucosa incubated with radio-iron in vitro were examined over a range of physiological iron concentrations. In normal control subjects and patients with iron overload, mucosal iron uptake showed saturation kinetics, thus providing evidence for a carrier-mediated transport process. In five patients with idiopathic haemochromatosis uptake at the lower concentrations of iron was increased up to 80 per cent when compared with 12 control subjects. Uptake at the highest medium iron concentration was not increased. These changes were related to a significant reduction in the apparent affinity constant (Kt) for iron transport and were independent of iron stores. In contrast, the uptake rates in six patients with secondary haemochromatosis were not different from controls except in a subgroup with severe iron-loading anaemias where uptake was modestly enhanced at the higher concentrations only. The studies point to a disorder of mucosal iron transport in idiopathic haemochromatosis which appears to be a primary abnormality rather than a consequence of the iron overload.
The ability of a range of homologous transferrin-like proteins to donate iron to pieces of human duodenal mucosa, was examined with an in vitro incubation technique. In contrast to serum transferrin and ovotransferrin, only lactotransferrin was able to yield its iron to intestinal tissue, but in an autologous system this protein was unable to donate iron to human reticulocyte preparations. Studies with 125I-labelled lactotransferrin and lactotransferrin dual-labelled with 59Fe and 125I, indicated that the intact protein is excluded from entry into the enterocytes. The experiments suggest that iron may be transported across the brush border after delivery to specific protein binding sites at the cell surface.
1. A method for determining initial rates of unidirectional radio-Fe uptake from a ferric chelate of nitrilotriacetic acid by human duodenal biopsy specimens in vitro has been devised. [57Co]cyanocobalamin was used as an extracellular fluid marker, and was shown to give results in close agreement with other markers. 2. Uptake was linear for up to 20 min and exhibited saturation kinetics over the concentration range 18--450 mumole/1. 3. In the presence of 2:4 dinitrophenol and fluoride, uptake was reduced by approxi-50%, indicating dependence on metabolic energy. 4. Uptake of Fe was markedly diminished at reduced incubation temperatures, demonstrating a high activation energy for the uptake process. 5. Many of the criteria for the demonstration that the initial uptake of Fe depends on an active transport mechanism have been fulfilled. 6. The apparent distribution volume of 14C-labelled nitrilotriacetate chelate did not exceed the extracellular fluid space, suggesting that Fe is transferred to specific receptors on the enterocyte. The findings are discussed in relation to the possibility that uptake may be a rate-controlling step for the regulation of net intestinal absorption of Fe in man.