Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse.

Iron is essential for many cellular functions; consequently, disturbances of iron homeostasis, leading to either iron deficiency or iron overload, can have significant clinical consequences. Despite the clinical prevalence of these disorders, the mechanism by which dietary iron is absorbed into the body is poorly understood. We have identified a key component in intestinal iron transport by study of the sex-linked anaemia (sla) mouse, which has a block in intestinal iron transport. Mice carrying the sla mutation develop moderate to severe microcytic hypochromic anaemia. Although these mice take up iron from the intestinal lumen into mature epithelial cells normally, the subsequent exit of iron into the circulation is diminished. As a result, iron accumulates in enterocytes and is lost during turnover of the intestinal epithelium. Biochemical studies have failed to identify the underlying difference between sla and normal mice, therefore, we used a genetic approach to identify the gene mutant in sla mice. We describe here a novel gene, Heph, encoding a transmembrane-bound ceruloplasmin homologue that is mutant in the sla mouse and highly expressed in intestine. We suggest that the hephaestin protein is a multicopper ferroxidase necessary for iron egress from intestinal enterocytes into the circulation and that it is an important link between copper and iron metabolism in mammals.

Alpha heating of indirect-drive layered implosions on the National Ignition Facility.

In order to understand how close current layered implosions in indirect-drive inertial confinement fusion are to ignition, it is necessary to measure the level of alpha heating present. To this end, pairs of experiments were performed that consisted of a low-yield tritium-hydrogen-deuterium (THD) layered implosion and a high-yield deuterium-tritium (DT) layered implosion to validate experimentally current simulation-based methods of determining yield amplification. The THD capsules were designed to reduce simultaneously DT neutron yield (alpha heating) and maintain hydrodynamic similarity with the higher yield DT capsules. The ratio of the yields measured in these experiments then allowed the alpha heating level of the DT layered implosions to be determined. The level of alpha heating inferred is consistent with fits to simulations expressed in terms of experimentally measurable quantities and enables us to infer the level of alpha heating in recent high-performing implosions.

Role of lung iron in determining the bacterial and host struggle in cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal genetic disorder in Caucasian populations. It is a multiorgan system disease that affects the lungs, gastrointestinal tract, liver, and pancreas. The majority of morbidity and mortality in CF relates to chronic airway infection with a variety of bacterial species, commencing in very early infancy, which results in lung destruction and ultimately organ failure (41, 43). This review focuses on iron homeostasis in the CF lung and its role in determining the success and chronicity of Pseudomonas aeruginosa infection. There have been previous excellent reviews regarding iron metabolism in the lower respiratory tract and mechanisms of P. aeruginosa iron acquisition, and we direct readers to these articles for further background reading (31, 53, 58, 77, 96). In this review, we have brought the "two sides of the coin" together to provide a holistic overview of the relationship between host and bacterial iron homeostasis and put this information into the context of current understanding on infection in the CF lung.

Incorporation of chylomicron fatty acids into the developing rat brain.

The developing brain obtains polyunsaturated fatty acids from the circulation, but the mechanism and route of delivery of these fatty acids are undetermined. 14C-labeled chylomicrons were prepared by duodenal infusion of [1-14C]16:0, [1-14C]18:2(n-6), [1-14C]18:3(n-3), or [1-14C]22:6(n-3) into adult donor rats, and were individually injected into hepatectomized 2-wk-old suckling rats. After minor correction for trapped blood in the brain, the incorporation of chylomicron fatty acids after 30 min was nearly half that of a co-injected free fatty acid reference. [1-14C]22:6(n-3)-labeled chylomicrons showed an average 65% greater incorporation than chylomicrons prepared from the other fatty acids. This apparent selectivity may have been partly due to lower oxidation of 22:6(n-3) in the brain compared to the other fatty acids tested, based on recovered water-soluble oxidation products. The bulk of the radioactivity in the brain was found in phospholipid and triacylglycerol, except that animals injected with [1-14C]22:6(n-3) chylomicrons showed considerable incorporation also into the fatty acid fraction instead of triacylglycerol. These data show that chylomicrons may be an important source of fatty acids for the developing rat brain.

The fission yeast ferric reductase gene frp1+ is required for ferric iron uptake and encodes a protein that is homologous to the gp91-phox subunit of the human NADPH phagocyte oxidoreductase.

We have identified a cell surface ferric reductase activity in the fission yeast Schizosaccharomyces pombe. A mutant strain deficient in this activity was also deficient in ferric iron uptake, while ferrous iron uptake was not impaired. Therefore, reduction is a required step in cellular ferric iron acquisition. We have cloned frp1+, the wild-type allele of the mutant gene. frp1+ mRNA levels were repressed by iron addition to the growth medium. Fusion of 138 nucleotides of frp1+ promoter sequences to a reporter gene, the bacterial chloramphenicol acetyltransferase gene, conferred iron-dependent regulation upon the latter when introduced into S. pombe. The predicted amino acid sequence of the frp1+ gene exhibits hydrophobic regions compatible with transmembrane domains. It shows similarity to the Saccharomyces cerevisiae FRE1 gene product and the gp91-phox protein, a component of the human NADPH phagocyte oxidoreductase that is deficient in X-linked chronic granulomatous disease.

Modulation of iron transport proteins in human colorectal carcinogenesis.

BACKGROUND AND AIMS: Total body iron and high dietary iron intake are risk factors for colorectal cancer. To date there is no comprehensive characterisation of iron transport proteins in progression to colorectal carcinoma. In this study, we examined expression of iron import (duodenal cytochrome b (DCYTB), divalent metal transporter 1 (DMT1), and transferrin receptor 1 (TfR1)) and export (hephaestin (HEPH) and ferroportin (FPN)) proteins in colorectal carcinoma. METHODS: Perl's staining was used to examine colonocyte iron content. Real time polymerase chain reaction (PCR) and western blotting were used to examine mRNA and protein levels of the molecules of interest in 11 human colorectal cancers. Semiquantitative immunohistochemistry was used to verify protein levels and information on cellular localisation. The effect of iron loading on E-cadherin expression in SW480 and Caco-2 cell lines was examined by promoter assays, real time PCR and western blotting. RESULTS: Perl's staining showed increased iron in colorectal cancers, and there was a corresponding overexpression of components of the intracellular iron import machinery (DCYTB, DMT1, and TfR1). The iron exporter FPN was also overexpressed, but its intracellular location, combined with reduced HEPH levels, suggests reduced iron efflux in the majority of colorectal cancers examined. Loss of HEPH and FPN expression was associated with more advanced disease. Iron loading Caco-2 and SW480 cells caused cellular proliferation and E-cadherin repression. CONCLUSIONS: Progression to colorectal cancer is associated with increased expression in iron import proteins and a block in iron export due to decreased expression and aberrant localisation of HEPH and FPN, respectively. This results in increased intracellular iron which may induce proliferation and repress cell adhesion.

Improved purification of the human placental transferrin receptor and a novel immunoradiometric assay for receptor protein.

A simplified method for the purification of human placental transferrin receptor is described. The procedure involves chromatography of a detergent extract of placenta on immobilized iron-loaded transferrin. Knowledge of the physiology of the interaction between transferrin and its receptor is applied to enable bound receptor to be eluted under mild conditions and essentially free of containing transferrin. Purified transferrin receptor and a monoclonal antibody to the receptor were used to develop a novel immunoradiometric assay for the receptor in which the monoclonal antibody is the radiolabelled species. A competition between two populations of receptor, one immobilized on a particulate support and the other in solution, provides the basis for the assay. Using this assay we have measured transferrin receptor levels in placental and hepatic tissue and in three cell lines during both the logarithmic and stationary phases of cell growth.

Reversibility of the effects of dietary fish oil on the fatty acid composition of the brain and retina of growing chicks.

Dietary fish oil increases levels of (n-3) fatty acids in the brain and retina of younger animals but has less effect in adults. The duration of the effects of fish oil in young animals, as well as the extent of reversibility of the effects, are unknown. Laying hens were fed either a fish oil diet or a soybean oil-based control diet. Resulting chicks were assigned to three diet groups: chicks from fish oil and soybean oil hens were continued on fish oil and soybean oil diets, respectively, for 0, 3, 6, or 9 weeks, and additional chicks from the fish oil hens were fed the fish oil diet for 0, 3, or 6 weeks and then reversed to the soybean oil diet for a period of 3 weeks. The fatty acid composition of the brain, retina, liver, and serum of the reversal chicks was compared with chicks fed the fish oil diet only or the soybean oil diet only. Brain levels of docosahexaenoic acid (22:6(n-3)) decreased substantially when reversal from the fish oil diet to the control diet was begun at hatching, but did not decrease when reversal was begun at later times. Other (n-3) fatty acids in the brain, docosapentaenoic acid (22:5(n-3)) and eicosapentaenoic acid (20:5(n-3)), decreased substantially at all ages, and to a greater extent than 22:6(n-3). Brain arachidonic acid (20:4(n-6)), which was low in fish oil chicks, rose to control after reversal at hatching, but recovered only partially when reversal was begun at later times. A similar patterns was observed in the retina. Serum and liver (n-3) fatty acids fell to control in all reversal chicks, and (n-6) fatty acids increased to control, except in chicks reversed at 6 weeks. This study demonstrates that by 3 weeks of age the chick brain strongly resists diet-induced lowering of high levels of 22:6(n-3).

Tissue-specific changes in iron metabolism genes in mice following phenylhydrazine-induced haemolysis.

Iron metabolism in animals is altered by haemolytic anaemia induced by phenylhydrazine (PHZ). In common with a number of other modulators of iron metabolism, the mode and the mechanisms of this response are yet to be determined. However, recent studies have shown increased expression of the ferrous transporter DMT1 in the duodenum and other tissues of mice administered PHZ. We examined the expression of the ferric reductase Dcytb, DMT1 and some other genes involved in Fe metabolism in tissues of mice dosed with PHZ. The expression of iron-related genes in the duodenum, liver, and spleen of the mice were evaluated using Northern blot analyses, RT-PCR and immunocytochemistry. Dcytb, and DMT1 mRNA and protein increased markedly in the duodenum of mice given PHZ. The efflux protein Ireg1 also increased in the duodenum of the treated mice. These changes correlated with a decrease in hepatic hepcidin expression. Dcytb, DMT1, Ireg1 and transferrin receptor 1 mRNA expression in the spleen and liver of mice treated with PHZ responded to the enhanced iron demand associated with the resulting stimulation of erythropoiesis. Enhanced iron absorption observed in PHZ-treated animals is facilitated by the up-regulation of the genes involved in iron transport and recycling. The probable association of the erythroid and the store regulators of iron homeostasis and absorption in the mice is discussed.

The effects of insulin and potassium on infarcting canine tissue: an in vitro study.

The electrophysiologic effects of insulin (40 mU/ml) and elevated potassium (4 to 6 mM) on glucose-superfused normal and infarcting tissue from 24 hour coronary artery ligated canine myocardium were studied. With standard intracellular microelectrode techniques, it was observed that insulin infusion for 30 minutes produced an increase in resting membrane potential and a prolongation of action potential duration. In the normal myocardium, the hyperpolarization and the repolarization delay were minimal, but in infarcting tissue with depressed electrophysiologic function, resting membrane potential and action potential duration were significantly improved. This was particularly evident in the presence of an increased potassium concentration (6 mM) when insulin hyperpolarized infarcting cells (n = 8) from 73 +/- 6 to 85 +/- 7 mV (p less than 0.01). In the same studies, action potential amplitudes were increased from 75 +/- 7 to 95 +/- 11 mV (p less than 0.01). In addition, action potential durations at 40 and 80% repolarization were extended from 64 +/- 25 and 141 +/- 46 ms to 132 +/- 34 and 198 +/- 27 ms, respectively (p less than 0.01). Thus, these data are in accord with the reduced ST segment elevation observed in patients treated with glucose-insulin-potassium and support the use of this intervention in the management of acute infarction.

Intramyocardial current flow in acute coronary occlusion in the canine heart.

Data from numerous experimental infarction studies indicate that rapid myocardial cell depolarization following ischemia causes the flow of injury currents. These currents were measured in the canine myocardium by monitoring voltage gradients across infarct boundaries using silver chloride plunge electrodes, followed by placement of a 100 omega resistor between the electrodes and again measuring the voltage gradients. Current flow was calculated from these measurements with the following results: 1) TQ currents developed within 15 seconds after occlusion and persisted for 120 to 150 minutes, often attaining a magnitude of 1 microA. 2) ST currents also developed within 15 seconds and attained 2 to 3 microA within 15 to 30 minutes, then usually subsided to some degree. 3) T currents were biphasic and attained 2 to 5 microA. Initially, current flowed from normal to ischemic myocardium but usually reversed within 30 minutes after occlusion. 4) The current flow was often disproportionate to the voltage gradient between 120 and 180 minutes after occlusion, possibly indicating electrical uncoupling of the infarcting cells from normal cells. These data indicate that intramyocardial current flow develops early after acute coronary occlusion. These currents may be sufficient to induce reexcitation.

Normalization of abnormal T waves in ischemia.

Inverted T waves due to coronary artery disease and previous myocardial infarction were observed to revert ot normal, upright position during ischemia in 38 patients. The normalization of inverted T waves was seen on the electroencephalograms of 19 patients during spontaneously occurring angina pectoris and of 11 patients when ischemia was provoked by treadmill exercise; for 8 patients, normalization occurred during the administration of isoproterenol hydrochloride and during the consequent episode of angina pectoris. The mechanism for normalization may be the algebraic sum of the extent of ST segment elevation and the amplitude of the T waves of acute ischemia plus the extent of preexisting ST segment depression and the degree of T wave inversion, to result in isoelectric ST segment and upright T wave. As with myocardial infarction, reciprocal changes may also be recorded. However, the reciprocal nature may be masked since either acute ST segment elevation of T wave inversion, or both, may not be recorded in the leads reflecting the ischemic area because of normalization.

Jagged1 (JAG1) mutation detection in an Australian Alagille syndrome population.

Alagille syndrome (AGS) is an autosomal dominant disorder characterized by abnormal development of the liver, heart, skeleton, eye, and face. Mutations in the Jagged1 gene (JAG1) have been found to result in the AGS phenotype and both protein truncating mutations and missense mutations have been identified. Using single stranded conformational polymorphism analysis we have screened 22 AGS affected individuals from 19 families for mutations within Jagged1. Twelve distinct Jagged1 mutations were identified in 15 (68.2%) of the 22 AGS cases, seven of which are novel. The mutations include three small deletions (25%), two small insertions (16.6%), three missense mutations (25%), two nonsense mutations (16.6%), and two splice-site mutations (16.6%). These mutations are spread across the entire coding sequence of the gene and most are localized to highly conserved motifs of the protein predicted to be important for Jagged1 function. One-half of the mutations found in this study are located between exons 9 and 12, a region constituting only 12% of the coding sequence. A splice-donor site mutation in intron 11 was shown to cause aberrant splicing of Jagged1 mRNA, consequently terminating translation prematurely in exon 12. The results of this study are consistent with the proposal that either haploinsufficiency for wild type Jagged1 and/or dominant negative effects produced by mutated Jagged1 are responsible for the AGS phenotype.

Transacylase activity of lactating bovine mammary fatty acid synthase.

An assay for the transacylation reaction catalyzed by fatty acid synthase was developed which does not require model substrates or labelled acyl-derivatives of CoA. It involves the transfer of the acyl group from unlabelled CoA to [3H]CoA. This assay shows the occurrence of transacylation at a relatively high rate with a variety of substrates that the enzyme is able to utilize. The activity is unaffected by dissociation of the enzyme or modification by iodoacetamide or 2-chloroacetyl-CoA.

Nuclear pre-mRNA splicing in Saccharomyces cerevisiae.

While there are some differences in the nuclear pre-mRNA splicing machineries of Saccharomyces cerevisiae and higher eukaryotic cells, it is apparent that the fundamental mechanism of this reaction is highly conserved. S. cerevisiae is, therefore, an attractive organism for the study of splicing, since it is amenable to classical and molecular genetics as well as traditional biochemical methods. Here we present an outline of some of the advances which have resulted from this powerful combination of approaches.

Accretion of n-3 fatty acids in the brain and retina of chicks fed a low-linolenic acid diet supplemented with docosahexaenoic acid.

Diets low in alpha-linolenic acid may not support normal brain accretion of n-3 fatty acids. An n-3 fatty acid-deficient diet was fed to laying hens and the resulting deficient chicks were fed a low-linolenic acid diet based on corn oil, or the same diet supplemented with docosahexaenoic acid. Control chicks from soybean oil-fed hens were fed a soybean oil-based diet. The fatty acid composition of the chick brains, retinas, livers, and serum was determined after 0-4 wk. The corn oil diet did not reverse the deficiency but the combination of corn oil and docosahexaenoic acid rapidly restored brain and retinal concentrations of docosahexaenoic acid. Supplemented chicks, however, showed a slight lowering of arachidonic acid in the brain and serum. This study demonstrates that a low-linolenic acid diet without docosahexaenoic acid fails to support accretion of n-3 fatty acids in the nervous tissue of chicks.

Cheek cell phospholipids in human infants: a marker of docosahexaenoic and arachidonic acids in the diet, plasma, and red blood cells.

BACKGROUND: Assessment of essential fatty acid status requires collection of blood or adipose tissue samples. However, these invasive techniques cannot always be used in studies involving infants, young children, or subjects from whom it is difficult to obtain blood. A body tissue that is easy to access is the buccal mucosa (cheek cells). OBJECTIVE: The objective was to investigate the degree to which fatty acids of cheek cells reflect the fatty acid content of plasma, red blood cells, and the diet. DESIGN: Thirty-one infants aged 12 mo were enrolled. Five infants were fed human milk and 26 infants received formulas that provided a wide range of arachidonic acid and docosahexaenoic acid (DHA) intakes. Cheek cells were collected on a small piece of gauze by gently swabbing the inside of the cheek 3 times. Lipids were extracted from the gauze and the phospholipid fatty acid content of the cheek cells was determined. RESULTS: Cheek cell DHA and arachidonic acid in phospholipids were significantly correlated with DHA and arachidonic acid in plasma [r = 0.61 (P < 0.001) and r = 0.37 (P <0.05), respectively], red blood cells [r = 0.58 (P < 0.001) and r = 0.37 (P < 0.05), respectively], and the diet [r = 0.65 (P < 0.001) and r = 0. 51 (P < 0.01), respectively]. CONCLUSIONS: Given these correlations and the ease and noninvasive nature of this technique, cheek cell fatty acids may serve as a marker of the essential fatty acid content, especially of DHA and arachidonic acid, in plasma, red blood cells, and the diet.

The significance of exercise-induced Q waves.

The significance of transient exercise-induced Q waves present during treadmill testing was prospectively evaluated and correlated with the findings at cardiac catheterization. Exercise-induced Q waves were present in 14 of 560 patients (2.5 per cent) undergoing treadmill exercise testing. Thirteen patients had Q waves in leads V1 through V3 and one patient had Q waves in leads II, III and aVF. Ten patients underwent cardiac catheterization; six did not have coronary artery disease as determined by angiography. Two patients had a documented anterior myocardial infarction and did not undergo cardiac catheterization. Exercise myocardial perfusion imaging was performed in 10 patients. Six patients without coronary artery disease had no evidence of exercise perfusion defects. In the four patients with coronary artery disease, three patients had an abnormal resting perfusion study without change with exercise and one patient had a new exercise myocardial perfusion defect. We conclude that exercise-induced transient Q waves are not diagnostic of underlying coronary disease. In patients without coronary artery disease the mechanism of exercise-induced Q waves is as yet unclear. In patients with coronary artery disease, the mechanism may also be undefined or secondary to myocardial ischemia.

Proton NMR lipid profile of Leishmania donovani promastigotes.

The proton nuclear magnetic resonance (NMR) lipid profile of Leishmania donovani was obtained in the one-dimensional and two-dimensional modes. Partial assignments of lipid classes and individual lipids were obtained purely from the proton NMR spectrum of the mixture. A more complete assignment and quantitative analysis was achieved by prior separation of the lipids by high pressure liquid chromatography (HPLC) followed by proton NMR analysis of the fractions. This work showed that proton NMR spectroscopy could facilitate lipid analysis and classification of various parasitic protozoa and serve as a basis for rapid studies of comparative lipid metabolism in parasites.

Effect of dietary N-3 fatty acids upon the phospholipid molecular species of the monkey retina.

PURPOSE: To characterize the molecular species composition of ethanolamine glycerophospholipids (EGP) in the primate retina and to examine the effects of different dietary fats, the authors fed rhesus monkeys diets containing widely ranging amounts of n-3 fatty acids. METHODS: From birth, infant monkeys were fed either a control soybean oil diet, containing 8% of total fatty acids as 18:3 (n-3), or a safflower oil-based n-3 fatty acid deficient diet containing < 0.4% 18:3 (n-3). A subset of the n-3 deficient group was later repleted with 1.6% ethyl docosahexaenoate, 22:6 (n-3), starting at 10 months of age. Tissues were taken from all monkeys upon termination at 21 to 51 months of age. The diacyl, alkenylacyl, and alkylacyl EGPs were quantitated by high-pressure liquid chromatography (HPLC). RESULTS: Twenty-eight molecular species were identified in the retina of control monkeys. Ether phospholipids comprised 36% of the retinal ethanolamine glycerophospholipids. Species containing polyunsaturated fatty acids in both the sn-1 and sn-2 positions (dipolyenes) were present only in the diacyl subclass and comprised 16% of the total species. Species having n-3 fatty acids in the sn-2 position contributed 59%, 36%, and 70% of total species in the diacyl, alkenylacyl, and alkylacyl subclasses, respectively. In the molecular species of the n-3 fatty acid deficient monkeys, the major change was the loss of most of the 18:0-22:6(n-3) species and its partial replacement with 18:0-22:5(n-6). In contrast, the species 18:1-22:6(n-3) decreased only slightly, from 6.2% to 4.8% of total diacyl species. Although the total concentration of dipolyenes (15% to 20% of the total species) was not affected by diet, their fatty acid compositions were changed drastically. The dipolyene species 22:6(n-3)-22:6(n-3) nearly disappeared in the n-3 deficient monkeys. Concomitantly, two new species, 22:5(n-6)-22:6(n-3) and 22:5(n-6)-22:5(n-6), appeared at 2.6% and 2.0%, respectively. Deficient monkeys given the ethyl ester of 22:6(n-3) in the diet recovered to a near-normal molecular species composition, except in the ether lipids, in which 16:0-20:4 remained low. CONCLUSION: Diets of differing n-3 fatty acid content had profound qualitative and quantitative effects on the molecular species of retinal phospholipids, and the replacement of 22:6(n-3) by 22:5(n-6) in the retinas of n-3 deficient monkeys was asymmetric and functionally incomplete.