Epicardial adipose tissue volume in patients with coronary artery disease or non-ischaemic dilated cardiomyopathy: evaluation with cardiac magnetic resonance imaging.

AIM: To compare the amount of epicardial adipose tissue (EAT) in patients with coronary artery disease (CAD) or non-ischaemic dilated cardiomyopathy (NIDCM) with that in patients with negative cardiac magnetic resonance imaging (CMR). MATERIALS AND METHODS: One hundred and fifty patients (median age 57 years, interquartile range [IQR] 46-66 years) who underwent CMR were evaluated retrospectively: 50 with CAD, 50 with NIDCM, and 50 with negative CMR. For each patient, the EAT mass index (EATMI) to body surface area, end-diastolic volume index (EDVI), end-systolic volume index (ESVI), stroke volume (SV), ejection fraction (EF) for both ventricles, and left ventricle (LV) mass index were estimated. Intra and inter-reader reproducibility was tested in a random subset of 30 patients, 10 for each group. Mann-Whitney U test, Kruskal-Wallis test, Spearman's correlation, and Bland-Altman statistics were used. RESULTS: The EATMI in CAD patients (median 15.7 g/m2, IQR 8.3-25.7) or in NIDCM patients (15.9 g/m2, 11.5-18.1) was significantly higher than that in negative CMR patients (9.1 g/m2, 6-12; p<0.001 both). No significant difference was found between CAD and NIDCM patients (p=1.000). A correlation between EATMI and LV mass index was found in NIDCM patients (r=0.455, p=0.002). Intra- and inter-reader reproducibility were up to 80% and 72%, respectively. CONCLUSION: Patients with NIDCM or CAD exhibited an increased EATMI in comparison to negative CMR patients. CMR can be used to estimate EAT with good reproducibility.

Providing straw to allow exploratory behaviour in a pig experimental system does not modify putative indicators of positive welfare: peripheral oxytocin and serotonin.

Numerous studies have shown that providing straw to pigs can reduce undesirable behaviours such as aggression, tail biting and stereotypy. The measurement of various neuromodulators can be helpful in assessing the development of positive behaviours and overall animal welfare. The oxytocin release is frequently linked to positive emotions and positive welfare. It has been suggested that oxytocin modulates the serotoninergic system. This study aims to investigate the potential effect of straw provision in pigs on peripheral levels of oxytocin and serotonin. In total, 18 mini-pigs were involved in an exploratory study conducted in two parallel groups, Enriched (n=10) and Control (n=8) groups. Pigs were divided by group and housed in pens of two individuals. Straw was provided continuously only in Enriched group and renewed each day for 2 weeks. Two blood samples were drawn from each animal 5 to 10 min before providing the straw, and 15 min after providing straw, during the 1st week, to analyse peripheral changes in oxytocin and serotonin before and after straw provision, and determine the existence of a putative short-term effect. The same procedure was carried out for Control group, without straw provision. Long-term effects of straw provision were also examined using blood samples drawn at the same hour from each animal in the 2nd and 3rd weeks. During this time, animals had the permanent possibility to explore the straw in Enriched group but not in Control group. At the end of each week, one animal-keeper completed two visual analogue scales for each mini-pig regarding the difficulty/ease to work with and handle it and its trust in humans. Results showed peripheral oxytocin increases in both groups after 2 weeks (P=0.02). Results did not demonstrate any effect of providing straw to allow exploratory behaviour on peripheral serotonin. Other results were not significant. This preliminary study explored the relationship between peripheral oxytocin and serotonin and the presence of straw that allow pigs to perform exploratory behaviour, suggesting that there was no relationship between them. Some future studies may include crossing oxytocin and serotonin with other parameters, such as behavioural measures, to obtain more information about the true state of the animal and any possible relationship with pig welfare.

Skin rash and arthritis a simplified appraisal of less common associations.

Skin and joint manifestations are part of the clinical spectrum of many disorders. Well-known associations include psoriatic arthritis and arthritis associated with autoimmune connective tissue diseases. This review focuses on less common associations where skin lesions can provide easily accessible and valuable diagnostic clues, and directly lead to the specific diagnosis or limit the list of possibilities. This may also affect health care resources as diagnostic tests are often low-specific, highly expensive and poorly available. This group of diseases can be divided into two subsets, based on the presence/absence of fever, and then further classified according to elementary skin lesions (macular, urticarial, maculo-papular, vesico-bullous, pustular, petechial and nodular). In most instances joint involvement occurs as peripheral migrating polyarthritis. Erythematosus macular or urticarial rashes occur in most febrile disorders such as monogenic autoinflammatory syndromes, Schnitzler's syndrome, Still's disease and rheumatic fever and afebrile diseases as urticarial vasculitis. Pustular rash may be observed in chronic recurrent multifocal osteomyelitis (CRMO) and pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) syndrome (both febrile) as well as in Behcet's disease and Synovitis, acne, pustulosis, hyperostosis and osteitis syndrome (both non-febrile). Papular lesions are typical of secondary syphilis, sarcoidosis, interstitial granulomatous dermatitis, papular petechial of cutaneous small-vessel vasculitis and nodular lesions of polyarteritis nodosa and multicentric reticulohistiocytosis all of which are afebrile. Differential diagnosis includes infections and drug reactions which may mimic several of these conditions. To biopsy the right skin lesion at the right time it is essential to obtain relevant histological information.

Long-lasting neuroprotection and neurological improvement in stroke models with new, potent and brain permeable inhibitors of poly(ADP-ribose) polymerase.

BACKGROUND AND PURPOSES: Thienyl-isoquinolone (TIQ-A) is a relatively potent PARP inhibitor able to reduce post-ischaemic neuronal death in vitro. Here we have studied, in different stroke models in vivo, the neuroprotective properties of DAMTIQ and HYDAMTIQ, two TIQ-A derivatives able to reach the brain and to inhibit PARP-1 and PARP-2. EXPERIMENTAL APPROACH: Studies were carried out in (i) transient (2 h) middle cerebral artery occlusion (tMCAO), (ii) permanent MCAO (pMCAO) and (iii) electrocoagulation of the distal portion of MCA in conjunction with transient (90 min) bilateral carotid occlusion (focal cortical ischaemia). KEY RESULTS: In male rats with tMCAO, HYDAMTIQ (0.1-10 mg·kg(-1)) injected i.p. three times, starting 4 h after MCAO, reduced infarct volumes by up to 70%, reduced the loss of body weight by up to 60% and attenuated the neurological impairment by up to 40%. In age-matched female rats, HYDAMTIQ also reduced brain damage. Protection, however, was less pronounced than in the male rats. In animals with pMCAO, HYDAMTIQ administered 30 min after MCAO reduced infarct volumes by approximately 40%. In animals with focal cortical ischaemia, HYDAMTIQ treatment decreased post-ischaemic accumulation of PAR (the product of PARP activity) and the presence of OX42-positive inflammatory cells in the ischaemic cortex. It also reduced sensorimotor deficits for up to 90 days after MCAO. CONCLUSION AND IMPLICATIONS: Our results show that HYDAMTIQ is a potent PARP inhibitor that conferred robust neuroprotection and long-lasting improvement of post-stroke neurological deficits.

Cellular iron status influences the functional relationship between microglia and oligodendrocytes.

Previously, we have reported that there is a spatiotemporal relationship between iron accumulation in microglia and oligodendrocytes during normal development and in remyelination following injury. This in vivo observation has prompted us to develop a cell culture model to test the relationship between iron status of microglia and survival of oligodendrocytes. We found that conditioned media from iron-loaded microglia increases the survival of oligodendrocytes; but conditioned media from iron loaded activated microglia is toxic to oligodendrocytes. In the trophic condition, one of the proteins released by iron-loaded microglia is H-ferritin, and transfecting the microglia with siRNA for H-ferritin blocks the trophic response on oligodendrocytes. Lipopolysaccharide (LPS) activation decreases the amount of H-ferritin that is released from microglia and increases the release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1. LPS activation of iron-enriched microglia results in the activation of NF-kB and greater release of cytokines when compared with that of control microglia; whereas treating microglia with an iron chelator is associated with less NF-kB activation and less release of cytokines. These results indicate that microglia play an important role in iron homoeostasis and that their iron status can influence how microglia influence growth and survival of oligodendrocytes. The results further indicate that ferritin, released by microglia, is a significant source of iron for oligodendrocytes.

Low serum tryptophan levels, reduced macrophage IDO activity and high frequency of psychopathology in HCV patients.

Indoleamine 2,3-dioxygenase (IDO), a key enzyme of tryptophan (TRP) metabolism, is induced in various tissues of patients with bacterial and viral infection or with neoplastic diseases. This induction is considered the main cause of the decreased serum TRP levels, the reduced brain serotonin synthesis and the occurrence of psychopathological disorders often detected in patients with chronic infections or different forms of cancer. We studied 89 subjects including: (a) 39 patients with chronic hepatitis C virus (HCV) infection and mild liver damage (b) 40 healthy controls, and (c) 10 patients with chronic hepatitis B virus (HBV) infection. We measured serum TRP and kynurenine levels and IDO activity in macrophages. Furthermore, each patient had an accurate psychopathological evaluation. HCV-infected patients had lower (-28%) serum TRP concentrations than healthy volunteers or HBV-infected patients with comparable liver damage. Depression and anxiety symptoms were particularly common in HCV patients. Unexpectedly, serum kynurenine levels and IDO activity in cultured macrophages (under both basal or stimulated conditions) were lower in HCV patients than in controls. Our study shows that HCV patients have reduced serum TRP levels and confirms that they frequently suffer from anxiety and depression-related symptoms. The reduced IDO activity found in the macrophages of these patients suggest that HCV infection may hamper macrophage functions.

Kynurenine 3-mono-oxygenase inhibitors reduce glutamate concentration in the extracellular spaces of the basal ganglia but not in those of the cortex or hippocampus.

Kynurenine 3-mono-oxygenase (KMO, kynurenine hydroxylase) inhibitors increase brain kynurenic acid (KYNA) synthesis and cause pharmacological actions possibly mediated by a reduced activity of excitatory synapses. We used in vivo microdialysis and passive avoidance to study the effects of local KYNA or systemic KMO inhibitor administration on glutamate (GLU) neurotransmission. Local application of KYNA (30-100 nM) through reverse microdialysis reduced GLU content in caudate and cortical dialysates by 75 and 55%, respectively. No changes were found in the hippocampus. Systemic administration of Ro 61-8048 (4-40 mg/kg) increased KYNA levels in dialysates obtained from the cortex (from 10.3 +/- 1.9 to 45.5 +/- 15 nM), caudate (from 2.4 +/- 0.8 to 9.5 +/- 0.9 nM) and hippocampus (from 7.7 +/- 1.7 to 19.2 +/- 3.5 nM). It also caused a parallel robust decrease in GLU levels in the dialysates collected from the caudate (from 2.2 +/- 0.5 to 0.63 +/- 0.05 microM) but not in those collected from the parietal cortex or the hippocampus. In a passive avoidance paradigm, the administration of the NMDA receptor antagonist MK-801 (0.1 mg/kg) reduced, while Ro 61-8048 (4-80 mg/kg) did not change the latency time of entering into the dark compartment on the recall trial. Our data show that KMO inhibitors increase brain KYNA synthesis and selectively reduce GLU extracellular concentration in the basal ganglia.

Assessment of ozone visible symptoms in the field: perspectives of quality control.

The second UN/ECE ICP-Forests Intercalibration Course on the Assessment of Ozone Injury on European Tree Species was carried out in August 2001 at Lattecaldo (Canton Ticino, CH) and Moggio (Lombardy, I). Forty-eight experts from several European countries participated in the exercises and assessed visible symptoms of ozone injury both in open-top chambers (OTC) (Lattecaldo) and under open field (Moggio) conditions. Evaluation of the results indicated a large variability among the teams and call for adequate training of the observers prior to symptom assessment for quality assurance purposes. Highest variability was found for the species developing unclear symptoms which could be confused with senescence processes; such species should not be used in the field. The authors provide suggestions to improve the reliability of the ozone injury assessment on forest plant species.

Acute cerebral focal ischaemia alters the adrenergic and NANC responses in the bisected rat vas deferens.

1. Disturbances of the autonomic nervous system are common in right hemisphere stroke patients, including a marked decline in male sexual functions. There is a lack of information on the influence of stroke on male secondary sex organs such as the vas deferens. 2. This study investigates the effect of right brain focal ischaemia on the adrenergic and purinergic responses in isolated epididymal and prostatic portions of rat vas deferens. 3. In both epididymal and prostatic portions the concentration-response curves to noradrenaline are flattened resulting in a reduction (up to 67 - 76%) of the maximum contractile response in the tissue from ischaemic rats compared to the controls. In the prostatic portion from ischaemic rats the concentration-response curve to alpha,beta-methylene ATP was also depressed. 4. The first purinergic and the second delayed adrenergic phase to single pulse was not modified by brain ischaemia. In contrast both phasic and tonic components of the electrically induced contractions by trains of stimuli at high frequencies (2 - 30 Hz) were significantly depressed in the epididymal and prostatic portions from ischaemic rats. 5. These results demonstrate an autonomic imbalance at the level of male sexual secondary organs which may contribute to sexual impairment after stroke.

Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia.

An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.

The expression of glutamate transporter GLT-1 in the rat cerebral cortex is down-regulated by the antipsychotic drug clozapine.

We show here that clozapine, a beneficial antipsychotic, down-regulates the expression of the glutamate transporter GLT-1 in the rat cerebral cortex, thereby reducing glutamate transport and raising extracellular glutamate levels. Clozapine treatment (25--35 mg kg(-1) day(-1) orally) reduced GLT-1 immunoreactivity in several brain regions after 3 weeks; this effect was most prominent after 9 weeks and most evident in the frontal cortex. GLT-1 protein levels were reduced in the cerebral cortex of treated rats compared with controls and were more severely affected in the anterior (71.9 +/- 4.5%) than in the posterior (53.2 +/- 15.4%) cortex. L-[(3)H]-glutamate uptake in Xenopus laevis oocytes injected with mRNA extracted from the anterior cerebral cortex of rats treated for 9 weeks was remarkably reduced (to 30.6 +/- 8.6%) as compared to controls. In addition, electrophysiological recordings from oocytes following application of glutamate revealed a strong reduction in glutamate uptake currents (46.3 +/- 10.2%) as compared to controls. Finally, clozapine treatment led to increases in both the mean basal (8.1 +/- 0.7 microM) and the KCl-evoked (28.7 +/- 7.7 microM) output of glutamate that were 3.1 and 3.5, respectively, higher than in control rats. These findings indicate that clozapine may potentiate glutamatergic synaptic transmission by regulating glutamate transport.

Presynaptic kynurenate-sensitive receptors inhibit glutamate release.

Kynurenic acid is a tryptophan metabolite provided with antagonist activity on ionotropic glutamate and alpha7 nicotinic acetylcholine receptors. We noticed that in rats with a dialysis probe placed in the head of their caudate nuclei, local administration of kynurenic acid (30-100 nM) significantly reduced glutamate output. Qualitatively and quantitatively similar effects were observed after systemic administration of kynurenine hydroxylase inhibitors, a procedure able to increase brain kynurenate concentrations. Interestingly, in microdialysis studies, methyllycaconitine (0.3-10 nM), a selective alpha7 nicotinic receptor antagonist, also reduced glutamate output. In isolated superfused striatal synaptosomes, kynurenic acid (100 nM), but not methyllycaconitine, inhibited the depolarization (KCl 12.5 mM)-induced release of transmitter or previously taken-up [3H]-D-aspartate. This inhibition was not modified by glycine, N-methyl-D-aspartate or subtype-selective kainate receptor agents, while CNQX or DNQX (10 microM), two AMPA and kainate receptor antagonists, reduced kynurenic acid effects. Low concentrations of kynurenic acid, however, did not modify [3H]-kainate (high and low affinity) or [3H]-AMPA binding to rat brain membranes. Finally, because metabotropic glutamate (mGlu) receptors modulate transmitter release in striatal preparations, we evaluated, with negative results, kynurenic acid (1-100 nM) effects in cells transfected with mGlu1, mGlu2, mGlu4 or mGlu5 receptors. In conclusion, our data show that kynurenate-induced inhibition of glutamate release is not mediated by glutamate receptors. Nicotinic acetylcholine receptors, however, may contribute to the inhibitory effects of kynurenate found in microdialysis studies, but not in those found in isolated synaptosomes.

Kynurenine 3-mono-oxygenase activity and neurotoxic kynurenine metabolites increase in the spinal cord of rats with experimental allergic encephalomyelitis.

Kynurenine 3-mono-oxygenase, one of the key enzymes of the "kynurenine pathway", catalyses the formation of 3-hydroxykynurenine and may direct the neo-synthesis of quinolinic and kynurenic acids. While 3-hydroxykynurenine and quinolinic acid have neurotoxic properties, kynurenic acid antagonizes excitotoxic neuronal death. Here we report that the expression and activity of kynurenine 3-mono-oxygenase significantly increased in the spinal cord of rats with experimental allergic encephalopathy, an experimental model of multiple sclerosis. As a consequence of this increase, the spinal cord content of 3-hydroxykynurenine and quinolinic acid reached neurotoxic levels. We also report that systemic administration of Ro 61-8048, a selective kynurenine 3-mono-oxygenase inhibitor, reduced the increase of both 3-hydroxykynurenine and quinolinic acid, and caused accumulation of kynurenic acid. In the brain and spinal cord of the controls, kynurenine 3-mono-oxygenase immunoreactivity was located in granules (probably mitochondria) present in the cytoplasm of both neurons and astroglial cells. In the spinal cord of rats with experimental allergic encephalopathy, however, cells with a very intense kynurenine 3-mono-oxygenase immunoreactivity, also able to express class II major histocompatibility complex and inducible nitric oxide synthase, were found in perivascular, subependymal and subpial locations. These cells (most probably macrophages) were responsible for the large increase in 3-hydroxykynurenine and quinolinic acid found in the spinal cords of affected animals. The results show that cells of the immune system are responsible for the increased formation of 3-hydroxykynurenine and quinolinic acid, two neurotoxic metabolites that accumulate in the central nervous system of rats with experimental allergic encephalomyelitis. They also demonstrate that selective kynurenine 3-mono-oxygenase inhibitors reduce the neo-synthesis of these toxins.

Expression, refolding, and ferritin-binding activity of the isolated VL-domain of monoclonal antibody F11.

Expression of the VL-domain of mouse monoclonal antibody F11 to human spleen ferritin in Escherichia coli cells is associated with the formation of insoluble protein aggregates (inclusion bodies). The aggregates were solubilized in the presence of guanidine hydrochloride and the recombinant VL-domain was purified by immobilized metal affinity chromatography (IMAC). Subsequent renaturation results in approximately 99% pure preparation with high yield. The VL-domain forms dimers at concentrations from 1 to 10 mg/ml. Monomeric form is detected only at protein concentrations below 0.5 mg/ml. Functional activity of the VL-domain was verified by two variants of ELISA. The affinity of the VL-domain ((0.2-1.2). 108 M(-1)) is similar to the affinity of the full-length parental antibody F11 because when the immobilized VL-domain was used, the binding constant of ferritin to the VL-domain was only 4-6-fold lower than that in the case of F11 antibody. In another ELISA system with immobilized ferritin, affinity was decreased 30-fold. The VL-domain of antibody F11 is the first example of the recombinant variable domain of the immunoglobulin light chain that preserves the antigen-binding activity in the absence of the partner VH-domain. The data indicate that the recombinant VL-domain can be used in construction of chimeric immunotoxins and other antigen-binding proteins in immunotherapy and in studies of correlations between folding, stability, and activity of immunoglobulins.

Antiferritin single-chain Fv fragment is a functional protein with properties of a partially structured state: comparison with the completely folded V(L) domain.

Differential scanning calorimetry and spectroscopic probes were applied to study folding and stability of the single-chain Fv fragment (scFv) of the anti-human ferritin antibody F11 and its isolated variable light-chain (V(L)) domain. The scFv fragment followed variable heavy-chain domain (V(H))-linker-V(L) orientation and contained (Gly(4)Ser)(3) linker peptide. The two proteins were produced in Escherichia coli and refolded from denaturant-solubilized inclusion bodies. The isolated V(L) domain demonstrated a typical immunoglobulin fold with well-defined secondary and tertiary structure and was capable of binding human ferritin with K(a) = 1.8 x 10(7) M(-)(1), approximately (1)/(30) of the affinity of the parent F11 antibody. Involvement of this V(L) domain into the two-domain scFv fragment yielded a distorted secondary and significantly destabilized tertiary structure in which neither of the two constituent domains attained complete folding. The thermal unfolding enthalpy of scFv F11 at pH 7.0 was as low as 5. 0 J.g(-)(1) versus 16.3 J.g(-)(1) obtained for the V(L) domain and 24.7 J.g(-)(1) for the parent F11 antibody (mouse IgG2a subclass). Intrinsic fluorescence and near-ultraviolet circular dichroic (CD) spectra, and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonate, confirmed partial loss of tertiary interactions in scFv. The spectroscopic and calorimetric properties of scFv F11 under physiological conditions are consistent with a model of a partially structured state with a distorted beta-sheet as a secondary structure and partial loss of tertiary interactions, which closely resembles the alternatively folded A-state adopted by an immunoglobulin at pH 2-3 [Buchner, J., Renner, M., Lilie, H., Hinz, H.-J., Jaenicke, R., Kiefhaber, T., and Rudolph, R. (1991) Biochemistry 30, 6922-6929]. However, scFv F11 demonstrated only an approximately 4-fold decrease in the antigen-binding affinity (K(a) = 1.3 x 10(8) M(-)(1)) versus the parent F11 antibody. The scFv fragment F11 provides the first description of a functional protein trapped under physiological conditions in a partially structured state. This state is either close to the native one in the antigen-binding affinity or, alternatively, initial weak binding of the antigenic epitope induces folding of scFv F11 into a more structured conformation that generates relatively high affinity.

Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity.

Transfectant HeLa cells were generated that expressed human ferritin H-chain wild type and an H-chain mutant with inactivated ferroxidase activity under the control of the tetracycline-responsive promoter (Tet-off). The clones accumulated exogenous ferritins up to levels 14-16-fold over background, half of which were as H-chain homopolymers. This had no evident effect in the mutant ferritin clone, whereas it induced an iron-deficient phenotype in the H-ferritin wild type clone, manifested by approximately 5-fold increase of IRPs activity, approximately 2.5-fold increase of transferrin receptor, approximately 1.8-fold increase in iron-transferrin iron uptake, and approximately 50% reduction of labile iron pool. Overexpression of the H-ferritin, but not of the mutant ferritin, strongly reduced cell growth and increased resistance to H(2)O(2) toxicity, effects that were reverted by prolonged incubation in iron-supplemented medium. The results show that in HeLa cells H-ferritin regulates the metabolic iron pool with a mechanism dependent on the functionality of the ferroxidase centers, and this affects, in opposite directions, cellular growth and resistance to oxidative damage. This, and the finding that also in vivo H-chain homopolymers are much less efficient than the H/L heteropolymers in taking up iron, indicate that functional activity of H-ferritin in HeLa cells is that predicted from the in vitro data.

Functional and immunological analysis of recombinant mouse H- and L-ferritins from Escherichia coli.

The production and characterization of recombinant mouse H- and L-ferritin chains from Escherichia coli are described. The proteins were efficiently expressed and purified with yields of 7-40 mg per liter of cell culture. They had the expected molecular mass and showed a physical stability analogous to that of the corresponding human ferritins. Mouse H- and L-ferritins had a very similar mobility on denaturing SDS-PAGE, but could be readily separated on nondenaturing PAGE because of the distinct slow mobility of mouse L-ferritin. Direct comparative experiments showed that mouse and human H-ferritins had the same iron incorporation activity, whereas mouse L-ferritin incorporated iron less efficiently than human L-ferritin. The difference was attributed to the substitution of a residue exposed on the cavity surface (Glu140 --> Lys) in mouse L-ferritin, a hypothesis confirmed by the finding that the mouse L-ferritin mutant Lys140-Glu incorporated iron as efficiently as human L-ferritin. Rabbit antisera elicited by the recombinant mouse ferritins were specific for the H- and L-chains and did not cross-react with the human ferritins. The antibodies and the derived specific ELISA assays allow the determination of H- and L-ferritins in mouse tissues.

Overexpression of the hereditary hemochromatosis protein, HFE, in HeLa cells induces and iron-deficient phenotype.

A transfectant HeLa cell clone expressing HFE under the control of a tetracycline-repressible promoter was generated. HFE expression was fully repressed by the presence of doxycycline, while it was strongly induced by growth in the absence of doxycycline. HFE accumulation was accompanied by a large (approximately 10-fold) decrease in H- and L-ferritin levels, by a approximately 3-4-fold increase in transferrin receptor, and a approximately 2-fold increase in iron regulatory protein activity. These indices of cellular iron deficiency were reversed by iron supplementation complexes. The overexpressed HFE immunoprecipitated together with transferrin receptor, indicating a physical association which is the likely cause for the observed approximately 30% decrease in 55Fe-transferrin incorporation after 18 h incubation. In the HFE-expressing cells the reduction in transferrin-mediated iron incorporation was partially compensated by a approximately 30% increase in non-transferrin iron incorporation from 55Fe-NTA, evident after prolonged, 18 h, incubations. The findings indicate that HFE binding to transferrin receptor reduces cellular iron availability and regulates the balance between transferrin-mediated and non-transferrin-mediated cellular iron incorporation.