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Biomedical subjects

S Levi

Publications and source records attributed to S Levi.

At least 19 recordsLinked to original sources
PubMed2003

Short-term outcome of isolated and associated congenital heart defects in relation to antenatal ultrasound screening.

OBJECTIVE: To investigate the outcome of fetuses affected by congenital heart defects (CHD), either detected or undetected at ultrasound screening, according to their complexity and severity. DESIGN: The study group comprised 3633 malformed fetuses entered into the Eurofetus database of which 798 had CHD. We compared the short-term outcome in cases where a CHD was detected by ultrasound screening with that in cases where a CHD was not detected. Isolated and associated CHD (ICHD and ACHD) and the degree of severity of defects were considered separately. Outcome data included termination of pregnancy (TOP), intrauterine fetal death, neonatal death (< 6 days after birth), gestational age at diagnosis and at delivery, mode of delivery and birth weight. RESULTS: Of the 798 fetuses with CHD, 595 had ICHD and 203 had ACHD. The diagnosis of an anomaly was made significantly earlier in ACHD cases. TOP was chosen in 28% of cases with a prenatal diagnosis of CHD, 20% for ICHD and 37% for ACHD (P < 0.001). The survival rate of antenatally diagnosed fetuses was lower in those with ACHD than in those with ICHD (P < 0.001) and lower for fetuses with antenatal diagnosis than with postnatal diagnosis (P < 0.001); this was due to significant differences in the complexity and severity of the defect. Premature delivery (< 32 weeks) was more frequent in fetuses in which an antenatal diagnosis of CHD had been made. Severe CHD were diagnosed earlier and were associated with a higher rate of TOP and spontaneous loss. CONCLUSIONS: The severity of CHD has a positive influence on the sensitivity of screening but a negative influence on the outcome. CHD that are not diagnosed antenatally include a high proportion of benign malformations. This explains the apparent paradox of a poorer outcome in fetuses in which a CHD was detected prenatally compared to those fetuses in which the defect was undetected prenatally. However, prenatal diagnosis provides parents with the option of TOP or of preventive care in specialized centers.

Female↗
PubMed2002

Fever of unknown origin in the elderly.

Fever of unknown origin (FUO) means fever that does not resolve itself in the period expected for self-limited infection and whose cause cannot be ascertained despite considerable diagnostic efforts. The differential diagnosis is often different in older patients, and presentation of disease is frequently nonspecific and symptoms are difficult to interpret. Multisystem disease has emerged as the most frequent cause of FUO in the elderly, and temporal arteritis is the most frequent specific diagnosis. Infections, particular tuberculosis, remain an important group. FUO is often associated with treatable conditions in this age group. Early recognition and prompt initiation of appropriate empirical therapy are cornerstones of the strategy.

Adolescent↗
PubMed2002

Sensitivity of ultrasound screening for congenital anomalies in unselected pregnancies.

OBJECTIVE: To assess the influence of heart defect frequency and severity on screening sensitivity of the entire spectrum of congenital anomalies (CA) and on detection rate of congenital heart defect (CHD) when performing routine ultrasound screening in unselected pregnant women. STUDY DESIGN: Secondary analysis was performed on data from Eurofetus, a large international collaborative study of ultrasound screening for CA in unselected populations. The present study addressed the relations (i) between the frequency of CHD in the screened population and the global sensitivity of ultrasound in detecting CA; (ii) between frequency of ventricular septum defect (VSD) and detection rate of CA and CHD; and (iii) between seriousness of CHD and CHD sensitivity. RESULTS: A negative correlation (p<0.001) between CHD frequency and CA sensitivity was observed in this study. Likewise, a negative correlation was also found between frequency of VSD and sensitivity for CA or CHD detection (p<0.001). In addition, a positive significant relationship (p<0.001) was observed between sensitivity and severity of CHD. CONCLUSION: A low CHD frequency introduces bias in CA and CHD sensitivity evaluation. Incomplete assessment of fetal and neonatal malformations brings on systematic error, leading to the pre-eminence of severe defects in the studied population sample and artificially improving sensitivity figures. On the other hand, from a clinician's viewpoint, the higher sensitivity for severe defect is welcomed, since their detection is most crucial for appropriate management.

Chi-Square Distribution↗
PubMed2001

A human mitochondrial ferritin encoded by an intronless gene.

Ferritin is a ubiquitous protein that plays a critical role in regulating intracellular iron homoeostasis by storing iron inside its multimeric shell. It also plays an important role in detoxifying potentially harmful free ferrous iron to the less soluble ferric iron by virtue of the ferroxidase activity of the H subunit. Although excess iron is stored primarily in cytoplasm, most of the metabolically active iron in cells is processed in mitochondria. Little is yet known of how these organelles regulate iron homeostasis and toxicity. Here we report an unusual intronless gene on chromosome 5q23.1 that encodes a 242-amino acid precursor of a ferritin H-like protein. This 30-kDa protein is targeted to mitochondria and processed to a 22-kDa subunit that assembles into typical ferritin shells and has ferroxidase activity. Immunohistochemical analysis showed that it accumulates in high amounts in iron-loaded mitochondria of erythroblasts of subjects with impaired heme synthesis. This new ferritin may play an important role in the regulation of mitochondrial iron homeostasis and heme synthesis.

Amino Acid Sequence↗
PubMed2001

Clinical, biochemical and molecular findings in a series of families with hereditary hyperferritinaemia-cataract syndrome.

Hereditary hyperferritinaemia-cataract syndrome (HHCS) is an autosomal dominant disease caused by mutations in the iron responsive element (IRE) of the l-ferritin gene. Despite the elucidation of the genetic basis, the overall clinical spectrum of HHCS has been less well studied as, to date, only individual case reports have been described. Therefore, we studied a total of 62 patients in 14 unrelated families, with nine different mutations. No relevant symptoms other than visual impairment were found to be associated with the syndrome. A marked phenotypic variability was observed, particularly with regard to ocular involvement (i.e. age range at which cataract was diagnosed in 16 subjects with the C39T: 6-40 years). Similarly, serum ferritin levels varied substantially also within subjects sharing the same mutation (i.e. range for the A40G: 700-2412 microg/l). We followed an HHCS newborn in whom well-defined lens opacities were not detectable either at birth or at 1 year. The lens ferritin content was analysed in two subjects who underwent cataract surgery at different ages, with different cataract morphology. Values were similar and about 1500-fold higher than in controls. These observations suggest that: (i) in HHCS the cataract is not necessarily congenital; (ii) in addition to the IRE genotype, other genetic or environmental factors may modulate the phenotype, especially the severity of the cataract.

Adolescent↗
PubMed2001

Double-gradient denaturing gradient gel electrophoresis assay for identification of L-ferritin iron-responsive element mutations responsible for hereditary hyperferritinemia-cataract syndrome: identification of the new mutation C14G.

BACKGROUND: Hereditary hyperferritinemia-cataract syndrome is an autosomic dominant disorder caused by heterogeneous mutations on the iron-responsive element (IRE) of ferritin L-chain mRNA. The mutations described to date were identified by direct sequencing of DNA from probands with hyperferritinemia often associated to bilateral cataracts. A direct genetic approach on a large population is useful to recognize polymorphisms in the DNA region and the prevalence of mutations associated with minor increases in serum ferritin and subclinical cataracts. We developed a rapid DNA scanning technique to detect mutations in a single electrophoretic analysis. METHODS: The double-gradient denaturing gradient gel electrophoresis (DG-DGGE) method consisted of PCR amplification of the target genomic DNA with GC-clamped oligonucleotides. The sequence encoded the 5' untranslated flanking region of ferritin L-chain mRNA, which includes an IRE stem-loop structure. The product was subjected to DG-DGGE (8.5-15% polyacrylamide and 50-95% denaturant) to separate the homo- and heteroduplexes. RESULTS: The method clearly identified all eight accessible mutations, including C-G transversions, which are the most difficult to detect. The method was applied to scan DNA samples from 50 healthy subjects and from 230 subjects with serum ferritin >400 microg/L. The new mutation G14C was identified. CONCLUSIONS: The DG-DGGE method detects all the mutations in the L-ferritin IRE sequence, is rapid and economical, and can be applied to scan large populations. The first population study indicated that the mutations are rare and may involve regions of the IRE structure not yet characterized.

Base Sequence↗
PubMed2001

Personal experience of the treatment of ruptured aortic aneurysms. The prognostic evaluation of some parameters.

BACKGROUND: The rupture of an aortic aneurysm is the most frequent and most severe complication, with an incidence of approximately 20-40/100,000 persons each year. The aim of this study was to identify the preoperative, intraoperative and postoperative factors that may influence the mortality rate. METHODS: Between January 1996 and December 1999 145 patients underwent emergency abdominal aortic repair surgery. One hundred and twenty-three patients represented a ruptured AAA and 22 a fissured aneurysm. One hundred and thirty-one patients were males and 14 were females; their mean age was 75+/-5.06 years. We selected a number of parameters after a review of the international literature and these were analysed in the two classes of survivors. No statistical analyses were performed on fissured aneurysms owing to the scant number of cases examined. RESULTS: Mortality was 41.4% (44.7% in ruptured aneurysms alone). The following were negative preoperative prognostic factors: old age, hypertension, ASA V, intraoperative anuria and platelet count <100,000. Intraoperative factors included: hypotension, cardiac arrest, onset of anuria, aortic clamping in more than one site, duration of surgery, quantity of RBC and plasma transfused. The main postoperative variables correlated to mortality were: hypotension, hypothermia, onset of anuria, need for further transfusions, high creatinine levels. CONCLUSIONS: Rapid intervention can minimise mortality in structures with expert teams of surgeons and anesthetists experienced in treating this type of pathology.

Age Factors↗
PubMed2000

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.

Anti-Bacterial Agents↗
PubMed2000

Early embryonic lethality of H ferritin gene deletion in mice.

Ferritin molecules play an important role in the control of intracellular iron distribution and in the constitution of long term iron stores. In vitro studies on recombinant ferritin subunits have shown that the ferroxidase activity associated with the H subunit is necessary for iron uptake by the ferritin molecule, whereas the L subunit facilitates iron core formation inside the protein shell. However, plant and bacterial ferritins have only a single type of subunit which probably fulfills both functions. To assess the biological significance of the ferroxidase activity associated with the H subunit, we disrupted the H ferritin gene (Fth) in mice by homologous recombination. Fth(+/-) mice are healthy, fertile, and do not differ significantly from their control littermates. However, Fth(-/-) embryos die between 3.5 and 9.5 days of development, suggesting that there is no functional redundancy between the two ferritin subunits and that, in the absence of H subunits, L ferritin homopolymers are not able to maintain iron in a bioavailable and nontoxic form. The pattern of expression of the wild type Fth gene in 9.5-day embryos is suggestive of an important function of the H ferritin gene in the heart.

Animals↗
PubMed2000

Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum.

We have constructed expression vectors for Dictyostelium discoideum which encode a green fluorescent protein (GFP) sequence upstream of a multicloning site for introduction of sequences of interest. Insertion of cDNAs into the multicloning site results in expression of fusion protein bearing an amino- or carboxyl-terminal GFP tag which can be used for fluorescent localization studies in Dictyostelium cells. A parallel construct fuses a FLAG epitope tag at the amino terminus of expressed protein. Each fusion cartridge was placed either in a G418-resistance vector allowing transactivated Ddp2-based extrachromosomal replication or in a vector allowing autonomous Ddp1-based replication. Distinct differences in expression stability were observed in the two vector types. When GFP-expressing cells were analyzed by fluorescence microscopy, significant cell-to-cell variability in expression level was observed when expression was based on the Ddp2 vector, while less cell-to-cell variation in expression level was observed when the Ddp1 backbone was used for expression.

Amino Acid Sequence↗
PubMed2000

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.

Amino Acid Sequence↗
PubMed2000

A new mutation (G51C) in the iron-responsive element (IRE) of L-ferritin associated with hyperferritinaemia-cataract syndrome decreases the binding affinity of the mutated IRE for iron-regulatory proteins.

Hereditary hyperferritinaemia-cataract syndrome is an autosomal dominant disorder characterized by a constitutively increased synthesis of L-ferritin in the absence of iron overload. The disorder is associated with point mutations in the iron-responsive element (IRE) of L-ferritin mRNA. We report a new mutation, G51C, identified in two members of a Canadian family, presenting a moderate increase in serum ferritin and a clinically silent bilateral cataract. Gel retardation assays showed that the binding of the mutated IRE to iron-regulatory proteins (IRPs) was reduced compared with the wild type. Structural modelling predicted that the G51C induces a rearrangement of base pairing at the lateral bulge of the IRE structure which is likely to modify IRE conformation.

Adolescent↗
PubMed2000

Relationship between TNF-alpha and iron metabolism in differentiating human monocytic THP-1 cells.

The human monocytic cell line THP-1 differentiates along the macrophage line after phorbol-12-myristate-13-acetate (PMA) supplementation and can be stimulated to secrete tumour necrosis factor alpha (TNF-alpha) by interferon gamma (IFN-gamma) addition. We found that, in the early stage of differentiation (1-48 h), PMA induction elicited an upregulation of intracellular H ferritin and H ferritin binding sites and a downregulation of transferrin receptor. In addition, we found that iron administration to PMA-differentiating cells induced the expression of TNF-alpha mRNA and TNF-alpha secretion to levels even higher than those induced by IFN-gamma alone. The iron chelator desferrioxamine showed the opposite effect and reduced TNF-alpha release. In contrast, preincubation of the cells with iron before PMA induction resulted in a decrease of the TNF-alpha secretion induced by IFN-gamma, whereas the opposite was true after preincubation with desferrioxamine. The data support a co-ordinate interaction between iron and TNF-alpha in monocyte macrophages, with an iron-mediated upregulation of TNF-alpha in the early phase of differentiation and an iron-mediated inhibition at later stages. This complex relationship has to be considered in evaluating the effects of iron on inflammation.

Binding Sites↗
PubMed2000

Immunohistochemistry of HFE in the duodenum of C282Y homozygotes with antisera for recombinant HFE protein.

BACKGROUND AND OBJECTIVE: HFE is a class-I MHC related protein which carries the C282Y mutation in most patients with hereditary hemochromatosis, an iron overload disease. HFE protein is expected to have a relevant role in the regulation of duodenal iron absorption, and HFE protein was immunohistochemically identified in the crypt cells. The aim of the work was to analyze whether the C282Y mutation affects HFE accumulation in the duodenum. DESIGN AND METHODS: We developed antisera for the extracellular portion of recombinant human HFE protein expressed in E. coli. The antisera were specific for HFE protein and the C282Y mutant in immunoblotting, immunoprecipitation and immunocytochemistry experiments of transfected cells, and they did not cross react with HLA antigens in various analyses. The antisera gave positive results in the staining of paraffin-fixed sections of duodenal slices of subjects with hemochromatosis. RESULTS: The antisera stained evident supranuclear granules in all enterocytes of 7 C282Y homozygous subjects, and a dark area in the same region in 3 other C282Y homozygotes. Granular bodies were absent from the duodenal sections of 8 C282Y negative subjects, from 2 C282Y heterozygotes and 3 C282Y homozygotes, with or without hemochromatosis. INTERPRETATION AND CONCLUSIONS: The detection of HFE-protein in granular bodies in the enterocytes of the large majority (77%) of C282Y homozygotes and not in other subjects suggests that the mutation facilitates protein accumulation in the duodenum.

Amino Acid Substitution↗
PubMed2000

[Fetal echocardiography volume mode (3 dimensions)].

About 10 out of 100 newborns are affected by congenital heart defects that remain the least easy to detect remain prenatally, despite the relatively high detection rate of fetal malformations. Antenatal diagnosis of congenital heart defects depends on operator experience and skill, but recent techniques of computer assisted ultrasound imaging should be useful by allowing easier recognition of fetal heart anomalies. New imaging procedures include a set of internal fetal views by volume rendering and post acquisition slice selection. Internal views are nearly similar to those obtained during open-heart surgery. In practice, from typical long axis and apical views, two volumes are stored for further examination. Fetal movement, fetal spine and maternal obesity are unfavorable circumstances for useful examination. The routine use of cardiac volume sampling for malformations screening should be possible with increasing speed in image acquisition and in data storage to display real-time 3D imaging.

Echocardiography↗
PubMed1999

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.

Apoferritins↗