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S Levi

Publications and source records attributed to S Levi.

At least 19 recordsLinked to original sources
PubMed1992

Evidence of H- and L-chains have co-operative roles in the iron-uptake mechanism of human ferritin.

The ability to incorporate iron in vitro was studied in homopolymers of human ferritin L-chain, human ferritin H-chain and its variants and in homopolymer mixtures. The H-chain variants carried amino acid substitutions in the ferroxidase centre and/or in carboxy residues on the cavity surface. Iron incorporation was examined by gel electrophoresis of the reaction products by staining for iron and protein. It was found that inactivation of the ferroxidase centre combined with the substitution of four carboxy groups on the cavity abolished the ability of H-chain ferritin to incorporate iron. Competition experiments with limited amounts of iron showed that, at neutral pH, L-chain ferritin is more efficient in forming iron cores than the H-chain variants altered at the ferroxidase activity or in the cavity. Competition experiments at pH 5.5 demonstrated that L-chain apoferritin is able to incorporate iron only when in the presence of H-chain variants with ferroxidase activity. The results indicate that L-chain apoferritin has a higher capacity than the H-chain apoferritin to induce iron-core nucleation, whereas H-chain ferritin is superior in promoting Fe(II) oxidation. The finding of cooperative roles of the H- and L-chains in ferritin iron uptake provides a clue to understanding the biological function of isoferritins.

Apoferritins↗
PubMed1992

Evidence that a salt bridge in the light chain contributes to the physical stability difference between heavy and light human ferritins.

Human ferritin, a multimeric iron storage protein, is composed by various proportions of two subunit types: the H- and L-chains. The biological functions of these two genic products have not been clarified, although differences in reactivity with iron have been shown. Starting from the hypothesis that the high stability typical of ferritin is an important property which may be relevant for its iron storage function, we studied ferritin homopolymers of H- and L-chains in different denaturing conditions. In addition we analyzed 13 H-chain variants with alterations in regions conserved within mammalian H-chains. In all the denaturation experiments H-chain ferritin showed lower stability than L-chain ferritin. The difference was greater in guanidine HCl denaturation experiments, where the end products are fully unfolded peptides, than in acidic denaturation experiments, where the end products are peptides with properties analogous to "molten globule." The study on H-chain variants showed: (i) ferritin stability was not affected by alterations of regions exposed to the inner or outer surface of the shell and not involved in intra- or inter-chain interactions; (ii) stability was reduced by alterations of sequences involved in inter-subunit interactions such as the deletion of the N-terminal extension or substitutions along the hydrophobic and hydrophilic channels; (iii) stability was increased by the substitution of 2 amino acids inside the four-helix bundle with those of the homologous L-chain. One of the residues is involved in a salt bridge in the L-chain, and we concluded that the stability difference between H- and L-ferritins is to a large extent due to the stabilizing effect of this salt bridge on the L-subunit fold.

Amino Acid Sequence↗
PubMed1992

Steroid ulcers.

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Animals↗
PubMed1992

Mutational activation of the N-ras oncogene assessed in primary clonogenic culture of acute myeloid leukemia (AML): implications for the role of N-ras mutation in AML pathogenesis.

The number of steps involved in the pathogenesis of acute myeloid leukemia (AML) is unclear. The initiating event would be expected to exist in all leukemic cells, but subsequent events may be subclonal. If several genetic events occur, they may cooperate within the same cell or be alternatively acquired by different subclones. These possibilities cannot be adequately analyzed in DNA prepared directly from patient specimens. In this study, N-ras mutations demonstrable in DNA prepared from peripheral blood of 10 patients with AML were examined in primary in vitro colonies (AML-colony-forming units [CFU]) grown from these patients. Both colonies containing the mutant gene and colonies containing normal allele only were obtained from each patient. The proportion of colonies containing no mutant allele varied among patients (5% to 57%). A subset of mutation containing colonies appeared to have lost the normal allele in nine of 10 AML cases analyzed. In the four cases with two N-ras mutations, the two mutations were found to exist in different subclones. In these cases, macroscopic colonies (AML-MCFU) were also obtained using an assay system designed to select for earlier clonogenic cells than in the AML-CFU assay. The N12cys mutation in AML10 was found in the CFU, but not in the MCFU, and the N12asp mutation in AML43 was found in the MCFU, but not in the CFU. These results suggest that N-ras mutation is a postinitiation event in AML that contributes to the outgrowth of more malignant subclones. Where two mutations are found in a case of AML, they appear to have been acquired by separate subclones, which may show different degrees of differentiation.

Base Sequence↗
PubMed1992

Binding and suppressive activity of human recombinant ferritins on erythroid cells.

We studied the relation between ferritin cellular binding and suppressive activity of recombinant H- and L-ferritin on human erythroid cells at different proliferation/differentiation phases. L-ferritin failed to show any suppressive activity or detectable binding to erythroblasts at any stage of maturation. In contrast, H-ferritin demonstrated binding to erythroblasts derived from peripheral BFU-E cells which increased steadily between 7-14 days of culture up to 15,000 molecules per cell. Reticulocytes and erythrocytes failed to bind either L- or H-ferritin. H-ferritin suppressed BFU-E colony formation and reduced K562 cell proliferation at nanomolar concentrations. This suggests that the expression of H-ferritin binding sites is modulated by cellular proliferation and differentiation, that cells expressing H-ferritin binding sites are sensitive to ferritin suppressive activity and that a causal relation exists between ferritin cellular binding and suppressive activity.

Binding Sites↗
PubMed1992

Effects of nonsteroidal anti-inflammatory drugs and misoprostol on gastroduodenal epithelial proliferation in arthritis.

In this study, quantitation of the epithelial proliferation status of gastric glands and duodenal crypts was performed using endoscopic biopsy specimens from patients with rheumatological conditions requiring long-term anti-inflammatory and analgesic therapy, testing the hypothesis that long-term administration of nonsteroidal anti-inflammatory drugs (NSAIDs) may lead to enhanced epithelial cell proliferation in the stomach and duodenum. After a 1-week washout period from NSAID administration, specimens were taken from endoscopically normal gastric antrum and duodenum, and microdissection was used to quantitate mitoses in these sites. Endoscopy and biopsy were then repeated after 2 weeks of NSAID administration, during which patients received in addition either the prostaglandin E1 analogue misoprostol (10 patients) or a placebo (10 patients). Mitotic counts in gastric glands increased similarly and significantly in the two groups from (mean +/- SEM) 4.45 +/- 0.57 to 7.69 +/- 1.08 (P less than 0.001) in the NSAID+placebo-treated group and from 4.31 +/- 0.53 to 7.68 +/- 1.08 (P = 0.006) in the NSAID+misoprostol-treated group. Similar changes took place in the duodenal crypts, from 5.44 +/- 0.68 to 8.60 +/- 1.28 (P = 0.013) in the NSAID+placebo-treated group and from 4.68 +/- 0.56 to 6.35 +/- 0.63 (P = 0.011) in the NSAIDs+misoprostol-treated group. NSAIDs increased the proportion of glands and crypts with bifid or more complex architectural forms. In a small group of patients, misoprostol alone did not alter mitotic rate in glands or crypts over 4 weeks. Thus, NSAIDs increase the rate of proliferation in endoscopically normal gastric and duodenal epithelium of patients with arthritis. This may form one of the mechanisms underlying gastric and duodenal adaptation to NSAIDs.

Adult↗
PubMed1992

Urinary excretion of glycosaminoglycans in patients with postmenopausal osteoporosis.

The organic bone matrix contains glycosaminoglycans (GAG) of which the precise function and importance in bone mineralisation are still unclear. We examined 85 persons--35 healthy women (25 premenopausal [preMP] mean aged 40.7 years; 10 menopausal [MP] mean aged 59.3 years) and 50 patients with postmenopausal osteoporosis [PMOP] at a mean age 60.4 years. The dynamic of urinary excretion of GAG was measured in 24-hour collected urine by precipitation with cetylpyridinum chloride and spectrophotometry at 560 nm, corrected for the level of excretion of creatinine. There was a significant increase in GAG excretion in patients with PMOP compared with healthy persons (8.25 mg/g and 9.53 mg/g vs 24.11 mg/g; p < 0.0001). A significant positive correlation was established between GAG and calcium urinary excretion and a negative one between GAG and serum estradiol levels. During the treatment with calcitonin the excretion of GAG was decreased which can be used for monitoring the changes of bone metabolism.

Adult↗
PubMed1992

Sucralfate diminishes basal acid output without affecting gastrin, H. pylori or gastritis in duodenal ulcer patients.

Twelve patients with active duodenal ulcer disease and Helicobacter pylori infection were treated with 1 g sucralfate q.d.s. for 1 month. Ulcers healed in 8 of the 12 patients without an alteration in the H. pylori-associated antral gastritis. Sucralfate produced a significant fall in basal acid output in all the patients, from a median of 4.8 (range 2.1-12.1) to 1.6 (0.4-8) mmol/h, P less than 0.01, whereas peak acid output was unchanged from 41 (21-59) before to 38 (24-55) mmol/h after treatment. Basal plasma gastrin concentrations and the meal-stimulated integrated gastrin response were not altered significantly by sucralfate: 8 (2-17) pmol/L and 732 (188-1045) pmol. min/L pre-treatment and 6 (2-17) pmol/L and 600 (140-1302) pmol. min/L post-treatment, respectively. The fall in basal acid output observed may contribute to prolonged duodenal ulcer remission after treatment with sucralfate.

Adult↗
PubMed1992

Antibodies for denatured human H-ferritin stain only reticuloendothelial cells within the bone marrow.

Human H-ferritin homopolymer was denatured in sodium dodecyl sulphate and injected in mice to obtain antibodies for dissociated H-subunit. The antisera and Moabs obtained were specific for the denatured H-chain with no cross-reactivity with assembled ferritins in immunoblotting experiments. In contrast the Moabs for native recombinant H-ferritin are specific for the assembled ferritin molecules with weak cross-reactivity with the denatured H-subunits. The epitope recognized by one of the anti-denatured H-chain Moabs was mapped on the C-terminal helix of ferritin. The antibodies were used to study H-ferritin conformation in cells. In immunocytochemistry experiments the antibodies for denatured H-ferritin stained HeLa and K562 cells weakly, with a different intensity and pattern to those obtained with anti-native H-ferritin antibody. In human bone marrow smears the anti-denatured ferritin antibodies stained only reticuloendothelial cells, and did not recognize the H-ferritin rich immature erythroblasts. It is concluded that assembled and denatured H-ferritins are immunogenically distinct, and that erythroid and reticuloendothelial cells within the bone marrow contain H-ferritin in different conformations.

Antibodies, Monoclonal↗
PubMed1992

Suppression of Helicobacter pylori reduces gastrin releasing peptide stimulated gastrin release in duodenal ulcer patients.

Helicobacter pylori increases gastrin release in duodenal ulcer patients. This may be through disruption or changes in the mucus layer affecting the access of luminal stimulants to gastrin releasing cells. The effect of suppressing H pylori on gastrin release stimulated by a non-luminal stimulus, gastrin releasing peptide (GRP), was examined. Eleven patients with active duodenal ulcer disease and colonised with H pylori received an intravenous infusion of GRP (2.9 pmol/kg/minute for 30 minutes) and the plasma gastrin response was measured. Basal and peak pentagastrin stimulated acid output were also determined. Patients were treated with tripotassium dicitratobismuthate (De-Nol) and metronidazole to suppress H pylori and the tests were repeated. Suppression of H pylori decreased plasma gastrin concentrations during GRP infusion, but acid output was not affected. Chromatographic analysis of the forms of gastrin in plasma showed a significant fall in gastrin 17, the predominant form found in the gastric antrum. Gastrin 34 did not fall significantly. This study shows that suppression of H pylori decreases the hypergastrinaemia caused by the nonluminal stimulant, GRP, mainly via decreasing gastrin 17.

Adult↗
PubMed1992

Non-steroidal anti-inflammatory drugs inhibit the processes of mucosal cell proliferation associated with duodenal ulcer healing.

We demonstrate that, in patients taking non-steroidal anti-inflammatory drugs (NSAIDs), there is inhibition of the proliferation of mucosal cells that normally leads to healing of duodenal ulcers. A microdissection technique was used to quantitate mitosis in duodenal crypts at the ulcer edge, giving a regeneration index of mitotic rate at that site, as compared to nearby mucosa. In patients with duodenal ulcers occurring in the absence of NSAID therapy, there was a brisk regenerative response (median index 2.48, range 1.55-9.81, n = 8), significantly greater than in patients taking NSAIDs (median index 1.10, range 0.73-2.16, n = 10, p = 0.014). Inhibition of the process of epithelial cell division normally involved in duodenal ulcer healing could contribute to the delay in ulcer healing which may explain the higher complication rate for duodenal ulcer during NSAID therapy.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1992

Low frequency of ras oncogene mutations in Philadelphia-positive acute leukemia and report of a novel mutation H61 Leu in a single case.

Activating ras mutations are frequent (25-60%) in chronic myelomonocytic leukemia (CMML) and in acute myeloid leukemia (AML) (30%), in contrast to chronic myeloid leukemia (CML) in which the incidence is very low (0-3%). This might reflect that the leukemic cell in CML is at a level of differentiation in which ras gene activation is not involved or, alternatively, might be due to the presence in CML of the bcrlabl fused gene. We have analyzed the presence of point mutations in codons 12, 13, 59, 61 and 63 of N-, K-, and H-ras genes, in 26 cases of Philadelphia-chromosome-positive, bcrlabl-positive acute leukemia (Ph+ AL), and in eight CMML cases by using the polymerase chain reaction. Aberrant ras genes were detected in a single Ph+ AL case, and in four out of eight CMML patients. The Ph+ AL showing altered ras allele had an unusual point mutation in H-ras gene, substituting leucine for glutamine. This mutation has not been previously found in any hematological disease. Our findings suggest that ras mutations are probably not involved in the pathogenesis of those leukemias in which blast cells contain bcrlabl oncogene activation.

Codon↗
PubMed1991

Influence of site-directed modifications on the formation of iron cores in ferritin.

The structure and crystal chemical properties of iron cores of reconstituted recombinant human ferritins and their site-directed variants have been studied by transmission electron microscopy and electron diffraction. The kinetics of Fe uptake have been compared spectrophotometrically. Recombinant L and H-chain ferritins, and recombinant H-chain variants incorporating modifications in the threefold (Asp131----His or Glu134----Ala) and fourfold (Leu169----Arg) channels, at the partially buried ferroxidase sites (Glu62,His65----Lys,Gly), a putative nucleation site on the inner surface (Glu61,Glu64,Glu67----Ala), and both the ferroxidase and nucleation sites (Glu62,His65----Lys,Gly and Glu61,Glu64,Glu67----Ala), were investigated. An additional H-chain variant, incorporating substitution of the last ten C-terminal residues for those of the L-chain protein, was also studied. Most of the proteins assimilated iron to give discrete electron-dense cores of the Fe(III) hydrated oxide, ferrihydrite (Fe2O3.nH2O). No differences were observed for variants modified in the three- or fourfold channels compared with the unmodified H-chain ferritin. The recombinant L-chain ferritin and H-chain variant depleted of the ferroxidase site, however, showed markedly reduced uptake kinetics and comprised cores of increased diameter and regularity. Depletion of the inner surface Glu residues, whilst maintaining the ferroxidase site, resulted in a partially reduced rate of Fe uptake and iron cores of wider particle size distribution. Modification of both ferroxidase and inner surface Glu residues resulted in complete inhibition of iron uptake and deposition. No cores were observed by electron microscopy although negative staining showed that the protein shell was intact. The general requirement of an appropriate spatial charge density across the cavity surface rather than specific amino acid residues could explain how, in spite of an almost complete lack of identity between the amino acid sequences of bacterioferritin and mammalian ferritins, ferrihydrite is deposited within the cavity of both proteins under similar reconstitution conditions.

Amino Acid Sequence↗
PubMed1991

Specific binding sites for H-ferritin on human lymphocytes: modulation during cellular proliferation and potential implication in cell growth control.

Interactions between human recombinant H- and L-ferritins and human lymphocytes were studied in vitro by direct binding assays and by flow cytometry. L-ferritin did not cause detectable specific binding, whereas H-ferritin showed a specific and saturable binding that increased markedly in phytohemagglutinin (PHA)-stimulated cells. This ferritin bound up to 30% of CD4+ and CD8+ T-lymphocytes and most B cells, indicating that expression of ferritin binding sites is not related to cell lineage or function. Dual-color flow cytometry experiments showed that ferritin binding sites were present on cells expressing the proliferation markers HLA-DR, MLR3, interleukin 2 (IL-2), and transferrin receptors (Tf-R). In addition, after PHA induction, the time course of the expression of H-ferritin binding sites was similar to those of the above proliferation markers. Ferritin binding sites were observed in lymphocytes at all cell cycle phases, including the early S-phase. H-Ferritin at nanomolar and picomolar concentrations had an inhibitory effect on PHA-induced blastogenesis. We propose that H-ferritin binding sites behave like proliferation markers, with the unusual function of downregulating proliferation.

Antigens, Surface↗
PubMed1991

Multiple K-ras codon 12 mutations in cholangiocarcinomas demonstrated with a sensitive polymerase chain reaction technique.

By using a modified polymerase chain reaction strategy, we have devised an approach to detect a K-ras oncogene mutated at codon 12 in the presence of 1000 normal alleles. This is a considerable improvement in sensitivity on previous assays. Application of this assay to 15 cholangiocarcinomas showed that all contained a K-ras mutation to codon 12 and that nine of the tumors contained two or more mutations. In 11 cases, mutations were present in less than 10% of the cells in the sample. In common with pancreatic adenocarcinomas, in which 75 to 95% of cases contain a mutation in K-ras, cholangiocarcinomas show a very high frequency of ras gene mutation, but within a tumor only a fraction of cells contain a ras mutation. The presence of multiple mutations and the low frequency of mutant alleles in the samples argue against K-ras mutations being the initiating genetic lesion in this tumor, but suggest that ras gene mutation is involved in the stepwise progression of neoplastic cells to full malignancy.

Adenoma, Bile Duct↗
PubMed1991

Solving the structure of human H ferritin by genetically engineering intermolecular crystal contacts.

Ferritin is important in iron homeostasis. Its twenty-four chains of two types, H and L, assemble as a hollow shell providing an iron-storage cavity. Ferritin molecules in cells containing high levels of iron tend to be rich in L chains, and may have a long-term storage function, whereas H-rich ferritins are more active in iron metabolism. The molecular basis for the greater activity of H-rich ferritins has until now been obscure, largely because the structure of H-chain ferritin has remained unknown owing to the difficulties in obtaining crystals ordered enough for X-ray crystallographic analysis. Here we report the three-dimensional structure of a human ferritin H-chain homopolymer. By genetically engineering a change in the sequence of the intermolecular contact region, we obtained crystals isomorphous with the homologous rat L ferritin and of high enough quality for X-ray diffraction analysis. The X-ray structure of human H ferritin shows a novel metal site embedded within each of its four-helix bundles and we suggest that ferroxidase activity associated with this site accounts for its rapid uptake of iron.

Amino Acid Sequence↗