Inhibition of uptake of transferrin-bound iron by human hepatoma cells by nontransferrin-bound iron

The liver acquires iron from transferrin by transferrin receptor-mediated (TR) and transferrin receptor-independent pathways (NTR) and from nontransferrin-bound iron (NTB-Fe). Iron uptake by the NTR processes involves an iron-carrier mediated step. Experiments, using human hepatoma cells (HuH7) transfected with TR antisense (sense for control) RNA expression vectors to suppress TR expression, were performed to examine the effect of unlabeled NTB-Fe as iron citrate on the uptake of 59Fe-125I-transferrin. This was to determine if the uptake of transferrin-bound iron (Tf-Fe) and NTB-Fe uptake is mediated by a common iron-carrier. Iron citrate inhibited the uptake of 59Fe-transferrin (2.5 micromol/L Fe) in a concentration-dependent manner with a maximum effect when the citrate-iron:Tf-Fe molar ratio was 10:1. Transferrin uptake was not affected. At a lower Tf-Fe concentration of (0.125 micromol/L) when uptake of iron is TR-mediated, a 10-fold molar excess of iron citrate had no effect on Tf-Fe uptake by HuH7 TR antisense and sense cells. However, at a higher Tf-Fe concentration (2.5 micromol/L), when uptake occurs mainly by the NTR-mediated process, there was a 40% reduction in the membrane-bound and intracellular uptake of iron. Iron citrate did not affect the maximum rate (Vmax) of Tf-Fe uptake but the Michaelis-Menten constant (Km) for Tf-Fe uptake by the NTR-mediated process was increased, indicating there was competitive inhibition of Tf-Fe uptake by iron citrate. These results suggest that the uptake of NTB-Fe and Tf-Fe by the NTR- mediated process occurs by the same cellular pathway, using a common iron-carrier.     

Aluminum taken up by transferrin-independent iron uptake affects the iron metabolism in rat cortical cells

The Symptom Check-List-90 (SCL-90) is a widely used psychiatric questionnaire which has not yet been validated in Finland. We investigated the utility of the translated version of the SCL-90 in the Finnish population, and set community norms for it. The internal consistency of the original subscales was checked and found to be good. Discriminant function analysis, based on the nine original subscales, showed that the power of the SCL-90 to discriminate between patients and the community is good. Factor analysis of the items of the questionnaire yielded a very strong unrotated first factor, suggesting that a general factor may be present. This together with the fact that high intercorrelations were found between the nine original subscales suggests that the instrument is not multidimensional. The SCL-90 may be useful in a research setting as an instrument for measuring the change in symptomatic distress, or as a screening instrument. The American community norms should be used with caution, as the Finnish community sample scored consistently higher on all subscales.     

Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of 59Fe2+ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was approximately 20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH4Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1.     

Dissimilar alterations of sodium-coupled uptake by platinum-coordination complexes in renal proximal tubular cells in primary culture

The potent anticancer drug cis-diamminedichloroplatinum (II) (CDDP) interferes early with electrolyte transport by the renal proximal tubule. To study the early effects of platinum coordination complexes on apical Na(+)-coupled transport systems, we examined the effect of increasing concentrations of CDDP, trans-diamminedichloroplatinum (II) (TDDP) and cis-diammine-1,1-cyclobutane-dicarboxylate platinum (II) (CBDCA) on Na(+)-coupled uptake of P(i), methyl-alpha-D-glucopyranoside (MGP) and L-alanine by rabbit proximal tubule cells in primary culture. At 17 microM CDDP and 540 microM CBDCA, 1) cell viability (lactate dehydrogenase release) and ATP content were unaffected, 2) Na(+)-K(+)-ATPase activity was reduced by 40%, 3) Na(+)-coupled uptake of MGP and P(i) was reduced, whereas 4) Na(+)-coupled uptake of alanine rose to twice the control value. Alterations of Na(+)-coupled uptake of P(i), MGP and alanine were due to changes in Km, with no significant change in Vmax. At 333 microM TDDP, Na(+)-dependent P(i) and MGP uptake decreased, whereas Na(+)-independent uptake increased markedly and was associated with a decline in cell viability and ATP content. We conclude that 1) the TDDP-induced decrease in Na+/P(i) and Na+/glucose cotransport was associated with reduced cell viability, 2) both CDDP and CBDCA had different effects on Na+/P(i), Na+/glucose and Na+/alanine cotransport, arguing against an alteration of the Na+ gradient due to reduced Na(+)-K(+)-ATPase activity and 3) CBDCA induced alterations of Na(+)-coupled uptake similar to those of CDDP at concentrations 20 to 30 times higher.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro modulation of L-arginine transport in trophoblast cells by glucose

BACKGROUND: The uptake of the semi-essential amino acid, L-arginine, into trophoblast cells was measured with the aim of determining the effect of different glucose concentrations on L-arginine influx kinetics. METHODS: This study used a novel superfused microcarrier culture system of BeWo cells (an established choriocarcinoma cell line with many characteristics of normal human trophoblast) and a rapid, paired-tracer dilution technique to measure unidirectional influx into the cells. RESULTS: At 10 mmol L-1 D-glucose, L-arginine unidirectional influx across the microvillous border of the cells was saturable with a Km of 1.14 +/- 0.14 mmol L-1 and a Vmax of 121. 36 +/- 5.89 nmol mg-1 protein min-1. When cells were preincubated for 24 h in the presence of 30 mmol L-1 D-glucose, there was a significant increase in the Vmax for L-arginine of nearly 30%. Similarly, preincubation in the presence of 1 mmol L-1 D-glucose and 12.5 mIU mL-1 human insulin reduced the Km for L-arginine influx by over 55%. CONCLUSION: These data suggest that the modulation of placental transport of L-arginine by glucose and insulin could contribute to the fetal macrosomia observed in diabetic mothers.     

Effect of various hormonal preparations on trophoblast growth in tissue culture

The application of different methods of tissue culture, in particular, investigations on trophoblast cells outside the mother's body, opens new possibilities for understanding pathogenesis of premature interruption of pregnancy caused by the hormonal and metabolic disorders. To study the effects of various hormones (Choriogonien, estrone and estradiol) on the growth and development of trophoblast cells the method of tissue culture was applied. 683 histological preparations, obtained after incubating trophoblast tissues with and without hormones were examined. It was established that choriogonine in doses of 5-100 IU/L stimulated growth in trophoblast cells but 200-400 IU/L of hormone did not increase this stimulating effect. Estrone and estradiol added to incubated tissues in doses of 60-120 IU/L brought about no changes or else had a weak effect on development of cultivated tissues. When choriogonine (or estrone) was added to the trophoblast tissues together with blood serum taken from women in danger of premature interruption of pregnancy the negative effect of the serum on the development of the trophoblast cells was prevented.     

Selective uptake of cholesteryl esters from high-density lipoprotein-derived LpA-I and LpA-I:A-II particles by hepatic cells in culture

Selective uptake of high-density lipoprotein (HDL)-associated cholesteryl esters (CE), i.e. lipid uptake independent of HDL particle uptake, delivers CE to the liver and steroidogenic tissues in vivo and in vitro. From human plasma HDL, two major subpopulations of particles can be isolated: one contains both apolipoprotein (apo) A-I and apo A-II (designated LpA-I:A-II) as dominant protein components, whereas in the other apo A-II is absent (LpA-I). In this study, selective CE uptake from LpA-I and LpA-I:A-II by cultured cells was investigated. LpA-I and LpA-I:A-II were isolated by immunoaffinity chromatography from human plasma high-density lipoprotein3 (HDL3, d = 1.125-1.21 g/ml) and both particles were radiolabeled in the protein (125I) as well as in the CE moiety ([3H]cholesteryl oleyl ether ([3H]CEt)). Several control experiments validated the labeling methodology applied. To investigate selective CE uptake, human Hep G2 hepatoma cells, human hepatocytes in primary culture and human skin fibroblasts were incubated in medium containing doubly radiolabeled LpA-I or LpA-I:A-II particles. Thereafter cellular tracer content was determined. For each cell type the rate of apparent lipoprotein particle uptake according to the lipid tracer ([3H]CEt) was in substantial excess over that due to the protein tracer (125I), demonstrating selective CE uptake from LpA-I as well as from LpA-I:A-II. This difference in uptake between [3H]CEt and 125I, i.e. the rate of apparent selective CE uptake, was significantly higher for LpA-I compared to LpA-I:A-II, and this was dose- as well as time-dependent. Thus in human hepatic cell and fibroblasts, CE are selectively taken up to a higher extent from LpA-I compared to LpA-I:A-II. These results may suggest that LpA-I particles of the human plasma HDL fraction may be those lipoproteins which more efficiently deliver CE to the liver via the selective uptake pathway whereas LpA-I:A-II may play a less important role.     

The non-expression of MHC class II in trophoblast cells

We report a rare case of neurobrucellosis in a 25-year-old woman with visual impairment, bilateral hearing loss, hyperprolactinaemia and meningitis. MRI revealed a sellar and suprasellar mass with enlargement of the optic chiasm.     

Cadmium uptake by Caco-2 cells. Effect of some milk components

The effect of some milk components on the cellular uptake of cadmium has been studied using a human intestinal cell line (Caco-2). Cadmium uptake by Caco-2 cells increased with the concentration of this metal in the culture medium, in a saturable way. These cells were exposed to different concentrations of cadmium and the synthesis of metallothionein was studied by a cadmium-saturation method. The levels of metallothionein increased with the cadmium concentration in the medium up to 20 microM of metal. Supplementation of the culture medium with 10% bovine milk caused a 25% decrease in the uptake of cadmium with respect to that internalized by the cells maintained in the culture medium alone. However, the uptake of cadmium from the medium supplemented with 10% human milk was similar to that with serum-free medium. beta-Lactoglobulin interacted with cadmium when studied by equilibrium dialysis, showing a stoichiometric binding constant of 5 x 10(4) l/mol. Interaction of lactoferrin with cadmium, however, was negligible. When Caco-2 cells were incubated in culture medium containing lactoferrin, cadmium uptake decreased with respect to that observed incubating the cells in a medium containing beta-lactoglobulin or in the free-protein medium. The inhibitory effect of lactoferrin on the uptake of cadmium might be due to a reduction of the cell surface charge, through its binding to the membrane.     

Modulation of sodium-coupled uptake and membrane fluidity by cisplatin in renal proximal tubular cells in primary culture and brush-border membrane vesicles

The proximal tubule appears to be the main target for the adverse effects of cis-diamminedichloroplatinum (II) (cDDP). We evaluated the early effects of cDDP at concentrations (3 to 67 microM) lower that those which alter cell viability, on three apical transport systems and on the physical state of the brush border membrane (BBM) in rabbit proximal tubule (RPT) cells in primary culture. The maximal effect, corresponding to a 30% decrease in Na(+)-coupled uptake of phosphate (Pi) and alpha-methylglucopyranoside (MGP) and a twofold increase in Na(+)-coupled alanine uptake, was obtained at 17 microM (5 micrograms/ml) cDDP and occurred through a modification of their affinity. At this concentration, cDDP increased BBM fluidity and decreased the BBM cholesterol content by 28%, without increasing the permeability of tight junctions. To clarify the role of cDDP-induced increase in BBM fluidity on alterations of Na(+)-coupled uptake, these parameters were also investigated in BBM vesicles isolated from rabbit renal cortex directly exposed to cDDP. cDDP induced a concentration-dependent inhibition of Na(+)-coupled uptake of MGP, Pi and alanine in BBM vesicles from the renal cortex, associated with a decrease in protein sulfhydryl content, without modifying BBM fluidity. Our findings strongly suggest that the cDDP-induced increase in BBM fluidity in RPT cells results from an indirect mechanism, possibly an alteration of cholesterol metabolism, and did not play a major role in the cDDP-induced inhibition of Na+/Pi and Na+/glucose cotransport systems that may be mainly mediated through a direct chemical interaction with essential sulfhydryl groups of the transporters.     

Molecular biology of iron and zinc uptake in eukaryotes

This study was designed to evaluate the feasibility of assessing myocardial viability using glucose loading followed by 201Tl SPECT. METHODS: First, the effect of insulin on the kinetics of 201Tl uptake was evaluated in isolated perfused rat hearts. Second, glucose-loading 201Tl myocardial SPECT was performed in 13 nondiabetic patients with histories of anterior myocardial infarction. Thirty minutes before acquiring rest 201Tl SPECT, 20 g of glucose were intravenously injected into the fasting subjects. Thallium perfusion defects were compared to those of conventional rest-redistribution SPECT images obtained within a 2-wk interval. SPECT images were divided into 21 segments, and a defect score in 17 segments was calculated as a sum of the semiquantitative defect scores (0 = normal; 1 = mildly decreased uptake; 2 = severely decreased uptake; 3 = absence of uptake). RESULTS: Thallium-201 uptake in isolated hearts showed a significant increase of 26% after insulin loading. Eleven (24%) of 45 segments showing perfusion defects on the conventional rest SPECT images demonstrated 201Tl uptake on glucose-loading SPECT imaging. Defect scores decreased significantly on the glucose-loading SPECT images (9.9 +/- 2.2 in early images; mean +/- s.e.) compared with the conventional rest-redistribution SPECT images (12.6 +/- 6.9 in delayed images, p < 0.05). CONCLUSION: Glucose-loading SPECT represents a superior method for assessing myocardial viability using 201Tl.     

MHC class II gene silencing in trophoblast cells is caused by inhibition of CIITA expression

A. baumannii is a multiresistant bacteria which is recognised as responsible for nosocomial infections and hospital outbreaks. The control of these outbreaks depends on the strain's typing and on the fight's policy against nosocomial infections. An outbreak of A. baumannii is occurred to patients who were hospitalized in Centre Hospitalier de Versailles. To investigate this outbreak, we have determined the biotype (Bouvet's method), the succeptibility pattern (disk diffusion and agar dilution results were analysed with the hierarchical classification and main component analysis) and the total DNA macrorestriction pattern (Pulse Field Gel Electrophoresis using SmaI restriction enzyme). A risk factors for A. baumannii acquisition were delineated in case-control study. During 2 years, 38 patients have been infected or colonized to A. baumannii. Thirty two patients were hospitalized in ICU. We studied 38 non repetitive clinical isolates and 9 strains of the patient's rooms. Four biotypes were defined by the Bouvet's typing method. Fourteen groups were obtained when succeptibility results were analysed with the hierarchical classification and 6 with the main composant analysis. The molecular typing permit us to define 4 epidemic and 6 sporadic strains. All the epidemic strains were isolated on ICU hospitalized patients. Our study has shown wide contamination in patient's rooms (Water tap, dry surfaces, patient's mattresses...). Environmental objects have been a major risk factor for A. baumannii acquisition. The control of this outbreak has been possible by application of hygienic measures (hands washing, isolment, meticulous cleaning of the ICU and environmental controls). No new case is occurred in the last year. Typing methods and case-control study are necessary to investigate cross-infections and take efficient measures against these outbreaks.     

Identification of prometalloproteinase-3 as a major protein secreted by human endometrial fibroblasts and inhibited by coculture with trophoblast cells

To assess endometrial fibroblast-cytotrophoblast interactions, we used a coculture system allowing analysis of the potential cell morphology modifications and protein secretion variations possibly involved in endometrial invasion arrest. Stromal cells and cytotrophoblasts were isolated from endometrial biopsies and first-trimester placental villi, respectively. In our culture conditions, a 57-kDa protein that was secreted by cultured fibroblasts but was absent in the 4-day coculture medium was found to be identical to prometalloproteinase-3 (proMMP-3) through determination of amino acid sequences of NH2-terminal and internal peptides. Northern blotting analysis of endometrial fibroblast total RNA showed a 38.6% metalloproteinase-3 (MMP-3) mRNA inhibition by 4-day 10(-6) M R5020 treatment. Inhibition of proMMP-3 secretion was weak when cytotrophoblasts were cultured for 4 days in a polycarbonate membrane insert over cultured fibroblasts without possible cell contact in spite of high levels of progesterone produced by cytotrophoblasts. Furthermore, cytotrophoblasts cultured on a monolayer of endometrial fibroblasts became syncytia, and most of the fibroblasts were decidualized. The closeness of the two cell types allowed paracrine relationships that might facilitate the progesterone action. Since MMP-3 is known to activate collagenases, inhibition of its secretion by cell contact might be a mechanism of invasion arrest for trophoblast cell migration.