PubMed Health⌕ Search

Biomedical subjects

J H Brock

Publications and source records attributed to J H Brock.

At least 19 recordsLinked to original sources
PubMed1992

Isolation of a ferritin from Bacteroides fragilis.

A ferritin was isolated from the obligate anaerobe Bacteroides fragilis. Estimated molecular masses were 400 kDa for the holomer and 16.7 kDa for the subunits. A 30-residue N-terminal amino acid sequence was determined and found to resemble the sequences of other ferritins (human H-chain ferritin, 43% identity; Escherichia coli gen-165 product, 37% identity) and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 20% identity). The protein stained positively for iron, and incorporated 59Fe when B. fragilis was grown in the presence of [59Fe]citrate. However, the isolated protein contained only about three iron atoms per molecule, and contained no detectable haem. This represents the first isolation of a ferritin protein from bacteria. It may alleviate iron toxicity in the presence of oxygen.

Amino Acid Sequence↗
PubMed1992

Effect of iron and retinoic acid on the control of transferrin receptor and ferritin in the human promonocytic cell line U937.

The effect of changes in iron availability and induction of differentiation on transferrin receptor expression and ferritin levels has been examined in the promonocytic cell line U937. Addition of iron (as 200 micrograms/ml saturated transferrin) or retinoic acid (1 microM) both caused approx. 70% reduction in the average number of surface transferrin receptors, while the iron chelator desferrioxamine caused an 84% increase. Comparable changes also occurred in the levels of transferrin receptor mRNA. Neither iron nor retinoic acid significantly altered the half-life of transferrin receptor mRNA in the presence of actinomycin D (approx. 75 min) but a 10-fold increase in stability occurred in the presence of desferrioxamine. Iron and retinoic acid both caused an increase in intracellular ferritin levels (approx. 4-and 3-fold, respectively), while desferrioxamine reduced ferritin levels by approx. two-thirds. The effect of iron and retinoic acid added together did not differ greatly from that of each agent alone. None of the treatments greatly affected levels of L-ferritin mRNA. Virtually no H-ferritin mRNA was detected in U937 cells. These results show that changes in ferritin and transferrin receptor caused by treatment with retinoic acid are similar to those induced by excess iron, and suggest that changes in these proteins during cell differentiation are due to redistribution of intracellular iron into the regulatory pool(s), rather than to iron-independent mechanisms.

Cell Differentiation↗
PubMed1992

Uptake and intracellular handling of iron from transferrin and iron chelates by mitogen stimulated mouse lymphocytes.

The ability of lymphocytes to utilise iron from different sources has been investigated. Iron uptake from transferrin by proliferating lymphocytes gradually increased as saturation of the protein with iron was increased up to 100%, but rose sharply when addition of further iron resulted in the presence of non-transferrin bound iron. Increasing the saturation of transferrin with iron caused an increased rate of proliferation up to about 100% saturation but when the level of iron present exceeded the binding capacity of the protein, proliferation decreased and at high levels of iron it was reduced below that seen in the absence of transferrin. Comparison of the degree of iron uptake from transferrin and from iron chelators showed that the hydrophilic chelator ferric nitrilotriacetate (FeNTA) donated larger amounts of iron to cells than did transferrin or the lipophilic chelator ferric-pyridoxal isonicotinoyl hydrazone (FePIH), but did not promote proliferation, and when present in high amounts caused inhibition. In contrast, FePIH supported proliferation as efficiently as transferrin. In cells cultured with FeNTA, iron was found predominantly in an insoluble form while in the cells cultured with Fe-transferrin or FePIH the largest proportion of iron was found in the non-ferritin high molecular weight fraction, which probably represents iron in enzymes and other metabolically-important proteins. In no case did iron associated with ferritin exceed 15% of the total uptake, and the cells showed no marked increase in synthesis of ferritin in response to any of the forms of iron. These results indicate that different forms of iron are handled in different ways by lymphocytes, and that iron delivered from hydrophilic chelates may be toxic and not readily available for metabolic use. Lymphocytes appear to be poorly equipped to sequester excess iron in ferritin, and this may account for abnormalities in the immune system reported in patients with iron overload.

Animals↗
PubMed1992

Effect of transferrin, lactoferrin and chelated iron on human T-lymphocytes.

The effect of different forms of iron and iron-binding proteins on the proliferative response of human lymphocytes to phytohaemagglutinin (PHA) has been studied. Transferrin enhanced proliferation, the effect being proportional to the degree of iron saturation up to 100%, but decreased if additional iron was present. The lipophilic complex ferric pyridoxal isonicotinoyl hydrazone (FePIH) also enhanced proliferation, but the hydrophilic complex ferric nitrilotriacetate (FeNTA) was inhibitory. Fe-lactoferrin could not substitute for Fe-transferrin, although iron-free (apo) lactoferrin abrogated the inhibitory effect seen when iron levels exceed the binding capacity of transferrin. Lymphocyte ferritin levels increased 4-fold as the iron saturation of transferrin increased from 0 to 90% but no further increase was seen at higher iron levels, suggesting that lymphocytes are poorly equipped to detoxify excess iron through stimulation of ferritin synthesis. The effect of iron on the CD4:CD8 ratio after 72 h culture with PHA was also examined. The ratio was approximately 2:1 for cells cultured with transferrin at iron saturations between 0 and 75%, with FePIH, or without either, but decreased to 1.1:1 when cells were cultured in the presence of FeNTA, regardless of whether or not saturated Fe-transferrin was present. These results show that iron can affect lymphocyte proliferation and subset ratios in different ways according to the form and amount present, and may help to explain some of the immunological disturbances associated with iron overload.

Adult↗
PubMed1992

Transferrin synthesis by macrophages: up-regulation by gamma-interferon and effect on lymphocyte proliferation.

We have investigated transferrin synthesis by human and mouse lymphoid and myeloid cells. It was found that transferrin synthesis is a property of mouse but not human macrophages, whereas in man T lymphocytes synthesised transferrin. Synthesis by mouse macrophages showed a dose-dependent increase in response to gamma-interferon (gamma-IFN), but iron added as ferric nitrilotriacetate had no effect. Macrophage-derived transferrin was found to contain iron already bound to it and was able to support Con A-stimulated mouse lymphocyte proliferation.

Animals↗
PubMed1992

Effect of pH and citrate on binding of iron and gallium by transferrin in serum.

Although both Al and Fe are bound to transferrin in plasma, they are metabolized differently. Aluminum is less tightly bound to transferrin than is Fe, and might be released in circumstances in which Fe remains bound. The effect of pH ana citrate on the binding of 67Ga (a radiotracer used as an analog of Al) to transferrin in normal human serum was tested in the presence of physiological concentrations of CO2. At pH less than 6.8, Ga started to dissociate from transferrin; at pH 6, greater than 50% of the added 67Ga was present in a low-M(r) form. In contrast, almost all Fe remained bound to transferrin at pH values as low as 4.7. Citrate at concentrations as great as 100 mmol/L had no effect on binding of Fe, but the binding of 67Ga was markedly reduced at citrate greater than 1 mmol/L. Being bound to transferrin less strongly than Ga is, Al could dissociate even more readily, and loss of Al from transferrin in the kidney might explain why Al but not Fe is excreted in urine.

Citrates↗
PubMed1991

Interaction of aluminium and gallium with human lymphocytes: the role of transferrin.

Aluminium-transferrin (Al-Tf) and gallium-transferrin caused a dose-dependent decrease in proliferation of human peripheral blood lymphocytes cultured for 3 days with phytohaemagglutinin (PHA). Addition of apotransferrin reduced the inhibitory effect. Al added as AlCl3 or aluminium citrate had no effect, and there was no significant difference in the response of cells from renal failure patients with or without high serum Al levels or controls. Lymphocytes cultured in the presence of Al-Tf showed a dose-dependent uptake of Al, whereas uptake from aluminium citrate was low and not dose-dependent. Uptake from AlCl3 was very high but probably involved a nonspecific uptake mechanism. Levels of Al in freshly isolated lymphocytes were approximately 1.6 ng/10(6) cells, there being no difference between cells from patients and controls. It is concluded that Al, when bound to transferrin, may have a detrimental effect on lymphocyte function and might contribute to the decreased immune responsiveness of renal failure patients on haemodialysis. However, lymphocyte Al levels are probably not useful as a marker of Al overload in such patients.

Aluminum↗
PubMed1991

Effect of ferric and ferrous iron chelators on growth of Bacteroides fragilis under anaerobic conditions.

Growth of Bacteroides fragilis under anaerobic conditions in the presence of either haemin or protoporphyrin IX was inhibited by the ferrous iron chelator bipyridyl. The ferric-iron chelator desferrioxamine inhibited growth in the presence of protoporphyrin but not haemin, suggesting that even under anaerobic conditions Fe3+ is involved in uptake of non-haem iron, which is required in the absence of haemin. However, the ferric iron chelators 1,2-dimethyl-3-hydroxy-pyrid-4-one (L1) and pyridoxal isonicotinoyl hydrazone (PIH) were only weakly inhibitory. Apotransferrin, which also binds Fe3+, inhibited growth, but this was not simply due to binding of iron in the medium, as under the reducing conditions present, transferrin was unable to bind iron. This study suggests that even under anaerobic conditions, uptake of non-haem iron by B. fragilis may involve conversion of Fe2+ to Fe3+.

Anaerobiosis↗
PubMed1991

The role of transferrin and citrate in cellular uptake of aluminium.

The ability of human erythroleukaemia K562 cells to take up aluminium from Al-transferrin and Al-citrate has been examined. Uptake from Al-transferrin was dose-dependent over the range 68-544 ng/ml of aluminium, and increased over a 12-day period. In contrast, uptake from Al-citrate was low even at an aluminium concentration of 6800 ng/ml and did not increase over time. Neither form of aluminium greatly affected cell growth. It is concluded that Al-transferrin, rather than Al-citrate, is the physiologically relevant form of this metal with respect to cellular uptake, but that any metabolic abnormalities induced by aluminium do not affect proliferation of this cell line.

Aluminum↗
PubMed1991

Role of iron metabolism in absorption and cellular uptake of aluminum.

The effect of iron status on aluminum (Al) absorption was investigated in this study in vivo using an animal model and in vitro using an intestinal mucosal cell line. In the in vivo model rats were rendered iron overloaded by intraperitoneal injection of iron dextran (5 mg/48 hr) or iron deficient by phlebotomy (2.5 to 3 ml blood/week). These rats, and normal controls, were then dosed with Al(OH)3 (40 mg/day) for 30 days. Urinary excretion of Al was significantly greater in the iron deficient group than in the other two groups throughout the study period, and brain Al at the end of the experiment was significantly increased in the iron depleted group (1.93 micrograms/g) and decreased in the iron overloaded group (0.73 microgram/g) compared with controls (1.42 micrograms/g). The brain Al levels in iron overloaded rats were no higher than those in normal rats that had not been dosed with Al(OH)3 (0.61 microgram/g). No significant differences were found in serum Al levels. In the in vitro experiments cultures of the rat intestinal cell line RIE1 were iron overloaded by addition of iron nitrilotriacetate (0.1 mM) or iron depleted with desferrioxamine (5 micrograms/ml) for 20 days prior to pulsing with Al transferrin (0.5 mg/ml) for 24 hours. Uptake of Al was significantly greater in the iron depleted cells (2.3 ng/micrograms cell DNA) than in iron overloaded (0.81 ng) or untreated (0.83 microgram) cells. These studies show that iron depletion markedly increases absorption and cellular uptake and suggest that susceptible individuals, such as renal failure patients, run an increased risk of toxicity if they are iron deficient.

Aluminum↗
PubMed1991

Iron uptake in aluminium overload: in vivo and in vitro studies.

In this study we have evaluated the haematological consequences of chronic aluminium (Al) overload. We have also investigated 'in vivo' whether aluminium overload may modulate gastrointestinal iron (Fe) absorption and 'in vitro' whether the presence of aluminium may influence the cellular uptake of iron. The in vivo studies were performed in rats with normal renal function and the in vitro experiments were done using the rat intestinal epithelial cell line RIE-1. The results demonstrate that aluminium deposit in tissues even with normal renal function. The intraperitoneal aluminium loading resulted in serum and tissue aluminium increases comparable with concentrations found in aluminium-intoxicated renal patients. The aluminium intoxication was accompanied by a microcytic anaemia with a haematological pattern similar to that observed in iron-deficiency anaemia. Nevertheless, iron absorption was significantly reduced despite an increased total iron binding capacity (TIBC). In addition, aluminium was also able to reduce in vitro cellular uptake of iron in the RIE-1 intestinal cell line. These experimental results demonstrate that aluminium interferes with iron absorption and iron transfer, and suggest that these mechanisms may be responsible for maintaining and even increasing the anaemia observed in aluminium overload.

Aluminum↗
PubMed1991

Aluminium uptake by intestinal cells: effect of iron status and precomplexation.

The investigation of different aluminium-binding agents and metabolic conditions that might act as critical factors in modulating aluminium absorption could yield valuable information in the understanding of aluminium gastrointestinal absorption. We evaluated the effect of iron depletion in aluminium uptake and the role of transferrin and citrate in aluminium incorporation by the intestinal epithelial cell line RIE1. Both complex, aluminium-citrate and aluminium-transferrin were prepared in a molar ratio of 2:1; both facilitated the aluminium uptake although a greater aluminium incorporation was found when aluminium was administered as aluminium-transferrin. This difference became even greater in the iron-depleted cells (normal cells vs iron-depleted cells): 7.7 +/- 1.4 versus 30 +/- 5.6 ng Al/micrograms DNA (P less than 0.05). From these and previous data it is possible to speculate that iron status could modulate the intestinal uptake of aluminium and that both transferrin and citrate would act as effective carriers in the incorporation of aluminium into mucosal cells.

Aluminum↗
PubMed1991

Expression of transferrin receptors by monocytes and peritoneal macrophages from renal failure patients treated by continuous ambulatory peritoneal dialysis (CAPD).

Approximately 20% of monocytes and peritoneal macrophages from renal failure patients undergoing continuous ambulatory peritoneal dialysis (CAPD) were transferrin-receptor (TfR) positive by immunofluorescence, whereas cells from normal controls were generally TfR negative, as were monocytes from rheumatoid arthritis patients and from renal failure patients treated by haemodialysis. There was a significant correlation between the length of time on CAPD and the proportion of TfR-positive blood monocytes. CAPD peritoneal macrophages possessed 6.7-37.1 x 10(3) transferrin binding sites per cell, with a Ka of 3-25 x 10(7) mol l-1. In culture, monocytes from CAPD patients showed a progressive decrease in TfR expression, while in contrast about 20% of monocytes from normal controls which were originally 100% TfR negative expressed TfR after 3 days in culture. These findings indicate that regulation of TfR in monocytes/macrophages is complex, and that frequent removal of peritoneal cells during dialysate exchange may place a strain on the bone marrow, resulting in the release of an increasingly immature population of TfR positive monocytes to the circulation in CAPD patients.

Ascitic Fluid↗
PubMed1991

Relative availability of transferrin-bound iron and cell-derived iron to aerobactin-producing and enterochelin-producing strains of Escherichia coli and to other microorganisms.

A method is described for determination of the relative availability of transferrin-bound iron and cell-derived iron to microbial iron-scavenging mechanisms. This involved incubation of parallel cultures of microorganisms in dialysis tubes placed in RPMI 1640 tissue culture medium containing 30%-iron-saturated transferrin and K562 erythroleukemia cells. In one culture the transferrin was labelled with 59Fe and in the other the cells were labelled, and the relative uptake of radioiron by the microorganisms determined. The results showed that Staphylococcus epidermidis and Staphylococcus aureus acquired iron predominantly from cells, while Candida albicans and the enteropathogenic Escherichia coli NCTC 8623 tended to acquire iron from transferrin. E. coli K-12 strains W3110 and LG1705, which (like NCTC 8623) produce the siderophore enterochelin but not aerobactin, acquired predominantly transferrin-bound iron, whereas the related E. coli strains LG1315 and LG1628, which produce aerobactin but not enterochelin, showed a preference for cell-derived iron. When the cells were incubated in the presence of 59Fe-labelled transferrin and 55Fe-labelled ferritin, no difference in relative availability of iron to E. coli was observed, suggesting that differences in the ability of aerobactin and enterochelin to remove iron from intracellular ferritin were not responsible for this preference. These results may help to explain why production of aerobactin, despite its relatively low affinity for iron, is more closely associated with invasiveness in E. coli than is enterochelin production. Reduced availability of cell-bound iron during inflammation may contribute to antimicrobial defenses.

Candida albicans↗
PubMed1990

Effect of aluminium on iron uptake and transferrin-receptor expression by human erythroleukaemia K562 cells.

Incubation of human erythroleukaemia K562 cells with Al-transferrin inhibited iron uptake from 59Fe-transferrin by about 80%. The inhibition was greater than that produced by a similar quantity of Fe-transferrin. Preincubation of cells for 6 h with either Al-transferrin or Fe-transferrin diminished the number of surface transferrin receptors by about 40% compared with cells preincubated with apo-transferrin. Al-transferrin did not compete significantly with Fe-transferrin for transferrin receptors and, when cells were preincubated for 15 min instead of 6 h, the inhibitory effect of Al-transferrin on receptor expression was lost. Both forms of transferrin also decreased the level of transferrin receptor mRNA by about 50%, suggesting a common regulatory mechanism. Aluminium citrate had no effect on iron uptake or transferrin-receptor expression. AlCl3 also had no effect on transferrin-receptor expression, but at high concentration it caused an increase in iron uptake by an unknown, possibly non-specific, mechanism. Neither Al-transferrin nor AlCl3 caused a significant change in cell proliferation. It is proposed that aluminium, when bound to transferrin, inhibits iron uptake partly by down-regulating transferrin-receptor expression and partly by interfering with intracellular release of iron from transferrin.

Aluminum↗
PubMed1990

Effect of novel 1-alkyl-3-hydroxy-2-methylpyrid-4-one chelators on uptake and release of iron from macrophages.

The effect of several iron chelators on iron uptake and release by mouse peritoneal macrophages has been investigated. The 1,2-dimethyl (L1) and 1-ethyl-2-methyl (L1NEt) derivatives of 3-hydroxypyrid-4-one markedly enhanced iron mobilisation from macrophages pulsed with 59Fe-transferrin-antitransferrin immune complexes and were more effective than desferrioxamine, maltol, or mimosine. Release increased with increasing chelator concentration. None of the chelators donated significant amounts of iron to macrophages, and none showed any cytotoxic effect. The synthetic alpha-ketohydroxypyridine chelators may therefore be active in removing iron from the reticuloendothelial system as well as from hepatocytes, and indeed may be superior to desferrioxamine.

Animals↗
PubMed1990

Tissue specific expression of mouse transferrin during development and aging.

Transferrin (TF) is a major plasma protein that binds ferric iron and transports it to all target tissues of the body. This study is the first step to identify the tissue specific expression of the transferrin gene in mice during development, into maturity and throughout the aging process. The transferrin gene expresses mainly in mouse liver, the cerebral hemispheres and cerebellum. In mouse, transferrin is expressed in peritoneal macrophages and in mouse macrophage cell line MO59. At 19 days of gestation, transferrin mRNA is detected in the fetal lung, heart, stomach and kidney. TF mRNA levels increase in liver throughout gestation with maximum expression occurring at 19 days. Transferrin mRNA was detected in placentas of pregnant mice, with levels progressively increasing throughout the term of pregnancy. The levels of liver TF mRNA in mouse vary in a cyclic manner during the development increasing with the aging processes. Because of the dynamic nature of tissue requirements for transferrin during homeostasis the TF gene serves as a promising system for analyzing tissue-specific regulation in vivo during development and aging. Results from this study designate periods in the life-span of the mouse where regulatory mechanisms interacting with the TF gene appear to dynamically alter its expression.

Aging↗