The major yolk protein in sea urchins is a transferrin-like, iron binding protein.

The major yolk protein (MYP) in sea urchins has historically been classified as a vitellogenin based on its abundance in the yolk platelets. Curiously, it is found in both sexes of sea urchins where it is presumed to play a physiological role in gametogenesis, embryogenesis, or both. Here we present the primary structure of MYP as predicted from cDNAs of two sea urchins species, Strongylocentrotus purpuratus and Lytechinus variegatus. The sequence from these two species share identity to one another, but bear no resemblance to other known vitellogenins. Instead the sequence shares identity to members of the transferrin superfamily of proteins. In vitro iron binding assays, including both (59)Fe overlay assays of MYP enriched coelomic fluid and immunoprecipitation of native iron-bound MYP from coelomic fluid, support this classification. We suggest that one of MYP's transferrin-like properties is to shuttle iron to developing germ cells.

Downregulation of a protective Actinobacillus pleuropneumoniae antigen during the course of infection.

The persistence of Actinobacillus pleuropneumoniae in convalescent pigs significantly contributes to the distribution of disease. The downregulation of protective antigens in vivo as one possible mechanism responsible for this phenomenon was investigated using the small iron-regulated transferrin binding protein (TbpB-protein) as exemplary protective antigen. From a total of 21 pigs experimentally infected with A. pleuropneumoniae serotype 7 in three trials, bronchoalveolar lavage fluid (BALF) was obtained on day 1 or 2, day 7, day 14 and day 21. Employing double immunofluorescence of BALF with a monoclonal anti-TbpB antibody and an A. pleuropneumoniae -specific anti-polysaccharide antiserum a statistically significant decrease of the percentage of A. pleuropneumoniae bacteria strongly expressing TbpB protein was observed during the course of infection. These results were supported by in vitro incubation of A. pleuropneumoniae in medium supplemented with BALF. In addition, it was found that TbpB-expression in BALF from day 7 after infection could not be inhibited by the substitution of iron. These results suggest (i) the downregulation of protective antigens is one possible mechanism allowing bacterial persistence, (ii) in vitro induction in the presence of BALF mimics the in vivo situation, and (iii) TbpB expression is additionally regulated by an iron-independent mechanism.

Effect of adjuvants in the isotypes and bactericidal activity of antibodies against the transferrin-binding proteins of Neisseria meningitidis.

Twenty-eight Neisseria meningitidis strains of different serogroups, serotypes, and TbpB isotypes were used to test the effect of five adjuvant formulations on the immune response to the meningococcal transferrin-binding proteins (Tbps) in mice. Levels of anti-Tbps antibodies were relatively low when purified TbpA-TbpB complexes were used for immunization, those obtained with the RAS adjuvant being the highest, and the isotype distribution reveals a prevalence of the non-bactericidal IgG1. Specific anti-Tbps antibody levels were five to 125 times higher immunizing with whole outer membrane vesicles, with bactericidal isotypes prevailing, which suggests that presentation of these antigens in their natural conformation is crucial to elicit a good response. Nevertheless, bactericidal activity did not correlate with these characteristics, confirming that it must be also influenced by other factors, and direct evaluation of the killing ability is necessary to draw conclusions about the efficacy of antigens or adjuvants in vaccine design.

Lack of expression of the global regulator OxyR in Haemophilus influenzae has a profound effect on growth phenotype.

A pBR322-based library of chromosomal DNA from the nontypeable Haemophilus influenzae TN106 was screened for the expression of transferrin-binding activity in Escherichia coli. A recombinant clone expressing transferrin-binding activity contained a 3.7-kb fragment of nontypeable H. influenzae DNA. Nucleotide sequence analysis of this insert revealed the presence of two complete open reading frames encoding proteins of approximately 26 and 34 kDa. Mini-Tn10kan transposon mutagenesis at different sites within the open reading frame encoding the 34-kDa protein resulted in the abolition of transferrin-binding activity in the recombinant E. coli clone. The deduced amino acid sequence of the 34-kDa protein had 70% identity with the OxyR protein of E. coli; this latter macromolecule is a member of the LysR family of transcriptional activators. When a mutated H. influenzae oxyR gene was introduced into the chromosome of the wild-type H. influenzae strain by allelic exchange, the resulting oxyR mutant still exhibited wild-type levels of transferrin-binding activity but was unable to grow on media containing the heme precursor protoporphyrin IX (PPIX) in place of heme. This mutant also exhibited reduced growth around disks impregnated with heme sources. Supplementation of the PPIX-based growth media with catalase or sodium pyruvate resulted in normal growth of the H. influenzae oxyR mutant. Provision of the wild-type H. influenzae oxyR gene in trans also permitted the growth of this mutant on a PPIX-based medium. Exogenously supplied catalase restored the growth of this mutant with heme sources to nearly wild-type levels. These results indicate that expression of a wild-type OxyR protein by H. influenzae is essential to allow this organism to protect itself against oxidative stresses in vitro.

Nutrition of uremic patients in the course of CAPD treatment.

Our aim was an evaluation of daily protein and energy intake, plasma protein, albumin, and cholesterol concentrations as well as total iron binding capacity in the course of continuous ambulatory peritoneal dialysis (CAPD) for up to 36 months. Our results indicate that CAPD patients, despite adequate clinical laboratory scores for up to 36 months of treatment, usually do not show optimal protein intake. When protein calorie malnutrition is prolonged, plasma proteins decrease. On the other hand, the greater the peritoneal permeability the lower the plasma protein intake.

Identification and characterization of genes encoding the human transferrin-binding proteins from Haemophilus influenzae.

Haemophilus influenzae, a strict human pathogen, acquires iron in vivo through the direct binding and removal of iron from human transferrin by an as yet uncharacterized process at the bacterial cell surface. In this study, the tbpA and tbpB genes of H. influenzae, encoding the transferrin-binding proteins Tbp1 and Tbp2, respectively, were cloned and sequenced. Alignments of the H. influenzae Tbp1 and Tbp2 protein sequences with those of related proteins from heterologous species were analyzed. On the basis of similarities between these and previously characterized proteins, Tbp1 appears to be a member of the TonB-dependent family of outer membrane proteins while Tbp2 is lipid modified by signal peptidase II. Isogenic mutants deficient in expression of Tbp1 or Tbp2 or both proteins were prepared by insertion of the Tn903 kanamycin resistance cassette into cloned sequences and reintroduction of the interrupted sequences into the wild-type chromosome. Binding assays with the mutants showed that a significant reduction in transferrin-binding ability resulted from the loss of either of the Tbps and a complete loss of binding was evident when neither protein was expressed. Loss of either Tbp2 or both proteins correlated with an inability to grow on media supplemented with transferrin-bound iron as the sole source of iron, whereas the Tbp1+ Tbp2- mutant was able to grow only at high transferrin concentrations.

Erythropoietin with oral iron in peritoneal and hemodialysis patients. A comparison in an inner city population.

Studies on the comparative efficacy of erythropoietin (rHuEPO) in chronic hemodialysis (HD) and peritoneal dialysis (PD) patients are scarce. The authors compared the use of rHuEPO in 74 stable patients on hemodialysis with 24 on chronic peritoneal dialysis. All patients were on oral iron supplements. In PD patients, hematocrits were 23.1 and 30.1%, rHuEPO dose 80.9 and 89.0 U/kg/wk, whereas in HD patients, hematocrits were 21.2 and 27.5 and rHuEPO dose was 140.2 and 165.0 U/kg/wk at initiation and 6 months, respectively. Serum iron and transferrin saturations were unchanged in peritoneal, but decreased in hemodialysis patients on rHuEPO therapy. These findings suggest that rHuEPO is more effective in peritoneal dialysis patients than in hemodialysis patients receiving oral iron. The improved efficacy of rHuEPO in peritoneal dialysis may be due to decreased blood loss, subcutaneous administration, or better removal of inhibitors of erythropoiesis. Peritoneal dialysis may be more cost effective and desirable than hemodialysis for rHuEPO dependent or resistant patients.

Nonspecific antibacterial factors in milk from cows immunized with human oral bacterial pathogens.

Both the immunoglobulins and non-specific antibacterial factors in milk from cows immunized with pathogenic oral bacteria have the potential to influence the oral microflora during passive immunization studies. The first six milks after calving were collected from 2 cows immunized with adjuvant and from 14 cows immunized with adjuvant and heat-killed strains of periodontopathic Actinomyces, Porphyromonas, Prevotella, and Fusobacterium. Analysis of the products from the first to the sixth milks revealed that the protein and lysozyme content decreased approximately 66 and 72%, respectively; the mean specific activity of the enzyme remained relatively constant. In contrast, the mean lactoperoxidase activity increased 2.3-fold in the second milking and increased further in the fourth and sixth milkings. The mean iron-binding activity increased 1.2-fold from the first to the second milkings and then decreased 3.6-fold through the sixth milking. Cows immunized with adjuvant alone showed similar responses. Per unit volume, the milk contained approximately 150 times less lysozyme than whole human saliva obtained from six subjects but higher concentrations of lactoperoxidase and iron-binding components. Purified bovine nonspecific factors prevented the growth of the bacteria used for immunization when bacteria were tested at concentrations similar to those found in saliva and milk. Because bovine nonspecific antibacterial factors could influence both the pathogenic target bacteria and the indigenous microflora in oral passive immunization studies with bovine immunoglobulins, the presence of these proteins should be considered.

Identification and purification of transferrin- and lactoferrin-binding proteins of Bordetella pertussis and Bordetella bronchiseptica.

Bordetella pertussis and Bordetella bronchiseptica were both able to grow in iron-deficient medium when supplemented with iron-saturated human lactoferrin or transferrin but not with human apotransferrin. Direct contact between the transferrins and the Bordetella cells did not appear to be required for growth but considerably improved the growth of the organisms. Analysis of B. pertussis and B. bronchiseptica whole-cell lysates from cultures carried out in iron-deficient or iron-replete media revealed iron-repressible proteins (IRPs) of 27 kDa in B. pertussis and of 30, 32, 73.5, and 79.5 kDa in B. bronchiseptica. Iron-inducible proteins of 16, 23.5, 36.5, and 92.5 kDa and of 17, 23.5, 70, 84, and 91 kDa were also identified in B. pertussis and B. bronchiseptica, respectively. By use of affinity chromatography with iron-saturated human lactoferrin or transferrin as ligands, the 27- and 32-kDa IRPs from B. pertussis and B. bronchiseptica, respectively, were specifically isolated. By using iron-chelated affinity columns, we showed that these proteins exhibit an affinity for iron. Cell fractionation experiments indicated that both of these proteins are probably associated with the outer membrane. Growth of the organisms under modulating conditions showed that the production of these IRPs is not under the genetic transcriptional control of vir or bvg, the general virulence regulon in Bordetella spp.

Bactericidal activity of M14659 enhanced in low-iron environments.

The bactericidal activity of M14659 against Escherichia coli in low-iron environments was investigated and compared with that of ceftriaxone and ceftazidime. The bactericidal activity of M14659 against E. coli in Mueller-Hinton broth was enhanced 30- to 20,000-fold by addition of transferrin, which is an iron-binding protein, whereas the activity of ceftriaxone or ceftazidime was much less strongly affected. This enhancement by transferrin was completely inhibited by saturating the iron-binding capacity of transferrin with FeCl3. M14659 was taken up markedly into bacterial cells in the presence of transferrin, and its uptake was inhibited by the protonophore dinitrophenol, which inhibits active-transport systems coupled to an energized membrane such as the iron transport systems of E. coli. The bactericidal activity of M14659, which chelates Fe3+, was also enhanced in the presence of other iron-binding compounds such as lactoferrin and alpha,alpha'-dipyridyl or in iron-deficient Mueller-Hinton broth (Fe3+ concentration, less than 2 nM) supplemented with FeCl3 at 0.1 to 1.0 microM, but not in unsupplemented iron-deficient Mueller-Hinton broth. The E. coli used in this study was confirmed to derepress iron transport systems in the presence of transferrin, lactoferrin, and alpha,alpha'-dipyridyl and in the iron-deficient Mueller-Hinton broth supplemented with FeCl3 at 0 to 1.0 microM. M14659 also showed an excellent antibacterial activity in vitro against other gram-negative bacteria in the low-iron environments. These findings indicate that M14659 may be actively taken up with Fe3+ into bacterial cells, probably through the iron transport systems under conditions of low iron and, thus, kills bacteria effectively.

Iron, iron-binding proteins and immune system cells.

In summary, the work reviewed in the present paper indicates that 1. Iron and the iron-binding proteins can act as regulators of immune function, and not only as a result of a nutritional dependence of lymphoid cells on transferrin and transferrin-iron. Subsets of cells of the immune system respond differently to increases in iron concentration in vitro and in vivo. 2. Macrophages and lymphocytes differ in the H and L subunit content of the ferritins synthesized in response to increases in iron concentration in vitro. 3. NK activity by adherent and nonadherent cells differ in their susceptibility to the enhancing effect of lactoferrin in vitro. 4. Responses to mitogen stimulation by PHA and Con A are diminished, while the PWM response remains unaffected by exposure to acidic ferritins or by increasing concentrations of iron in vitro and in vivo. 5. Pretreatment of effector but not target cells with iron results in diminished responses in the MLR, an effect that appears to be related to the HLA-A locus. 6. In situ hybridization studies indicate that transferrin is synthesized by a specific subset of the T lymphocytes. 7. Transient increases in serum iron concentration above the full saturation of transferrin, reproducing the clinical situation frequently seen in hereditary hemochromatosis, are followed by a series of cellular changes in the synovium that can be correlated to changes in the course of an experimental model of arthritis in the rat.

E-0702, a new cephalosporin, is incorporated into Escherichia coli cells via the tonB-dependent iron transport system.

E-0702, a new cephalosporin with a potent antipseudomonal action, was synthesized. In the study of the mode of action of this antibiotic in Escherichia coli, it was found that mutants which acquired resistance to E-0702 were isolated spontaneously and could be shown to be susceptible to its closely related derivatives, E-0702-060 and E-0702-061, and other representative beta-lactam antibiotics. In these mutants, no increased production of beta-lactamase was detectable. No apparent differences between the resistant mutants and the parental strains were observed in the affinity of E-0702 for penicillin-binding proteins. Furthermore, no significant reduction in or loss of both OmpF and OmpC porin proteins in the outer membrane was observed. The mutation was mapped to the tonB gene, which is known to be essential for the iron transport system of bacteria. The bactericidal action of E-0702 was rapidly expressed against iron-starved cells in which the iron transport system was induced, whereas the bactericidal action against iron-supplemented cells was ineffective. It is suggested that E-0702 is incorporated into bacterial cells as a chelator of iron via the tonB-dependent iron transport system, after which its strong and rapid bactericidal action is manifested.

Iron, infection, and neoplasia.

In nearly all forms of life, the number and diversity of enzymes that contain iron or that depend on the presence of this metal for activity are impressive. Not surprisingly, chemical mechanisms have been evolved by many organisms that permit them to solubilize and acquire iron while at the same time depriving their competitors or their pathogens of this element. Proteins such as transferrin and lactoferrin that are employed by vertebrate hosts for iron transport and acquisition can, to some extent, withhold the metal from the siderophores of invading bacteria and fungi. Attempts also are made by animal hosts to withhold iron from protozoa and neoplastic cells. Unfortunately, pathogenic microorganisms have developed a variety of counter measures that are especially dangerous in hosts stressed by iron overload in specific fluids, tissues, or cells. In recent years, however, a number of possible methods and agents for strengthening iron-withholding defense have become apparent. Nearly 3,000 papers on various aspects of iron withholding are contained in the 18-year Medline Database and numerous reviews have been published since 1966. The present paper will focus on developments that have been reported within the past 2 1/2 years.

Levels of iron, vitamin B12, folic acid and their binding proteins during pregnancy.

One hundred pregnant women, not anemic and not receiving iron or vitamin supplements, were chosen at random among the three trimesters of pregnancy to determine the incidence of unexpected iron, plasma folate and/or cobalamin deficiency, and the significance of fluctuating levels of their respective binding proteins. Pregnant females had a nonsignificant fall in serum iron and a fourfold decline in serum ferritin in the 3rd trimester compared with 1st trimester values. There was a steady decrease in plasma cobalamin and cobalophilin levels in every trimester but no difference in transcobalamin II values. Unsaturated cobalamin (UBBC) and unsaturated transcobalamin binding capacity (UTCBC) were lower in the 1st trimester fully recovering afterwards. Plasma folate levels were not lower, although there was a steady reduction in total and unsaturated folate binding capacity throughout pregnancy.

Enhanced mobilization of iron from body stores in malnourished patients during intravenous nutritional support.

Abnormal iron metabolism, characterized by low serum iron concentration and diminished saturation of iron-binding capacity, is known to occur in patients with malignant or inflammatory diseases. In this study, serum iron concentration and iron-binding capacity were measured in 21 patients with malignant disease, trauma or inflammatory illness before and during intravenous nutritional support without iron supplementation. Prior to nutritional support, the serum iron value averaged 26.0 +/- 3.5 micrograms per deciliter and the iron-binding capacity, 160.0 +/- 14.0 micrograms per deciliter. After ten to 21 days of parenteral nutrition, the serum iron-binding value serum iron concentration rose to an average of 68 +/- 9 micrograms per deciliter and the iron-binding capacity to 200 +/- 15 micrograms per deciliter, and increased reticulocytosis was observed. The rise in serum iron value reflected a shift of iron from body stores to plasma, since the saturation of iron-binding capacity increased from 18.0 +/- 3.5 to 36.0 +/- 4.0 per cent. Fever, granulocytosis, malignant disease, pulmonary infiltrates or open wounds persisted in all patients throughout the observation, ruling out resolution of a chronic process as the cause for rising serum iron levels. Changes in serum ceruloplasmin and ascorbate values also could not account for the rise. We propose the provision of adequate nutritional substrates to establish an anabolic state during total parenteral nutrition is associated with improved mobilization of iron from body stores.