Immunohistochemical localization of the transferrin receptor in the seminiferous epithelium of the rat

The transferrin receptor has been immunohistochemically localized in the seminiferous epithelium of the rat with a monoclonal antibody, MRC OX26, which recognizes the transferrin receptor glycoprotein. The receptor was detectable on mitotically and meiotically dividing germ cells and, less abundantly, on round spermatids. It was lost from germ cells during spermatid elongation and was undetectable on immature spermatozoa. The transferrin receptor was also present on Sertoli cells in the testes of immature animals and on Sertoli cells in the testes of aspermatogenic animals that had been irradiated in utero. It was not detectable on Sertoli cells in the testes of cryptorchid animals. These studies demonstrate that the transferrin receptor is abundant on dividing germ cells as well as dividing somatic cells.     

Extracellular H2O2 and not superoxide determines the compartment-specific activation of transferrin receptor by iron regulatory protein 1

Iron regulatory protein 1 (IRP1) functions as translational regulator that plays a central role in coordinating the cellular iron metabolism by binding to the mRNA of target genes such as the transferrin receptor (TfR)—the major iron uptake protein. Reactive oxygen species such as H₂O₂ and that are both co-released by inflammatory cells modulate IRP1 in opposing directions. While H₂O₂—similar to iron depletion—strongly induces IRP1 via a signalling cascade, inactivates the mRNA binding activity by a direct chemical attack. These findings have raised the question of whether compartmentalization may be an important mechanism for isolating these biological reactants when released from inflammatory cells during the oxygen burst cascade. To address this question, we studied cytosolic IRP1 and its downstream target TfR in conjunction with a tightly controlled biochemical modulation of extracellular and H₂O₂ levels mimicking the oxygen burst cascade of inflammatory cells. We here demonstrate that IRP1 activity and expression of TfR are solely dependent on H₂O₂ when co-released with from xanthine oxidase. Our findings confirm that extracellular H₂O₂ determines the functionality of the IRP1 cluster and its downstream targets while the reactivity of is limited to its compartment of origin.     

Change of composition of intermediate filaments in rat telencephalon during early postnatal period of ontogenesis

The goal of the present work was to study composition and spatial-temporal distribution of cells containing various proteins of intermediate filaments (nestin, vimentin, GFAP) in various brain areas at the early postnatal period of rat ontogenesis. By using methods of immunohistochemical determination of proteins of intermediate filaments, it has been found that at the early postnatal period of development, in the course of maturation of the nervous tissue, in the cells of cortex, hippocampus, and subventricular area, there occurred changes of immunohistochemical profile of intermediate filaments (ratio of immunopositive (+) and immunonegative (?) cells): nestin⁺/vimentin⁺/GFAP^? cells become nestin^?/vimentin^?/GFAP⁺.     

Action of novocain on cardiac and somatomotor autorhythmical functions in early postnatal ontogenesis in rats

In conscious rat pups aged 2–3, 10–11, and 22–23 postnatal days, it has been shown that intraperitoneal administration of 0.5% Novocain at a dose of 25 mg/kg body mass leads to phasic changes of the level of spontaneous periodic motor activity—from increased at once after the administration to depression and subsequent restoration. They are accompanied by the cardiac rhythm fluctuations that change with age their direction on the background of an increased motor activity from brady to tachycardia. All the changes are the most pronounced in newborns. At comparison of the results with the afferent impulsation level fluctuations recorded under the same conditions in the peripheral vagus segment, it is suggested that the revealed reactions are associated with changes of the interoceptive afferentation flow. The conclusion is made that at early stages of ontogenesis, interoception plays an important role in regulation of autorhythmical functions and that this role decreases with age.     

Direct correlation between the R2 component of the early receptor potential and the formation of Metarhodopsin II in the excised bovine retina

The formation of Metarhodopsin II, measured spectroscopically in the excised bovine retina at 37 C, can be described by the sum of two exponentials. The R2 response of a simultaneously recorded early receptor potential can be matched by the difference of two weighted exponentials, whose time constants are identical to those obtained for the spectroscopic signal.     

About the formation of an image in the inverted retina of the eye

Possible distortions of images of an object by the layer of nerve cells have been analyzed. It has been shown that, based on microoscillations of the visual apparatus, it is possible to propose the procedures of processing the arising diffraction scattering spectra and isolating from these spectra initial images that do not contradict the available morphological and neurophysiological data.     

Cloning, characterization, and modeling of a monoclonal anti-human transferrin antibody that competes with the transferrin receptor.

In this report we describe the isolation and characterization of a monoclonal antibody against human serum transferrin (Tf) and the cloning and sequencing of its cDNA. The antibody competes with the transferrin receptor (TR) for binding to human Tf and is therefore expected to bind at or very close to a region of interaction between Tf and its receptor. From the deduced amino acid sequence, we constructed a 3-dimensional model of the variable domains of the antibody based on the canonical structure model for the hypervariable loops. The proposed structure of the antibody is a first step toward a more detailed characterization of the antibody-Tf complex and possibly toward a better understanding of the Tf interaction with its receptor. The model might prove useful in guiding site-directed mutagenesis studies, simplifying the experimental elucidation of the antibody structure, and in the use of automatic procedures to dock the interacting molecules as soon as structural information about the structure of the human Tf molecule will be available.     

Involvement of transferrin in the reduction of iron by the transplasma membrane electron transport system

Nonpermeable electron acceptors can be reduced by a transplasma membrane electron transport system in suspensions of intact cells. Here we report that diferric transferrin is reduced by HeLa S3 cells. The reduction is recorded spectrophotometrically as the formation of the ferrous complex of bathophenanthroline disulfonate. Ferric ammonium citrate can also be used as an electron acceptor, and the presence of low concentrations of diferric transferrin greatly stimulates the reduction of trivalent iron under these conditions. Likewise very low concentrations of ferricyanide, which does not give rise to a ferrous bathophenanthroline disulfonate complex formation, have a strong stimulatory effect on the complex formation when ferric ammonium citrate is the source of ferric iron. Apotransferrin is a potent inhibitor of the reaction. The inhibition occurs at the concentration necessary for complete occupancy of the transferrin receptors. The inhibition can be demonstrated also when high concentrations of ferricyanide are used as electron acceptor. The possible mechanism behind the reported phenomena is discussed, and it is concluded that the transplasma membrane electron transport system can be involved in the process of cellular iron uptake.     

Dynamics of absorption in gut and reabsorption in kidney of yellow fluorescent protein in rat postnatal ontogenesis

In experiments on rats aged 5, 12, and 25 days and on adult rats, absorption of yellow fluorescent protein (YFP) in small intestine was shown, with its subsequent entry to kidney with blood flow and accumulation in cells of the nephron proximal segment. With age, intensity of the YEP absorption in the gut decreased; the YEP accumulation in kidney was somewhat more active in the rat pups of younger age groups than in adult animals. No accumulation of YEP was revealed in liver. The obtained results indicate an intensive absorption in the rat pup small intestine in early postnatal ontogenesis and an important role of kidney in protein metabolism and proteolysis of alien proteins.     

Transferrin receptor expression and the regulation of placental iron uptake

Placental transferrin receptors, located at the apical side of syncytiotrophoblast, mediate placental iron uptake. Regulation of transferrin receptors on the fetal-maternal exchange area could be a major determinant in the regulation of trans-placental iron transport.Transferrin receptor expression in cultured human term cytotrophoblasts is on a much lower level than in choriocarcinoma cells, with a higher proportion of receptors located on the cell surface. Differentiation of cells, either due to longer culture periods or to 8-bromo-cAMP treatment does not lead to an increase of transferrin receptor expression. In vitro, the level of expression is largely regulated by the cellular density in the culture dishes. Low cellular occupancy of the dish leads to a high level of transferrin receptors. Treatment with iron-sources results in a down regulation of transferrin receptors.Thus, though the level of transferrin receptors in cultured normal trophoblast is at a constant level, unaffected by differentiation, high levels of maternal transferrin-iron availability can lead to a decrease in placental iron uptake. This feed-back mechanism makes placental iron uptake independent of maternal iron stores.Abbreviations hCG human Chorionic Gonadotrophin - TfR Transferrin Receptor     

Role of seasonal factors in pre-and postnatal ontogenesis in etiology of type 1 diabetes mellitus

Type 1 diabetes mellitus is a chronic disease characterized by autoimmune degradation of insulinproducing β-cells. Seasonality of birth of children with type 1 diabetes has been shown in a number of epidemiological studies. It could testify that autoimmune process began during fetal and postnatal development. No such studies were carried out in the former Soviet Union countries. The aim of the present study is to compare the seasonal birth month pattern in patients with type 1 diabetes (10780 men and 9337 women) born in 1960–2003 to that in the total population of Ukraine (14 995768 men and 14 109792 women) born during the same period. Significant differences were found between these two populations: χ ² = 103.97, p < 0.0001 and 135.17, p < 0.0001 in men and women, respectively. The results of cosinor analysis showed similar sinusoidal birth patterns of patients with type 1 diabetes in all subgroups, irrespective of the age of clinical disease manifestation: 0–9, 10–19, or 20–29 years. In all cases, the highest and lowest predispositions to type 1 diabetes were inherent in the people born in spring and autumn, respectively. In all groups, the highest incidence rate was in persons born in spring and early summer and the lowest ones, in those born in autumn and early winter. We propose that seasonality of birth of patients with type 1 diabetes in the two above populations could be due to long-term programming of glucose-insulin metabolism caused by the effect of certain seasonal factors during early ontogenesis.     

The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP‐1B‐deficient epithelia

Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial‐specific clathrin adaptor AP‐1B. Some native epithelia lack AP‐1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP‐1B‐deficient epithelia to relocate AP‐1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP‐1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP‐1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus‐end kinesin KIF16B and non‐centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a‐dependent TfR recycling pathway of non‐polarized cells. They define a transcytotic pathway important for the physiology of native AP‐1B‐deficient epithelia and report the first microtubule motor involved in transcytosis.     

Ferroportin 1 and hephaestin expression in BeWo cell line with different iron treatment

The process of placental iron transfer is an important physiological process during pregnancy. However, the molecular mechanism of placental iron transport has not been completely elucidated until now. Ferroportin 1 (FPN1) and hephaestin (Heph) have been identified as the important molecules involved in duodenal iron export. However, whether they participate in the placental iron efflux has been undefined until now. In this study, the BeWo cells were treated with desferrioxamine and Holo-transferrin human in different concentrations and harvested at 48 and 72 h. The mRNA expression of FPN1 and Heph was detected with quantitative real-time polymerase chain reaction, and the protein expression was detected with western blots. The results showed an up-regulated FPN1 expression with desferrioxamine treatment and down-regulated expression with Holo-transferrin human supplementation. However, the change of FPN1 expression at protein level was limited. Heph expression enhanced when cells were treated with desferrioxamine although the quantity of Heph expression was low. Heph expression showed no significant change with Holo-transferrin human supplementation. It indicates that FPN1 may participate in placental iron transport, and placental FPN1 expression is obviously not dependent on the iron regular element/iron regular protein regulation. An alternatively spliced FPN1 isoform that lacks an iron regular element may be the predominant expression in BeWo cells. It also demonstrates that Heph is active in placenta but may not play a key role in placental iron transport because it is not the main part of placental copper oxidase.     

Calcium channels and GABA receptors in the early embryonic chick retina

The properties of calcium channels were studied at the period of neurogenesis in the early embryonic chick retina. The whole neural retina was isolated from embryonic day 3 (E3) chick and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). The retinal cells were depolarized by puff application of high-K⁺ solutions. Increases in intracellular Ca²⁺ concentrations were evoked by the depolarization through calcium channels. The type of calcium channel was identified as l-type by the sensitivity to dihydropyridines. The Ca²⁺ response was completely blocked by 10 μM nifedipine, whereas it was remarkably enhanced by 5 μM Bay K 8644. Then we sought a factor to activate the calcium channel and found that GABA could activate it by membrane depolarization at the E3 chick retina. Puff application of 100 μM GABA raised intracellular Ca²⁺ concentrations, and this Ca²⁺ response to GABA was also sensitive to the two dihydropyridines. Intracellular potential recordings verified clear depolarization by bath-applied 100 μM GABA. The Ca²⁺ response to GABA was mediated by GABA_A receptors, since the GABA response was blocked by 10 μgM bicuculline or 50 μM picrotoxin, and mimicked by muscimol but not by baclofen. Neither glutamate, kainate, nor glycine evoked any Ca²⁺ response. We conclude that l-type calcium channels and GABA_A receptors are already are already expressed before differentiation of retinal cells and synapse formation in the chick retina. A possibility is proposed that GABA might act as a trophic factor by activating l-type calcium channels via GABA_A receptors during the early period of retinal neurogenesis. © 1993 John Wiley & Sons, Inc.     

Expression of iron-related proteins during infection by bovine herpes virus type-1

Bovine herpesvirus 1 (BHV-1), a dsDNA animal virus, is an economically important pathogen of cattle and the aetiological agent of many types of disease. The efficient replication of a DNA virus is strictly dependent on iron since this metal plays a crucial role in the catalytic center of viral ribonucleotide reductase. Consequently, iron metabolism is an important area for virus/host interaction and a large body of evidence suggests that viral infection is potentially influenced by the iron status of the host. The aim of the present study was to address the effects of BHV-1 on iron metabolism in Madin-Darby bovine kidney (MDBK) cells at different times of post-infection. For this purpose, cell viability, iron regulatory proteins (IRPs) activity and levels, transferrin receptor 1 (TfR-1), ferritin expression and LIP were evaluated. Our data demonstrate that a productive BHV-1 infection in MDBK cells determines an overall decrease of IRPs RNA-binding activity without affecting their expression. As consequence of this modulation, an increased ferritin mRNA translation and a decreased TfR-1 mRNA translation were also observed. Moreover, the LIP level was decreased following viral infection. These results are consistent with the hypothesis that by reducing the iron up-take and by enhancing the sequestration of free iron, animal cells will limit the iron availability for virus proliferation. Therefore, the results presented herein support the view that iron metabolism could be critical for the interaction between DNA viruses, such as BHV-1, and mammalian cells. Delineation of the interplay among pathogen and host may provide new antimicrobial agents.     

Genetic polymorphisms in bovine transferrin receptor 2 (TFR2) and solute carrier family 40 (iron-regulated transporter), member 1 (SLC40A1) genes and their association with beef iron content

Beef is considered to be an excellent source of dietary iron. However, little is known about the genetic control of beef iron content. We hypothesized that genetic polymorphisms in transferrin receptor 2 (TFR2) and solute carrier family 40 (iron‐regulated transporter), member 1 (SLC40A1) could influence skeletal muscle iron content. The objective of this study was to use Angus cattle to identify single‐nucleotide polymorphisms (SNPs) in the exons and flanking regions of the bovine TFR2 and SLC40A1 genes and to evaluate the extent to which genetic variation in them was associated with bovine longissimus dorsi muscle iron content. Ten novel SNPs were identified in TFR2, of which one SNP tended to be associated (P < 0.013) with skeletal muscle iron content. Nine novel SNPs in SLC40A1, NC007300: rs133108154, rs137140497, rs135205621, rs136600836, rs134388440, rs136347850, rs134186279, rs134621419 and rs137555693, were identified, of which SNPs rs134388440, rs136347850 and rs137555693 were significantly associated (P < 0.007) with skeletal muscle iron content. High linkage disequilibrium was observed among SLC40A1 SNPs rs134388440, rs136347850 and rs137555693 (R² > 0.99), from which two haplotypes, TGC and CAT, were defined. Beef from individuals that were homozygous for the TGC haplotype had significantly (P < 0.001) higher iron content than did beef from CAT homozygous or heterozygous individuals. The estimated size of effect of the identified haplotypes was 0.3% of the phenotypic variance. In conclusion, our study provides evidence for genetic control of beef iron concentration. Moreover, SNPs identified in SLC40A1, rs134388440, rs136347850 and rs137555693 might be useful markers for the selection of Angus cattle for altered iron content.