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PubMed1997

Regulation of cellular iron metabolism by erythropoietin: activation of iron-regulatory protein and upregulation of transferrin receptor expression in erythroid cells.

Erythropoietin (Epo) is the central regulator of red blood cell production and acts primarily by inducing proliferation and differentiation of erythroid progenitor cells. Because a sufficient supply of iron is a prerequisite for erythroid proliferation and hemoglobin synthesis, we have investigated whether Epo can regulate cellular iron metabolism. We present here a novel biologic function of Epo, namely as a potential modulator of cellular iron homeostasis. We show that, in human (K562) and murine erythroleukemic cells (MEL), Epo enhances the binding affinity of iron-regulatory protein (IRP)-1, the central regulator of cellular iron metabolism, to specific RNA stem-loop structures, known as iron-responsive elements (IREs). Activation of IRP-1 by Epo is associated with a marked increase in transferrin receptor (trf-rec) mRNA levels in K562 and MEL, enhanced cell surface expression of trf-recs, and increased uptake of iron into cells. These findings are in agreement with the well-established mechanism whereby high-affinity binding of IRPs to IREs stabilizes trf-rec mRNA by protecting it from degradation by a specific RNase. The effects of Epo on IRE-binding of IRPs were not observed in human myelomonocytic cells (THP-1), which indicates that this response to Epo is not a general mechanism observed in all cells but is likely to be erythroid-specific. Our results provide evidence for a direct functional connection between Epo biology and iron metabolism by which Epo increases iron uptake into erythroid progenitor cells via posttranscriptional induction of trf-rec expression. Our data suggest that sequential administration of Epo and iron might improve the response to Epo therapy in some anemias.

Animals↗

PubMed1996

Telomere changes in human hepatocellular carcinomas and hepatitis virus infected noncancerous livers.

BACKGROUND: Telomeres, at the ends of eukaryotic chromosomes, are defined functionally as necessary for chromosome stability. Chromosome instability induced in part by loss of telomeric DNA has been considered to play a significant role in the development of human cancers. However, little is known about the status of telomeres per se during human hepatocellular carcinoma (HCC) development. METHODS: The study was conducted to determine the length of terminal restriction fragments (TRFs) at the telomeric ends in 23 HCCs and 23 corresponding noncancerous liver tissues from the same patients harboring hepatitis virus infections, and in 5 samples of noncancerous livers without the hepatitis virus. The latter samples had been obtained as controls at surgery for metastatic liver tumors. RESULTS: All 23 HCCs demonstrated reduction in TRF length compared with the corresponding noncancerous liver tissues with viral infection. The TRF in the latter cases also demonstrated a significant shortening as compared with the virus free control tissue, the degree being calculated to represent an average of 42 cell divisions. Reduction in TRF length in HCC samples tended to increase with the tumor diameter, although this failed to show statistical significance. CONCLUSIONS: These results indicate that telomere shortening occurs during HCC development. The fact that this change is already evident in noncancerous liver tissues from patients with viral hepatitis suggests that it may play an important role in the associated generation of tumors.

Adult↗

PubMedDate not supplied

Comparative studies on the interaction of proteins with a polydimethylsiloxane elastomer. I. Monolayer protein capture capacity (PCC) as a function of protein pl, buffer pH and buffer ionic strength.

Polydimethylsiloxane (PEP) is widely used in medical prostheses and therefore is in contact with plasma and secretory proteins. Two pair of globular proteins, lactoferrin (Lf) and transferrin (Trf), and bovine IgG1 and IgG2a, which differ substantially between pair members in their pl, were used to study the interaction of a PEP widely used in breast implants and soluble protein. Studies were done using iodinated proteins over a concentration range that resulted in an apparent protein monolayer. Secondary incubations with dilute protein solutions were needed to form the monolayer on PEP, possibly as a consequence of micro air bubbles trapped on its highly textured surface as shown by atomic force microscopy. Immunoassay quality polystyrene microtiter wells were used as controls. Adsorption studies were routinely performed at pH 4, 7 and 10 and at ionic strengths corresponding to 0.95, 9.5 and 90.0 mS. The protein capture capacity (PCC) of PEP for Lf and Trf was optimal at physiological pH and ionic strength and comparable under these conditions to that of Immulon 2 (Imm 2) microtiter wells. While increasing the ionic strength and pH further increases the PCC of Imm 2 for Lf and Trf, this markedly lowered the PCC of PEP for these proteins suggesting that initial polar interactions may precede subsequent hydrophobic bonding to PEP. This was tested using a hydrophilic variant of PEP, which when tested in a 90.0 mS buffer, showed a > five-fold lower PCC at neutral and alkaline pH. The greatly reduced PCC of the hydrophilic variant might also suggest that hydrophilic variants of silicone would be more biocompatible than those currently used. The PCC of PEP for the IgGs was less than that of Imm 2 but still optimal at physiological conditions. Consistent with the data on Lf/Trf, PCC progressively decreased with increasing ionic strength at alkaline pH. Differences in pl between the protein pairs had only a marginal effect on the PCC of PEP. Monolayer adsorption on both PEP and Imm 2 was slowly reversible and greater in the presence of free ligand (< 2% in 16 h) suggesting that the process follows Mass Law principles. However, even in the presence of non-ionic detergent and free ligand, 85-90% remained bound on either surface. Thus, desorption of proteins in the monolayer should not complicate subsequent immunochemical studies conducted on adsorbed monolayers.

Adsorption↗

PubMed1994

Time-resolved fluorometry in the diagnosis of Leber hereditary optic neuroretinopathy.

We have applied time-resolved fluorometry (TRF) to construct a DNA hybridization assay for the diagnosis of Leber hereditary optic neuroretinopathy (LHON). A rapid and reliable detection of the most prevalent mitochondrial DNA (mtDNA) point mutation associated with LHON is demonstrated. In addition, the TRF-method can be used in the quantification of heteroplasmy, a phenomenon commonly present in mtDNA mutations. The assay includes PCR amplification of a fragment encompassing the mutation site followed by hybridization reactions with allele-specific europium (Eu)-labelled oligonucleotide probes. A time-resolved fluorometer is used to measure the bound label. The TRF assay was successfully used to demonstrate the ND4/11778 mutation in patient samples. For quantification of heteroplasmy, synthetic target oligonucleotide mixtures with known ratios of wild-type and mutated sequences were used as standards to control the hybridization step. The assay allowed the detection of heteroplasmy ranging from 5 to 95%. This was also shown in a family with several heteroplasmic members.

Base Sequence↗

PubMed2005

Telomere length analysis in goat clones and their offspring.

Incomplete epigenetic reprogramming of the donor genome is believed to be the cause behind the high rate of developmental mortality and post-natal anomalies observed in animal clones. It appears that overt phenotypic abnormalities are not transmitted to their progeny suggesting that epigenetic errors are corrected in the germline of clones. Here, we show variation in telomere lengths among Nigerian dwarf goat clones derived from different somatic cell types and that the offspring of two male clones have significantly shorter telomere lengths than age-matched noncloned animals. Telomere lengths were significantly shorter in skin biopsies of goat clones derived from adult granulosa cells compared to those measured for controls. Telomere lengths were highly variable in male goat clones reconstructed from fetal fibroblasts but their mean terminal repeat fragment (TRF) length was within normal range of normal goats. However, in the progeny of two male clones, mean TRF lengths were considerably shorter than age-matched controls for both skin and leukocyte samples. Evidence for possible inheritance of shortened telomeres was obtained by measuring telomere lengths in testicular biopsies obtained from the clones, which when compared with those from noncloned animals of a similar age were significantly shorter. The offspring exhibited telomere lengths intermediate to the TRF values obtained for their cloned fathers' and age-matched control testes. These results demonstrate that telomere length reprogramming in clones is dependent on the type of donor cell used and that the progeny of clones may inherit telomere length alterations acquired through the cloning procedure.

Aging↗

PubMed2000

A comparison of biodistribution of liposomal and soluble IL-2 by a new method based on time-resolved fluorometry of europium.

A novel method was developed to determine the pharmacokinetics and biodistribution of cytokines and lymphokines based on time-resolved fluorometry (TRF) of europium (Eu). The comparison of two formulations of IL-2 was used to illustrate the sensitivity and applicability of this method as well as to extend the information on the pharmacokinetics of liposomal IL-2 and soluble IL-2. The blood kinetics and biodistribution of liposomal and soluble IL-2 in lymphoid organs and kidneys as measured by TRF were similar to those determined by the radioisotopic method. In both instances, the formulation of IL-2 into liposomes increased its serum half-life and accumulation in reticuloendothelial and lymphoid organs. The increased sensitivity of the Eu/TRF method permitted the extension of observational time points and the analysis of biodistribution in organs such as lymph nodes and bone marrow. These results suggest that Eu-labelled proteins in conjunction with TRF offer a suitable alternative to radiolabelled proteins for pharmacokinetics and tissue distribution studies in animals. This method offers distinct advantages over traditional techniques employing radioistopes since it has greater sensitivity, no half-life limitations and no radioactive or hazardous waste disposal.

Animals↗

PubMed1998

Dissociation of telomere dynamics from telomerase activity in human thyroid cancer cells.

Prevention of telomere erosion through acquisition of telomerase activity is thought to be an essential mechanism in most human cancer cells for avoidance of cellular senescence and crisis. It has been generally assumed that once telomerase has been activated, no further telomere shortening should ensue. We show here, however, that a much more complex pattern of telomere dynamics can exist in telomerase-positive immortal cancer cells. Using a panel of subclones derived from a human thyroid cancer cell line, K1E7, we found that some clones show persistent decline in mean telomere restriction fragment (TRF) length by up to 2 kb over 450 population doublings (pd), despite sustained high telomerase activity (as assessed by the in vitro "TRAP" assay). TRF length subsequently stabilized at around 5 kb, but with no corresponding increase in telomerase activity. One clone showed an even more unexpected biphasic time course, with the mean TRF length initially increasing by 1.5 kb over 90 pd, before "plateauing" and then returning over a similar period to its original value, again without any correlation to TRAP activity. Such dissociations between telomere dynamics and telomerase activity support the existence of additional controls on telomere length in the intact cell. Our observations are consistent with current negative-feedback models of telomere length regulation by telomere binding proteins and these cell lines should prove useful experimental tools for their further evaluation.

Humans↗

PubMed1999

Telomere length in fibroblasts and blood cells from healthy centenarians.

Several lines of evidence indicate that telomere shortening during in vitro aging of human somatic cells plays a causal role in cellular senescence. A critical telomere length seems to be associated with the replicative block characterizing senescent cells. In this paper we analyzed the mean length of the terminal restriction fragments (TRF) in fibroblast strains from 4 healthy centenarians, that is, in cells aged in vivo, and from 11 individuals of different ages. No correlation between mean TRF length and donor age was found. As expected, telomere shortening was detected during in vitro propagation of centenarian fibroblasts, suggesting that in fibroblasts aged in vivo telomeres can be far from reaching a critical length. Accordingly, chromosome analysis did not show the presence of telomeric associations in early passage centenarian fibroblasts. In blood cells from various individuals, the expected inverse correlation between mean TRF length and donor age was found. In particular, a substantial difference (about 2 kb) between telomere length in the two cell types was observed in the same centenarian. Expression analysis of three senescence-induced genes, i.e., fibronectin, apolipoprotein J, and p21, revealed for only the fibronectin expression levels a clear positive correlation with donor age. Our results suggest that (1) telomere shortening could play a different role in the aging of different cell types and (2) the characteristics of fibroblasts aged in vitro might not be representative of what occurs in vivo.

Adolescent↗

PubMed1976

An electrophoretic polymorphism in the serum transferrins of Praomys (Mastomys) natalensis.

Three serum transferrin phenotypes of Praomys (Mastomys) natalensis, and African rodent having a high incidence of many varieties of spontaneous neoplasms, were detected by electrophoresis and autoradiography. On electrophoresis, transferrin of Trf-K was found to be a protein that migrates slowly toward the cathode, whereas that of Trf-M migrates rapidly. Trf-KM is a heterozygous phenotype of the above two. Progeny testing demonstrated that these three serum transferrin phenotypes in Mastomys are probably controlled by two codominant allelic genes, Trfk and Trfm, at a single autosomal locus. The genotypes of these phenotypes may be Trfk/Trfk, Trfm/Trfm, and Trfk/Trfm.

Animals↗

PubMed1995

Behavioral and emotional problems among Chinese and American children: parent and teacher reports for ages 6 to 13.

This study compared behavioral and emotional problems reported by parents and teachers in Chinese urban and rural samples and demographically similar American samples. Parents of 469 6-to-13-year-old children of each nationality completed the Child Behavior Checklist (CBCL). Teachers completed the Teacher's Report Form (TRF). Cross-cultural differences were generally modest in magnitude. Chinese children scored higher on TRF Delinquent Behavior and Anxious/Depressed syndromes, and on Internalizing. American children scored higher on CBCL Aggressive Behavior and TRF Attention Problems syndromes. Boys exhibited more externalizing behaviors across both cultures. The mean correlation between parent and teacher ratings was .36 in the Chinese sample and .29 in the American sample, a nonsignificant difference. Findings indicate considerable similarity between problems reported for children in very different societies.

Adolescent↗

PubMed1996

Cross-cultural generalizability of the Youth Self-Report and Teacher's Report Form cross-informant syndromes.

Exploratory factor analyses on 569 Youth Self-Reports and 1,221 Teacher's Report Forms of clinically referred Dutch children revealed six and eight factors respectively, very similar to the eight YSR and TRF cross-informant syndromes derived by Achenbach (1991c, 1991d). Mean cross-cultural correlations were .89 for YSR syndromes and .95 for TRF syndromes. In confirmatory factor analyses of the Dutch and American YSR and TRF factor models in cross-validation samples of 570 YSRs and 1,221 TRFs, goodness-of-fit indices were only slightly better for Dutch factor models. The American cross-informant Social Problems and Attention Problems syndromes had the poorest fit. The application of the eight American cross-informant syndromes to Dutch self- and teacher reports was supported.

Adolescent↗

PubMed1996

Accelerated loss of telomeric repeats may not explain accelerated replicative decline of Werner syndrome cells.

The Werner syndrome (WS) is characterized by the premature onset and accelerated rate of development of major geriatric disorders, including atherosclerosis, diabetes mellitus, osteoporosis, ocular cataracts, and various neoplasms. Cultures of WS skin-fibroblastlike cells have been previously shown to undergo accelerated rates of decline of the replicative potentials and to exhibit variegated chromosomal translocations and deletions. Since the replicative decline of normal somatic cells is associated with a loss of telomeric repeats, we investigated the kinetics of telomeric repeat loss in WS cells. The mean length of telomere restriction fragments (TRF) from the earliest passages of WS cells studied was not shorter than those of controls, possibly reflecting selective pressure for subsets of cells with relatively high residual replicative capacity. Statistical evidence indicated an accelerated shortening of TRF length in serially passaged WS cultures, but the mean TRF lengths of WS cultures that had ceased replicating were significantly longer than those of senescent controls. Thus, while accelerated loss of telomeric repeats could potentially explain the rapid decline in proliferation of WS cells, it is possible that WS cells exit the cell cycle via mechanisms that differ from those of replicatively senescent cells from control subjects.

Adult↗

PubMed2000

Antiproliferative and apoptotic effects of tocopherols and tocotrienols on normal mouse mammary epithelial cells.

Studies were conducted to determine the comparative effects of tocopherols and tocotrienols on normal mammary epithelial cell growth and viability. Cells isolated from midpregnant BALB/c mice were grown within collagen gels and maintained on serum-free media. Treatment with 0-120 microM alpha- and gamma-tocopherol had no effect, whereas 12.5-100m microM tocotrienol-rich fraction of palm oil (TRF), 100-120 microM delta-tocopherol, 50-60 microM alpha-tocotrienol, and 8-14 microM gamma- or delta-tocotrienol significantly inhibited cell growth in a dose-responsive manner. In acute studies, 24-h exposure to 0-250 microM alpha-, gamma-, and delta-tocopherol had no effect, whereas similar treatment with 100-250 microM TRF, 140-250 microM alpha-, 25-100 microM gamma- or delta-tocotrienol significantly reduced cell viability. Growth-inhibitory doses of TRF, delta-tocopherol, and alpha-, gamma-, and delta-tocotrienol were shown to induce apoptosis in these cells, as indicated by DNA fragmentation. Results also showed that mammary epithelial cells more easily or preferentially took up tocotrienols as compared to tocopherols, suggesting that at least part of the reason tocotrienols display greater biopotency than tocopherols is because of greater cellular accumulation. In summary, these findings suggest that the highly biopotent gamma- and delta-tocotrienol isoforms may play a physiological role in modulating normal mammary gland growth, function, and remodeling.

Animals↗

PubMed1999

Achenbach's Child Behavior Checklist and Teachers' Report Form in a normative sample of Greek children 6-12 years old.

Achenbach's Child Behavior Checklist (CBCL) and Teachers' Report Form (TRF) were administered to 6-12 year old school children comprising a large random community sample (n = 1200) drawn from the whole of Greece. These are the first data on the TRF in Greece and the first nation-wide data on the CBCL. Appropriate cutoff points for the behavioral problems and competence scales of both questionnaires were obtained for boys and girls. These were considerably higher than USA cutoffs for the CBCL but not for the TRF. Analysis of scores in relation to degree of urbanization showed that it was not necessary to define different cutoffs in different strata. Parents' and teachers' ratings of the same child were most highly correlated for Externalizing and Aggressive behavior for boys and for Attention problems for both sexes.

Child↗

PubMed1975

Postreplication DNA repair in ultraviolet-irradiated Micrococcus luteus.

Postreplication DNA repair was studies in three strains of Micrococcus luteus having different sensitivity to ultraviolet light: a wild type ATCC 5698, a ultraviolet-sensitive mutant G7, deficient in the incision step of repair and in ultraviolet-resistant transformant obtained from G7 by treatment with DNA of wild type cells, Trf(G7). It is shown that the G7 mutant has a low capacity for repair of postreplication DNA gaps compared with the wild type or Trf(G7). It seems to be that postreplication repari capacity contributes significantly to the ultraviolet resistance of M. luteus in addition to the excision repair. In contrast with G7 the size of the DNA fragments synthesized immediately after ultraviolet irradiation in the wild type (and Trf(G7)) seems to be much higher than that expected if each dimer produces one DNA gap in the daughter strand. Since this cannot only be explained by the excision of dimers from parental DNA we have suggested that a rapid repair of postreplication DNA gap occurs in M. luteus.

Centrifugation, Density Gradient↗

PubMed1984

Dependence of T-cell-replacing factor and immunogenic dose for the production of monoclonal antibodies using the in vitro immunization technique.

Antigen-specific hybridomas can be produced using spleen cells that have been immunized in culture as parental cells in the hybridization step. The in vitro immunization of non-immune mouse spleen cells was supported by a lymphokine preparation that contained T-cell-replacing factor (TRF). The influence of TRF, produced by a mixed-thymocyte reaction, on immunization in culture has been investigated using bacterial cells as the immunogen. The cell vols of stimulated splenocytes were monitored and it was found that the induction of antigen-specific blast cells, which could subsequently be immortalized by fusing them with myeloma cells, was completely abolished in immunizations which were not supported by TRF. If serum-free conditions were used during the in vitro immunization step, the frequency of antigen-specific blast cells increased, which resulted in a higher yield of specific hybridomas. This was due to the reduced background stimulation achieved by omitting serum proteins. The relationship between immunogenic dose and response, measured as the specific efficiency obtained in hybridization experiments with in vitro immunized cells, was recorded using different amounts of sperm whale myoglobin as antigen. An antigen-specific response was recorded with as low as 1 ng antigen/ml.

Animals↗

PubMed1988

Peptide injections into the amygdala of conscious rats: effects on blood pressure, heart rate and plasma catecholamines.

The central nucleus of the amygdala (Ce) mediates cardiovascular and autonomic changes associated with defense or fear responses. At least 16 different neuropeptides have been identified within nerve terminals within the Ce. The role that these peptides play in the Ce regulation of cardiovascular and autonomic function has been assessed. Neuropeptides were microinjected into the region of the Ce and mean arterial pressure (MAP), heart rate (HR) and plasma catecholamine concentrations were measured. Five of the 16 peptides caused changes of MAP and HR. Thyrotropin releasing factor (TRF) and calcitonin gene-related peptide (CGRP) induced increases of MAP and HR. Angiotensin-II (A-II) and somatostatin-28 (SS-28) injection produced increases of MAP and decreases of HR. Bombesin (Bom) injections into the Ce induced an increase of MAP but did not alter HR. Corticotropin releasing factor (CRF), TRF and CGRP were the only peptides found to increase plasma catecholamine concentrations. These results support the conclusion that the Ce contains several peptides that could be involved in the regulation of cardiovascular and autonomic nervous system function. A role of the amygdala in mediating the observed effects of CRF, TRF, CGRP, A-II, SS-28, and Bom is suggested by these studies.

Amygdala↗

PubMed2025

A general framework to over-express tRNA-derived fragments from their parental tRNAs in mammalian cells.

tRNA-derived fragments (tRFs), generated from the cleavage of mature or precursor tRNAs are a category of regulatory noncoding RNAs with diverse functions in physiological or pathophysiological conditions. Here we describe a framework for the over-expression of tRFs from their parental tRNAs in mammalian cells. The process involves bioinformatics analysis to identify specific tRNAs that produce the tRF, PCR amplification of corresponding tRNA genes, and insertion into expression vectors. Transfection is carried out in HEK293T cells and detection of tRFs is achieved through northern blotting and dual luciferase reporter assays. In the latter, a complementary sequence to the tRF of interest is inserted into the luciferase reporter. By observing the reduction in luciferase activity, we can validate the expression of tRFs. This method enables precise study of tRF functions and their roles in cellular processes.

Humans↗