Specific Binding Characteristics Research Articles

Consideraciones sobre la citogenética y los factores de proliferación celular en la oncogénesis de los astrocitomas anaplásicos y glioblastoma multiforme

All currently available therapies on anaplastic astrocytomas and glioblastoma multiforme are palliative. The goal of treatment is to provide the longest period of good quality life but the setup treatment achieve discouraging results.Now, by the neurogenetics and the molecular biology of human brain tumors studies new approaches of research activity shows future treatment strategies. So, the initiation and progression of a neuroepithelial tumor is the result of inappropiate expresion of protooncogen, which encoding systems with biological activities that influence cell differentiation and proliferation.With the production of monoclonal antibodies with high specific binding characteristics, inmunohistology, tumoral cytocinetics studies became the preferable method by which to evaluate the prognosis and treatment options. In this article, the pathogenesis and diagnosis technics are reviewed and we can appreciate Iike in cell cycle, all oncogenic influences must converge.

Characterization of two nuclear proteins that interact with cytochrome P-450 1A2 mRNA. Regulation of RNA binding and possible role in the expression of the Cyp1a2 gene.

Regulation of the expression of the cytochrome P-450 la2 gene (cyp1a2) occurs mainly at the transcriptional level, but the molecular events involved in the induction process are partly unknown. Some reports have proposed involvement of post-transcriptional mechanisms [Adesnik, M. & Atchison, M. (1986) Crit. Rev. Biochem. 19, 247-305; Silver, G. & Krauter, K. S. (1990) Mol. Cell. Biol. 10, 6765-6768]. Here we report the identification of two proteins in the nuclear fraction of mouse liver, with specific binding characteristics towards CYP1A2 mRNA. The proteins have apparent molecular masses of 37 kDa and 46 kDa and exhibit a high affinity for a poly(U) motif in the 3' untranslated region of CYP1A2 mRNA. This motif seems to be important for their specific and apparently competitive binding to CYP1A2 mRNA. Treatment of mice with an inducer of CYP1A2, 3-methylcholanthrene, increases the binding of the 46-kDa protein and decreases the binding of the 37-kDa protein to the mRNA, suggesting that changes in the binding of the proteins to the mRNA could play a role in the upregulation of CYP1A2 mRNA by 3-methylcholanthrene. Phosphorylation of the 46-kDa protein, or of an intermediary factor, may play a role in its binding activity. Furthermore, the 46-kDa but not the 37-kDa protein is recognized by a monoclonal antibody against the heterogeneous nuclear ribonucleoprotein C, a nuclear protein probably involved in pre-mRNA processing. While more work is needed to understand the function of the proteins that bind to the 3' untranslated region of CYP1A2, it is possible that the 37-kDa protein has a role in the maintenance of uninduced levels of CYP1A2 mRNA, while the 46-kDa protein could be important in the maturation of elevated levels of CYP1A2 pre-mRNA, during induction.

Antibody immunoglobulin G (IgG) against human prostatic specific antigen (PSA) as a carrier protein for chemotherapeutic drugs to human prostate tumors: Part 1. A double immunofluorescence analysis.

Adenocarcinoma of the prostate (CaP) is the the second highest cause of cancer deaths in U.S. males. Current chemotherapeutic and/or endocrine treatments do not specifically and selectively target tumor cells of prostate cancer and benign prostatic hyperplasia (BPH). We hypothesized that because of the specific binding characteristics of antibody immunoglobulin G (IgG) to human prostatic-specific antigen (PSA), PSA-IgG could function as a carrier protein for conjugated chemotherapeutic drugs and that the immunoconjugate would selectively bind to prostatic epithelial cells and their tumors, but not to epithelial cells of unrelated organs. Our objective was to test the hypothesis using human prostatectomy specimens. WE used several derivatives of 5'-fluorouracil, namely, 5'-fluoro- 2'-deoxyuridine (5'-Fu-2'-d), 5'-fluoro-2'-deoxyuridine-5' monophosphate (5'-Fu-2'-d-5'-mp), 5'-fluoro-2'-deoxyuridine-5'-(p-aminophenyl) monophosphate (5'-Fu-2'-d'-5'-amp), to conjugate with rabbit anti-PSA-IgG together with fluorescent markers (such as rhodamine and fluorescein or fluorescein isothiocyanate: FITC). Prostate specimens were obtained from prostatectomy patients who had not been treated with cytotoxic drugs before surgery. We evaluated formalin-fixed and paraffin-embedded sections as well as cryostat sections of frozen specimens for localization of PSA-IgG alone and PSA-IgG-drug immunoconjugate using immunoperoxidase (IP) and single and/or double immunofluorescence (IF) localization techniques. Our study showed that the immunoconjugate (PSA-IgG-5'-Fu-2'-d) bound to PSA (molecular size of approximately 34 KDa) on nitrocellulose sheets in Western immunoblots of extracts of BPH and CaP tissues. This binding of immunoconjugate to PSA on immunoblots was similar to that of the unconjugated PSA-IgG. Immunostaining patterns for rabbit anti-PSA-IgG and PSA-IgG-5'-Fu-2'-d immunoconjugate were similar and specific for prostate epithelial cells and their tumors, as revealed by IP techniques. To demonstrate that both the antibody and drug localized in the same group of prostatic epithelial cells, we used an immunoconjugate in which the PSA-IgG was labeled with rhodamine and 5'-Fu-2'-d-5'-amp with FITC. Our study showed that fluorescence for rhodamine and FITC was present in the same group of prostatic epithelial cells. Phase contrast microscopy demonstrated details of prostatic glandular epithelium and connective tissues. Our study showed that fluorescence for rhodamine and FITC and immunostaining by IP techniques were not observed in prostate sections incubated with normal rabbit serum. We have shown that conjugation of 5'-Fu derivatives to PSA-IgG did not affect either the selectivity or specificity of the antibody for prostatic epithelial cells. Differential immunofluorescence study has shown that PSA-IgG may function as a carrier protein for chemotherapeutic drugs to prostate epithelial cells and their tumors. Furthermore, FITC-labeled 5'-Fu-2'-d did not specifically localize in prostatic glands, kidney, lungs, bladder, or colon. Because of the specificity and selectivity of the immunoconjugate for prostatic epithelial cells and their tumors, the immunoconjugate could be used in small dosages to treat prostatic tumors and such treatment would greatly reduce many unpleasant side effects in patients. This is the first report to show that PSA-IgG can function as an organ specific carrier protein for chemotherapeutic drugs to human prostate epithelium and its tumors.

Regional characteristics of [125I]cholecystokinin octapeptide specific binding to rat brain membranes following 6-hydroxydopamine treatment.

The relative changes in the amount of specific [125I]cholecystokinin octapeptide (CCK8) bound to regional brain membrane preparations after 6-hydroxydopamine (6-OHDA) treatment were examined. The specific binding in the frontal cortex and striatum decreased and reached a minimum on the 3rd day after 6-OHDA treatment. Thereafter, the specific binding recovered to 60% and 65% of control values in the frontal cortex and striatum respectively on the 28th day. On the other hand, in the nucleus accumbens, where CCK8 co-exists in the dopamine neuron, the specific binding decreased gradually, and its recovery was delayed compared with that of the frontal cortex and striatum. In the hippocampus, 6-OHDA treatment had no effect on the specific binding throughout the experimental period. The decrease of [125I]CCK8-specific binding could be caused by enhanced release of CCK8 in the frontal cortex, striatum, and nucleus accumbens, and the recovery of specific binding could be induced by depletion of CCK8 in nerve terminals. Particularly in the nucleus accumbens, the delayed recovery of specific binding suggests the loss of the pre-synaptic binding site, which exists in the CCK8/DA co-existing neuron, together with a change in CCK8 release.

A 16mer peptide of the human autoantigen calreticulin is a most prominent HLA-DR4Dw4-associated self-peptide

The human Ca 2+-binding (storage) protein calreticulin, located in the lumen of the endoplasmic reticulum, is proposed to play a role as autoantigen: anticalreticulin autoantibodies occur in the sera of patients with SLE and patients with onchocerciasis (calreticulin shows a high sequence homology to the Onchocerca volvulus antigen RAL-1). Here we present sequencing data of a HLA-DR4Dw4-associated calreticulin peptide fragment, Cal(295–310), purified from a DR4Dw4 self-peptide pool. Cal(295–310) proved to be one of three commonest self-peptides associated with DR4Dw4 molecules that were isolated from the EBV-transformed B-cell line BSM (DR4Dw4, DRw53). We tested the binding of Cal(295–309) and the analogous RAL-1 peptide to HLA-DR molecules: Cal(295–309) exhibited specific binding characteristics for DR4Dw4. Binding assays using self-peptide analogues with replaced amino acids led us to a DR4Dw4-binding motif with anchor residues at relative positions 1 and 6. The sequencing data suggest that calreticulin is a frequently processed intracellular protein. The abundance of calreticulin makes the presentation of different calreticulin peptides associated with HLA-D molecules likely to occur, supporting the immunologic relevance of this molecule.

Separation of proteins using cetyltrimethylammonium bromide discontinuous gel electrophoresis.

The gel electrophoresis system presented here allows the separation of proteins with the concomitant retention of detectable native activities. The system, referred to as CAT gel electrophoresis, uses the detergent cetyltrimethylammonium bromide in combination with a discontinuous gel matrix to resolve protein mixtures into discrete bands. Many proteins retain detectable levels of native activity after CAT electrophoresis, and gel bands can be easily identified using assays based on specific enzymatic activities or binding characteristics. The ability to identify protein bands based on Both M(r) and activity in a single gel makes the CAT system a powerful adjunct to existing biochemical techniques.

Immunocytochemical verification of the insulin receptor's specificity in the nuclear envelope of Tetrahymena. Comparison with receptors of the plasma membrane

The unicellular Tetrahymena possess hormone receptors in the nuclear envelope similarly to higher rank animals. These receptors bind insulin and their specificity is detectable by monoclonal antibodies developed to insulin. The hormonal (insulin) pretreatment (imprinting) of the cell did not alter the binding capacity of the nuclear membrane, demonstrated by antibody-technique. The specific binding characteristics of the plasma membrane was demonstrated and this was significantly increased following imprinting. In the nucleus of Tetrahymena presence of insulin was not detected by immunocytochemical method.

Bauhinia purpurea lectin (BPA) binding spectra in hyperplastic human tonsil and in peripheral blood: immunohistochemical, immunoelectron microscopic, and flow cytometric analyses.

We studied the binding of Bauhinia purpurea lectin (BPA) to human tonsils and peripheral blood mononuclear cells by immunohistochemical, immunoelectron microscopic, and flow cytometric techniques. The effect of several fixatives (acetone, ethanol, buffered and non-buffered formalin, B5, Bodian 11, Bouin's, Carnoy's, Zenker's, and glutaraldehyde) was also examined. BPA was reactive with germinal center lymphocytes, macrophages/histiocytes, follicular dendritic cells, squamous epithelial cells, and a subset of endothelial cells. Mantle zone and paracortical lymphocytes were non-reactive with BPA. The profile of the specific binding characteristic of BPA lectin was found to be influenced by the fixatives. Most significantly, formalin fixation greatly reduced overall binding intensity, particularly making germinal center lymphocytes totally non-reactive. The reaction intensity was most prominent in frozen sections or those fixed in Carnoy's or ethanol solution. The combination of heavy metal salt-containing fixatives with acetic acid usually did not enhance BPA binding. Glutaraldehyde solution used for immunoelectron microscopic study also preserved BPA receptors fairly well, and BPA was confined to the membrane in lymphocytes and to both the membrane and cytoplasm in macrophages/histiocytes and follicular dendritic cells. Neuraminidase treatment of tissues resulted in binding of BPA to lymphocytes that were non-reactive before treatment. Double-staining studies on cell suspensions from tonsils with FITC-BPA and PE-conjugated anti-CD3 or SIg reagents revealed that 28.4% of CD3+ cells and 61.3% or SIg+ cells were BPA reactive. In PBL, 65.6% and 81.4% of CD3+ and SIg+ cells, respectively, were BPA reactive. Neuraminidase treatment also increased the percentage of BPA-reactive lymphocytes. In conclusion, BPA is a marker for macrophages/histiocytes and germinal center lymphocytes, provided that the tissues are unfixed or fixed with suitable fixatives such as ethanol or Carnoy's solution.

Quantitative analysis of regions of adenovirus E1A products involved in interactions with cellular proteins.

Human adenovirus E1A proteins and oncogene products of several other DNA tumour viruses derive much of their oncogenic potential from interactions with cellular polypeptides. E1A proteins form complexes with p105Rb and a related p107 polypeptide, and with at least three other proteins (p60cycA, p130, and p300); all may be required for cell transformation. Using a series of E1A deletion mutants, we have carried out a quantitative analysis of the binding patterns of cellular proteins to E1A products. Binding of most of the proteins was affected at least partially by mutations within the amino terminal 25 residues, amino acids 36-69 within conserved region 1 (CR1), and residues 121-138 in conserved region 2 (CR2). However, the specific binding characteristics of each protein varied considerably. p300 was the only species for which binding was totally eliminated by deletions at the amino terminus. Removal of regions within CR1 eliminated binding of all species except p107 and p60cycA. Deletion of portions of CR2 reduced or eliminated binding of all proteins except p300. Thus, whereas cellular polypeptides generally were found to interact with the same three regions of E1A proteins, specific interactions varied considerably.

Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus.

The antidiuretic effect of arginine vasopressin (AVP) is mediated by renal-type (V2) receptors linked to adenylyl cyclase. We report here the cloning of the rat kidney V2 AVP receptor complementary DNA that encodes a 370-amino-acid protein with a transmembrane topography characteristic of G protein-coupled receptors, and with similarity to the V1a (hepatic) AVP receptor in its seven membrane-spanning domains. Expression of the cloned cDNA in mammalian cells showed specific ligand binding and activity characteristic of the native V2 AVP receptor. The receptor messenger RNA is detected only in the kidney. The human V2 receptor gene has been localized to the long arm of the X chromosome close to the locus for nephrogenic diabetes insipidus, an X-linked recessive disorder characterized by renal resistance to the antidiuretic action of AVP.

Recent results of oral cancer research in Kerala, India.

Findings from a research program in oral cancer at Regional Cancer Centre, Trivandrum, Kerala, India are reviewed. There is evidence of immune impairment in oral cancer patients. Plant lectins are being investigated for specific binding characteristics in various oral precancers and different histological subtypes of oral cancer. Tobacco and alcohol have been identified as the major risk factors for oral cancer. The chemopreventive potential of carotenoids and retinoids have been evaluated in oral leucoplakias. Beta-carotene and vitamin A in heavier doses induced remission of oral leukoplakias in 25%-50% of trial participants who continued with their tobacco and alcohol habits during the trial. The remission could be maintained with lower doses. Less than 20% of our patients with oral cancer were initially seen in localized stages. Various community-oriented programs for prevention of primary and secondary oral cancers are being evaluated. These include antitobacco health education, oral self-examination, and oral examination by trained volunteers. The results of radical radiotherapy and surgical salvage of radiation failure are also discussed. The 5-year disease-free survival rate of 34% is a reflection of the advanced stages of the disease when initially seen. Salvage rates with surgery for radiation failures were encouraging.

The molecular biology of retinoic acid receptors: orphan from good family seeks home.

A variety of c-DNAs coding for nuclear retinoic acid receptors (RARs) have recently been cloned. These receptors are members of the steroid/thyroid receptor superfamily and are believed to act as ligand-inducible transactivating factors; retinoic acid induces changes in receptor configuration that allows DNA binding and increased gene transcription from specific genes to occur. The retinoic acid receptor family itself may consist of up to 20 separate receptors each with a specific distribution and ligand binding characteristics. The RAR-gamma in the adult is found almost exclusively in the skin but other receptors which are found in a variety of other tissues are also present in skin. Associations of cutaneous disease states with receptor mutants have not yet been reported although some cases of leukaemia may be secondary to retinoic acid receptor gene rearrangements. A variety of approaches to identify the biological function of these receptors based on recombinant DNA technology are already underway.

Tumor necrosis factor-alpha affects growth hormone secretion by a direct pituitary interaction.

Administration of 50, 250, and 1,250 ng/kg iv of recombinant bovine tumor necrosis factor-alpha (RBTNF) did not affect basal plasma concentrations of growth hormone (GH) or thyroid-stimulating hormone in male calves. However, when administered 30 min before challenge with 1 microgram/kg iv of thyrotropin-releasing hormone (TRH), 250 ng/kg of RBTNF increased the subsequent incremental GH response. At 1,250 ng/kg of RBTNF, GH response to TRH was significantly blunted. For each dose of RBTNF administered, the incremental change in plasma thyroid-stimulating hormone following TRH was not significantly different from control. To examine direct effects of RBTNF on pituitary function, fresh bovine pituitaries were sliced into 1-mm cubes and incubated with 0 or 10(-8), 10(-9), or 10(-10) M RBTNF. Additional cultures were treated with 10(-8) or 10(-9) M GH-releasing factor or 10(-8) M TRH and 0 or 10(-8) M RBTNF. Media GH increased in cultures with 10(-10) M RBTNF and declined linearly as RBTNF concentration increased. RBTNF blocked GH release from GH-releasing factor- and TRH-challenged pituitary slices. Membranes prepared from homogenized bovine pituitaries had specific saturable binding characteristics for monomeric 125I-RBTNF. Membranes treated with 4 M MgCl2 for 10 min and washed free of Mg2+ produced Scatchard plots fit to a two-site model (high affinity site Kd = 6.6 nM), while Scatchards of non-Mg(2+)-treated membranes fit a single site (Kd = 8.9 nM). Polyacrylamide gel electrophoresis separation of 125I-RBTNF cross-linked pituitary membranes showed specific binding of monomeric 125I-RBTNF to protein components ranging in molecular weight from 19,000 to 77,000. The data suggest that RBTNF has modulatory effects on the regulation of GH secretion acting directly at the pituitary through specific receptors.

3‐[(3‐Cholamidopropyl)dimethylammonio]‐1‐Propane Sulfonate‐Solubilized Binding Sites for 2‐[125I]Iodomelatonin in Chick Brain Retain Sensitivity to Guanine Nucleotides

Binding of 2-[125I]iodomelatonin to 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS)-solubilized sites from chick forebrain was rapid. reversible, saturable, of high affinity, and of pharmacological selectivity. Scatchard analyses showed that 2-[125I]iodomelatonin binds to a single site with equilibrium dissociation constant (KD) values of 328 +/- 22 (n = 4) and 302 +/- 26 pM (n = 3) and a maximal number of binding sites (Bmax) of 36.2 +/- 2.0 and 49.5 +/- 6.6 fmol/mg of protein in solubilized and membrane fractions, respectively. The KD values obtained from the ratio of kinetic constants (k2/k1) in solubilized and membrane preparations were 228 and 216 pM, respectively. Inhibition studies indicated the following order of pharmacological affinities for both membrane and solubilized sites: 2-iodomelatonin greater than melatonin greater than 6-chloromelatonin much greater than prazosin greater than N-acetylserotonin much greater than serotonin greater than metergoline greater than ketanserin greater than propranolol greater than phentolamine greater than cyproheptadine. Guanyl nucleotides inhibited binding of 2-[125I]iodomelatonin to solubilized and membrane fractions, by converting binding sites from a high-affinity to a low-affinity state. These findings show that solubilized binding sites for melatonin exhibit the specific binding and pharmacological characteristics present in membrane-bound sites. Moreover, the retention of sensitivity to guanine nucleotides in fractions solubilized with CHAPS suggests that this solubilization procedure is suitable for further studies aimed at the isolation, purification, and molecular characterization of active melatonin binding sites.

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The interindividual relationship between unbound fraction of valproic acid and some factors such as total drug concentration, level of albumin, free fatty acids (FFA) and total bilirubin was investigated after repeated oral doses in 7 healthy adults. All serum samples (n=63) were collected just prior to the last dose and the following times there-after.The correlation between unbound fraction and both corresponding each variable value and ratio of FFA/albumin and total bilirubin/albumin was determined in all samples (method (1)). Secondly, the correlation between individual average unbound fraction and both average values for each variable and ratio of FFA/albumin and total bilirubin/albumin of individual was also determined (method (2)).For applied method (1), there was significant positive correlation between unbound fraction and both total drug concentration (P<0.001) and FFA/albumin ratio (P<0.01). On the other hand, for applied method (2), there was significant positive correlation only between average unbound fraction and average value for total drug concentration (P<0.001).In the evaluation on these results by using in vivo binding parameters (K for association constant, and (P) for concentration of free protein) of the Scatchard plot, we observed the specific binding characteristics between valproic acid and serum albumin within the investigated range of total concentration (<420μmol/l). Additionally, we showed that the mechanism of altered binding characteristics depended onn both changes in K (P) affected by average total concentration and K by average FFA/albumin ratio.

Molecular cloning and expression of the gene for a human D1 dopamine receptor

The diverse physiological actions of dopamine are mediated by its interaction with two basic types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Alterations in the number or activity of these receptors may be a contributory factor in diseases such as Parkinson's disease and schizophrenia. Here we describe the isolation and characterization of the gene encoding a human D1 dopamine receptor. The coding region of this gene is intronless, unlike the gene encoding the D2 dopamine receptor. The D1 receptor gene encodes a protein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Transient or stable expression of the cloned gene in host cells established specific ligand binding and functional activity characteristic of a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. Northern blot analysis and in situ hybridization revealed that the messenger RNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart. Several observations from this work in conjunction with results from other studies are consistent with the idea that other D1 dopamine receptor subtypes may exist.

Changes in acetylcholine receptor distribution and binding properties at the neuromuscular junction during aging

Junctional and extrajunctional acetylcholine receptors were characterized in diaphragm muscle obtained from mature adult and aged rats. Rhodamine-conjugated α-bungarotoxin was used to visualize receptor localization. At this level of resolution, there were no major changes in receptor distribution, and nerve terminals were consistently associated with receptors and vice versa. Specific binding characteristics were assayed by measuring 125I-α-bungarotoxin binding. Maximal binding to intact junctional and extrajunctional tissue samples was greater in the older rats. The association rate constant in minced tissue decreased in the older animals. Retardation of the initial rate of toxin binding by d-tubocurarine was described by a two-component nonlinear Hofstee plot; values of Ki were about the same for both age groups, but there was a significant shift towards the low-affinity values in the aged rats. Miniature end-plate currents (m.e.p.c.s) were recorded under voltage-clamp conditions before and after AChE inhibition. When AChE activity was inhibited m.e.p.c. amplitudes and decay time-constants increased in both age groups. The magnitude of these increases was larger in the older animals. Inhibition of AChE did not affect mean channel open time, which was estimated from spectral analyses of ACh-induced membrane noise. Lipid composition was assayed in whole muscle and isolated sarcolemma. Muscle cholesterol concentration rose 15–20 percent, but phospholipid concentrations were maintained. However, neither cholesterol, phospholipid levels, nor membrane fluidity changed significantly with age in isolated sarcolemmal membrane fractions.These data indicate that the numbers of junctional and extrajunctional receptors increase with age. In the junctional region, this is quite likely due to an expanded field of receptors and not an increased density. This is associated with an increased fraction of receptors with lower binding affinity during aging. These changes apparently are not caused by major changes in membrane fluidity or lipid composition.

Epidermal growth factor receptors in rat placenta, amnion and yolk sac: characteristics of specific binding are dependent on gestational age.

Studies of [125I]-EGF binding to the rat placenta, amnion and yolk sac were carried out on days 14, 17 and 20 of gestation. In the placenta EGF binding was detectable on all 3 days; in the amnion EGF binding was undetectably low on day 14 but was present on days 17 and 20, while in the yolk sac EGF binding was undetectably low on all 3 days. Although Scatchard analysis of EGF binding to placental tissue raised the possibility of high and low affinity receptors, a statistical analysis of the ligand binding data was consistent with the presence of only one type of EGF receptor. The overall affinity of the receptors did not change with stage of gestation. However, the concentration of EGF receptors was lower in placental tissue on day 17 than on days 14 or 20 of gestation; the receptor concentrations were similar on days 14 and 20. It is suggested that EGF binding to the placenta, amnion, and yolk sac may reflect the levels of cell proliferation in those tissues in the latter part of gestation.

Epidermal growth factor receptors in rat placenta, amnion, and yolk sac: Characteristics of specific binding are dependent on gestational age

Epidermal growth factor receptors in rat placenta, amnion, and yolk sac: Characteristics of specific binding are dependent on gestational age

Erythropoietin: Receptor characteristics during the ontogeny of hamster yolk sac erythroid cells

Erythropoietin (Epo) binds specifically to receptors on the surface membrane of responsive erythroid cells. In search of ontogenic changes in Epo receptor behavior, we studied characteristics of specific binding to hamster yolk sac erythroid cells during hamster ontogeny. We detected receptors specific for Epo on these cells throughout the duration of their intravascular existence (hamster gestational days 8 through 13). These receptors are saturable at an Epo concentration of 1.2 nM in the incubation medium. Attainment of equilibrium of binding prior to hormone internalization, a requirement for receptor binding assays, was possible at 10 degrees C but not at 37 degrees C. Hence, all incubations of cells with Epo were carried out at 10 degrees C. Data on specific binding analyzed by the method of Scatchard demonstrated that yolk sac erythroid cells possess a single class of Epo receptors at each stage of gestation examined. Binding affinity and numbers of receptors per cell change as ontogeny progresses: Kd (the dissociation constant) increases, a phenomenon observed in other differentiating cell systems, whereas the number of receptors per cell peaks on gestational day 10. The variability in number of receptors per cell is consonant with up and down regulation controlled by Epo availability. We propose that the progressive increase in Kd might be best explained by ontogenic changes in cell membrane structure contiguous to the receptors themselves.