Characterization of Insulin-Like Growth Factor Binding to Human Granulosa Cells Obtained During in Vitro Fertilizationd

Abstract

Insulin and IGF-I affect in vitro ovarian stromal and follicular cell function in several species. We previously characterized insulin receptors on human granulosa cells obtained from in vitro fertilization procedures but were unable to demonstrate specific binding of IGF-I.

Following modification of the assay conditions, we now report specific, high affinity IGF-1 binding sites on human granulosa cells. Substitution of equimolar concentrations of sucrose for sodium chloride in the buffer solution increased binding of IGF but not insulin in equilibrium assays. Maximal specific IGF-I binding was 2.69 ± 0.30%/10⁵ cells (SEM, n=9) with half-maximal inhibition of binding at 2 ng/ml IGF-I. Unlabeled insulin recognized the type I IGF receptor with low affinity. An IGF-I receptor monoclonal antibody (αIR-3) inhibited ¹²⁵I-IGF-I but not ¹²⁵I-insulin binding. Affinity crosslinking followed by SDS/PAGE under reducing conditions revealed IGF-I binding at a molecular weight compatible with the αsubunit of the type I IGF receptor and with a pattern of inhibition by various ligands that paralleled the equilibrium binding assays.

IGF-I receptors are present on freshly isolated human ovarian granulosa cells obtained following pharmacologic stimulation with gonadotrophin according to the protocols of in vitro fertilization. The biologic function of these receptors currently is being investigated.     

Binding of 125I-epidermal growth factor in human uterus

Summary Quantitative light-microscopic autoradiography was used on five human uteri at two different phases of the menstrual cycle to ascertain the cell types with binding sites for epidermal growth factor (EGF). The results revealed that stromal cells, glandular epithelium of endometrium, elongated and circular muscle cells of myometrium, smooth muscle and endothelial cells of arterioles in the basal endometrium and myometrium contained numerous silver grains following incubation with ¹²⁵I-EGF. Coincubation with 100-fold excess unlabeled EGF resulted in a complete disappearance of silver grains from all cell types. Quantitative grain analysis indicated that stromal cells contained the highest number of EGF-binding sites (P<0.05) with no significant differences among the others (P>0.05). There was no cyclic variation of EGF-binding to any of the uterine cell types. The present data demonstrate that all the cell types of human uterus, including arterioles, contain EGF-binding sites. This suggests that all the cells in human uterus subserving different functions are targets of EGF action.     

Binding of3H-phenamil,an irreversible amiloride analog,to toad urinary bladder: effects of aldosterone and vasopressin

Summary Phenamil, an analog of amiloride, has previously been shown to bind specifically to sodium channels in toad bladder (J.L. Garvin et al.,J. Membrane Biol. 87:45–54, 1985). In this paper,³H-phenamil was used to measure sodium channel density in both isolated epithelial cells and intact bladders. From the specific binding to intact bladders, a channel density of 455±102 channels/m² was calculated. No correlation between specific binding and the magnitude of irreversible inhibition of shortcircuit current was found. Pretreatment of intact bladders with 1 mg/ml trypsin reduced specific binding to isolated cells by 82±5%. In isolated cells, neither aldosterone nor vasopressin had any significant effect on specific phenamil binding. It is inferred that phenamil binds to both open and closed channels which may be either in the mucosal membrane or in the submembrane space. Finally, and rather surprisingly, we found that³H-phenamil binds irreversibly to the basolateral membrane at concentrations as low as 4×10^–7 m. Therefore, care must be used in interpreting binding studies with amiloride or its analog at such concentrations.     

Insulin binding in cultured Chinese hamster kidney epithelial cells: The effect of serum in the medium

Summary [¹²⁵I]Insulin (porcine) binding to an epithelial cell line established from a Chinese hamster kidney, CHK-AC_E−100, showed an optimum at pH 8.0 and reached a maximum after 2.5 h incubation at 25°C. Dissociation of bound [¹²⁵I]insulin was facilitated by the addition of unlabeled insulin in the dilution buffer. Porcine insulin effectively competed for [¹²⁵I]insulin binding to the cultured cells and was 30 and 90 times as potent as guinea pig insulin and porcine proinsulin in causing 50% inhibition of [¹²⁵I]insulin binding; glucagon was completely ineffective. Scatchard analysis of the binding data yielded a curvilinear plot and a capacity of 0.6 ng/10⁶ cells; the average affinity of the empty receptor, , was calculated to be 1.78×10⁸ M ⁻¹ and that of the filled receptor, , 0.57×10⁸ M ⁻¹. Substitution of fetal bovine serum (FBS) in the culture medium with bovine calf, bovine newborn, or bobby calf serum altered insulin binding characteristics in the cells and reduced cell growth. Insulin binding characteristics of cells grown in hormone-supplemented medium containing 0 to 0.1% FBS were similar to those of cells grown in minimum essential medium (MEM) containing 2 to 5% FBS. The data indicated that the established Chinese hamster kidney epithelial cell line CHK-AC_E−100 possessed specific insulin receptors and the characteristics of the receptors could be manipulated by changing the serum in culture medium.     

Characterization of endoglin on mouse uterine stromal cells

During the oestrous cycle and early pregnancy, the uterus undergoes a variety of morphological and physiological modifications involving uterine cell proliferation and differentiation as well as extensive tissue remodelling. Transforming growth factor beta (TGF-beta) has powerful effects on these events and thus is thought to have a critical role in uterine physiology. Endoglin is a transmembrane glycoprotein that binds TGF-beta 1 and -beta 3 and interacts with TGF-beta signalling receptors to modulate many effects of this growth factor in different types of cell. Studies in mice revealed the highest concentrations of endoglin in the reproductive tract, notably on stromal cells of cyclic and pregnant uteri. The objective of the present study was to investigate the role of endoglin expressed on uterine stromal cells in binding TGF-beta and in the cellular responses induced by this growth factor. Highly purified populations of uterine stromal cells were isolated by cell affinity to the monoclonal antibody MJ7/18, which is specific to mouse endoglin. Affinity labelling of these cells with 125I-labelled TGF-beta followed by immunoprecipitation with endoglin-specific polyclonal 1256:4b antiserum indicated that endoglin expressed at the surface of uterine stromal cells binds TGF-beta 1 and interacts with TGF-beta signalling receptors. Treatment of uterine stromal cells with different concentrations of TGF-beta 1 induced a biphasic proliferative response and addition of MJ7/18 as well as neutralizing TGF-beta antibodies showed endoglin to be a modulator of TGF-beta-induced stromal cell proliferation. Given the importance of TGF-beta in the regulation of uterine physiology, these results indicate a role for endoglin during uterine tissue remodelling and decidualization.     

Inhibition of Helicobacter pylori adherence by a peptide derived from neuraminyl lactose binding adhesin

Helicobacter pylori, like many other gut colonizing bacteria, binds to sialic acid rich macromolecules present on the gastric epithelium. NLBH (neuraminyl lactose binding haemagglutinin) a 32 kDa adhesin located on the surface of H. pylori has been shown to have specific affinity towards NeuAc2,3Gal1,4Gluc(3SL). This sialic acid moiety is over-expressed in an atrophic stomach undergoing parietal cell depletion. Antibodies against a lysine rich peptide fragment of NLBH inhibit agglutination of human erythrocytes. This lysine rich sequence from NLBH was proposed to be the receptor-binding site. In order to elucidate the binding of NLBH to gastric epithelium, a peptide (D-P-K-R-T-I-Q-K-K-S) was synthesized. A series of experiments were performed involving adherence inhibition assays, 2D-NMR, molecular modelling and measurement of modulation in acid secretion. Results indicated that the peptide fragment could be involved in receptor recognition, which is important for the binding of H. pylori to gastric epithelium. The binding is possibly through hydrogen bonding. Two lysines and a threonine residue seem to be within the hydrogen bonding distance of NeuAc2,3Gal1,4Gluc. Further, in vitro assays were performed to evaluate the role of the peptide on acid secretion by parietal cells isolated from human fundal biopsies. Interestingly, the peptide increases acid secretion only in H. pylori negative and in treated patients but not in H. pylori positive patients. This highlights the role of NLBH in acid secretion and could be of some consequence in the prognosis of the disease.     

cGMP modulates transport across the ciliary epithelium

cGMP reduced the short-circuit current (I _SC) when applied to the aqueous surface of isolated rabbit and cat ciliary epithelia. cGMP either stimulated (in the rabbit) or had no effect (in the cat) on I _SC when applied to the stromal surface. Addition of the cGMP-mediated hormone atrial natriuretic peptide (ANP) to the stromal (but not the aqueous) surface, or the nitrovasodilator sodium nitroprusside to the stromal surface, inhibited I _SC across rabbit ciliary epithelium.The response to stromal cGMP was partly mediated by K⁺ channels at the stromal surface of the rabbit pigmented epithelial (PE) cells, since the effect was inhibited by stromal Ba²⁺, and was unaffected by Cl^– replacement, by bumetanide, or by DIDS. In contrast, the response to aqueous cGMP was not likely mediated by changing either K⁺ or Cl^– channels, based on transepithelial measurements of rabbit ciliary epithelium and complementary whole-cell patch clamping of cultured human nonpigmented ciliary epithelial (NPE) cells. The possibility of interacting effects between cGMP and cAMP in targeting the Na⁺,K⁺-exchange pump was also considered. Strophanthidin blocked the responses to either aqueous or stromal cGAMP. Applying 10 m forskolin to generate endogenous cAMP enhanced the subsequent response to aqueous cGMP by 80%.We conclude that cGMP has at least two actions on the ciliary epithelium. The major effect may be to reverse cAMP-mediated inhibition of the NPE Na⁺ pumps at the aqueous surface of both rabbit and cat ciliary epithelia. The second effect is likely mediated by increasing K⁺-channel and pump activity of the rabbit PE cells at the stromal surface.Supported in part by research grants from the National Institutes of Health [EY10691 and EY00785 (for core facilities)] and from the American Health Assistance Foundation. We are grateful to Dr. Miguel Coca-Prados for graciously providing us with the ODM/SV40 NPE cells, and thank Prof. Rainer Greger (Albert-Ludwigs-Universitat, Freiburg, FRG) for kindly providing the NPPB.     

Binding of insulin and insulin-like growth factor I in bovine chromaffin cells in primary culture

• 1.1. Insulin and insulin-like growth factor I (IGF-I) receptors were studied in bovine chromaffin cells isolated from the medulla by collagenase digestion and kept in primary culture.
• 2.2. Specific ¹²⁵I-labelled insulin binding increased with time in culture with no significant change in the dissociation constant, K_d~0.3nM. Insulin was nearly 100-fold more potent than IGF-I in displacing ¹²⁵I-labelled insulin.
• 3.3. Affinity crosslinking and SDS gel electrophoresis revealed increased binding of ¹²⁵I-labelled insulin and ¹²⁵I-IGF-I with time in culture, the densities of the labelling indicating relatively a much higher expression of IGF-I than insulin receptors in the cells. The apparent molecular weight of both the hormone binding subunits were 135,000, suggesting that the insulin and IGF-I receptors in the adrenal medulla are of the peripheral types.
• 4.4. Both receptors thus appeared to be affected by the collagenase treatment but with a subsequent recovery when cells were kept in culture.

     

Identification of differences between the surface proteins and glycoproteins of normal mouse (Balb/c) and human erythrocytes

Summary The topography of the external surface of the Balb/c mouse erythrocyte has been investigated and compared to the human erythrocyte by using a series of protein radiolabeling probes. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the pattern of Coomassie Blue stained proteins was very similar for mouse and human erythrocyte ghosts, as was the distribution of radioactivity in protein bands after lactoperoxidase catalyzed radioiodination. The mouse erythrocyte glycoproteins identified by periodic-acid-Schiff and Stains-All reagents, sialic acid analysis of gel slices, binding of¹²⁵I-wheat germ agglutinin and¹²⁵I-concanavalin A to the gels, and glycoprotein radiolabeling techniques, differed markedly from the sets of proteins labeled by radioiodination, and also differed from the human erythrocyte glycoproteins. Instead of the PAS I to PAS IV series of sialoglycoproteins characteristic of human erythrocytes, the mouse erythrocyte possesses a broad band of sialoglycoproteins with several peaks ranging in mol wt from 65,000 to 32,000. The same group of sialoglycoproteins were labeled by the periodate/B³H₄ ^– technique specific for terminal sialic acid, and the galactose oxidase/B³H₄ ^– method (plus neuraminidase) specific for galactosyl/N-acetylgalactosaminyl residues penultimate to sialic acid. These results emphasize the necessity to employ a variety of protein radiolabeling probes based on different labeling specificities, to study the membrane topography of cells which are poorly understood compared to the human erythrocyte membrane.     

Atrial and brain natriuretic peptides share binding sites on cultured cells from the rat trachea

Summary We examined the distribution of binding sites for alpha-atrial natriuretic peptide (¹²⁵I-ANP_1–28) and the recently discovered porcine brain natriuretic peptide (¹²⁵I-pBNP) on immunocytochemically identified cells in dissociated culture preparations of the rat trachea. Specific binding sites for both ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP were evenly distributed over distinet subpopulations of smooth muscle myosin-like immunoreactive muscle cells, fibronectin-like immunoreactive fibroblasts and S-100-like immunoreactive glial cells. Neither keratin-like immunoreactive epithelial cells nor protein gene product 9.5-like immunoreactive paratracheal neurones expressed natriuretic peptide binding sites, although autoradiographically labelled glial cells were seen in close association with both neuronal cell bodies and neurites. The binding of each radiolabelled peptide was abolished by the inclusion of either excess (1 M) unlabelled rat ANP or excess unlabelled porcine BNP, suggesting that ANP and BNP share binding sites in the trachea. Furthermore, the ring-deleted analogue, Des-[Gln¹⁸, Ser¹⁹, Gly²⁰, Leu²¹, Gly²²]-ANF_4–23-NH₂, strongly competed for specific ¹²⁵I-ANP_1–28 and ¹²⁵I-pBNP binding sites in the tracheal cultures; this suggests that virtually all binding sites were of the clearance (ANP-C or ANF-R₂) receptor subtype.     

Milking the stroma in triple-negative breast cancer

Comment on: Witkiewicz AK, et al. Cell Cycle 2012; 1108–1117Investment in the post-genomic molecular dissection of breast cancer has resulted in an emphasis on prognostic and predictive markers, signatures derived to stratify the disease and the drive to generate targeted therapies. However, there remain significant challenges to individualize therapeutic targeting and improve the prognosis for the thousands of women who die each year from the heterogeneous range of breast cancers. This is particularly true for poor prognosis “triple-negative” breast cancers (TNBC), most prevalent in young and African American women, lacking the established therapeutic targets of estrogen receptor, progesterone receptor or HER2.Research has largely focused on the epithelial component of breast cancer rather than the tumor microenvironment, now recognized as a key hallmark of cancer.¹ In vitro, animal models and observations on clinical material² are now moving to consider physiological mechanisms by which stromal cells may influence breast epithelial and carcinoma cells.Witkiewicz et al.³ build on published evidence from the Lisanti group that cancer cells secrete hydrogen peroxide, initiating oxidative stress and aerobic glycolysis in tumor stroma, with L-lactate secretion from cancer-associated fibroblasts fueling oxidative mitochondrial metabolism in epithelial cancer cells: the “reverse Warburg effect.”They demonstrate stromal monocarboxylate transporter 4 (MCT4), detected by immunohistochemistry, as a functional marker of stromal hypoxia, oxidative stress, aerobic glycolysis and L-lactate efflux. High stromal MCT4 expression (but, critically, not epithelial MCT4) was associated with poor prognosis in TNBC patients. Combined high stromal MCT4 and loss of stromal caveolin-1 identify particularly poor prognostic TNBC.Thus, development of cancer may not lie solely in genetic or epigenetic epithelial changes, but with acquired functional changes in the stromal infrastructure of the breast. This supports the concept of epithelial malignant changes consequent with ecological and evolutionary opportunity.⁴ The “parasitic” character of tumor cells feeding off stromal cells highlights the need to seriously consider both ecological and biophysical concepts.⁵ We need to think beyond “intraspecific” competition among clonal subpopulations in the tumor and to consider tumor and stromal cells as distinct populations in a cancer ecosystem, with a range of “interspecific” competitive, exploitative and opportunistic interactions. Furthermore, the reverse Warburg effect relies on the inefficient diffusion of nutrients from stromal cells to tumor cells in a complex three-dimensional space. The extracellular space is brought to the foreground, and physical properties of molecular transport in this space may have as much impact on tumor growth as intricate cellular processes. The importance of the spatial arena is also apparent when contrasting the reverse Warburg effect with angiogenesis. In the former, tumor cells are exploiting their local environment, which will presumably be of limited yield, whereas angiogenesis taps the nutrients of the entire organism—­an effectively infinite reservoir for a growing tumor. In the reverse Warburg effect, a balance of ecological and biophysical factors underpins the sustainability of this mode of cancer nutrition. A two-compartment model coupling oxidative epithelial cells with glycolytic fibroblasts reflects increased expression of hypoxia-associated genes as a component part of prognostic stromal signatures.⁶ Further evidence of stromal/epithelial interaction comes from evidence that the effects of radiation on normal breast epithelium in vivo is at least partially dependent on the stromal context.⁷Manipulation of the tumor microenvironment to promote an anticancer phenotype challenges the cancer treatment paradigm. The long-established antidiabetes biguanide drugs offer a low-toxicity opportunity to disrupt the reverse Warburg effect. Metformin may target the cancer mitochondria³ and phenformin induce stromal sclerosis, at least in a breast cancer xenograft model,⁸ in addition to in vivo AMPK pathway and insulin-mediated systemic effects of metformin in women with breast cancer.⁹ The reverse Warburg effect challenges our therapeutic focus on breast cancer epithelium. Stromal MCT4 expression with caveolin-1 loss identifies poor prognostic TNBC patients and emphasizes the roles of the tumor microenvironment and ecological interactions between distinct populations of cells. The challenges now revolve around therapeutic manipulation of the stroma/epithelial interaction and the extracellular space, and testing these concepts in pre-invasive and metastatic settings where stromal changes may provide tissue niches of evolutionary opportunity for malignant cells.     

Autoradiographic localization of platelet-activating factor (PAF) binding sites in the rabbit endometrium during the peri-implantation period

Summary This communication describes the use of in-vivo and in-vitro autoradiography to map specific platelet-activating factor (PAF) receptors in the rabbit uterus. Specific [³H]PAF uptake was predominantly localized on epithelial, but not on stromal or myometrial cells. Very few silver grains were associated with the luminal epithelial cells in the uterus of the estrous rabbit, primarily because of the non-differentiated state of the epithelium. In the differentiated pregnant uterus, significantly more [³H]PAF was bound to the glandular epithelial cells, with the stromal cells binding consistently significantly less. The highest density of silver grains was observed at the implantation sites on day 7 of pregnancy. There was no apparent difference in [³H]PAF C18:0 uptake between the epithelial cells at the inter-implantation zone on day 7 and on day 6. Bound [³H]PAF was displaceable by lyso-PAF, U66985, CV3988, but not U66982, L652,731, SRI 63,441 or the inactive PAF isomer, oleoyl PAF. Bovine serum albumin (BSA) significantly inhibited tissue uptake of [³H]PAF C18:0. Intraluminally administered [³H]PAF C18:0 and intravenously injected [³H]methylcarbamyl-PAF, a non-metabolizable PAF analog, penetrated the implanted blastocyst and bound to the embryoblast. This event was reproducible in vitro with pre-implantation blastocysts from day-6 pregnant rabbits, which suggests that uterine-derived PAF may translocate into the blastocyst after attachment.     

Modulation of sialic acid-binding proteins of rat uterus in response to changing hormonal milieu

A group of sialic acid binding (SAS) agglutinins has been isolated from the rat uteri at different stages [Proestrus (P), estrus (E) and diestrus (D)] of estrous cycle. Studies of biochemical properties indicate that SAS agglutinins are glycoprotein in nature having molecular weights between 28–31 Kd and microheterogenous pI. Function-based characterization revealed that inspite of the fact that all three proteins exhibit sialic acid binding property, the sialic acid binding affinities, calculated from Scatchard analysis, using 4-methylumbelliferyl sialic acid as a ligand, varied in stage specific manner (Ka:D-SAS-9.03×10⁵ M^–1, P-SAS-2.33×10⁵ M^–1, E-SAS-2.13×10⁵ M^–1). Circular dichroism spectra of these three agglutinins suggested that differences exist in the secondary structures of the proteins isolated from different stages. Removal of carbohydrate moiety by trifluoromethane sulfonic acid treatment and CNBr cleavage studies showed some homology between these proteins, however, the variation in the carbohydrate moiety was apparent from the sugar analysis data. Functionally and immunologically these proteins can be grouped as estrogenic and progestogenic SAS agglutinins.     

A histochemical study of the binding of 125I-HCG to the rat ovary throughout the estrous cycle

Summary Changes in the distribution of the in vitro uptake of ¹²⁵I-HCG by the ovaries of adult rats were examined histochemically throughout the estrous cycle.Only in follicles wider than 500 m, occurring mainly at diestrus and proestrus, could granulosa cells bind the labelled hormone. The labelling increased with follicular size and decreased in intensity from the peripheral granulosa cells inwards. No uptake occurred in the oocytes, in the cells of the cumulus oophorus nor in the granulosa cells of the atretic follicles.The binding capacity of the newly-formed corpora lutea of estrus was less than that of preovulatory follicles. The uptake of ¹²⁵I-HCG by corpora lutea during the first cycle reached its maximum at diestrus but fell sharply by proestrus. The uptake was patchy in the corpora lutea of the second cycle and not significant in the older ones.The uptake of ¹²⁵I-HCG by thecae increased with follicular size and was greater in the thecae of atretic follicles than in the thecae of growing follicles of like size. There was a greater uptake in the last formed interstitial tissue than there was in older tissue.At proestrus, the uptake of ¹²⁵I-HCG was unaffected by the LH surge at 18.00h but had decreased slightly at 24.00 h.The implications of these data in relation to the regulation of receptor sites, is discussed.     

Isolation of membrane bound light-harvesting-complexes from the dinoflagellates Heterocapsa pygmaea and Prorocentrum minimum

We have isolated Chl a-Chl c-carotenoid binding proteins from the dinoflagellates Prorocentrum minimum and Heterocapsa pygmaea grown under high (500 mol m^–2 s^–1, HL) and low (35 mol m^–2 s^–1, LL) light conditions. We compared various isolation procedures of membrane bound light harvesting complexes (LHCs) and assayed the functionality of the solubilized proteins by determining the energy transfer efficiency from the accessory pigments to Chl a by means of fluorescence excitation spectra. The identity of the newly isolated protein-complexes were confirmed by immunological cross-reactions with antibodies raised against the previously described membrane bound Chl a-c proteins (Boczar et al. (1980) FEBS Lett 120: 243–247). Spectroscopic analysis demonstrated the relatedness of these proteins with the recently described Chl-a-c ₂-peridinin (ACP) binding protein (Hiller et al. (1993) Photochem Photobiol 57: 125–131; Iglesias Prieto et al. (1993) Phil Trans R Soc London B 338: 381–392). The water-soluble peridinin-Chl a binding-protein (PCP) was not detectable in P. minimum. Two functional forms of ACP with different pigmentation were isolated. A variant of ACP which was isolated from high-light grown cells, that specifically binds increased amounts of diadinoxanthin was compared to the previously described ACPs that bind proportionately more peridinin.Abbreviations ACP Chl a-Chl c-peridinin binding protein - AEBSF 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride - DDM dodecyl -d maltoside - Deriphat 160 N-lauryl-beta-iminopropionic acid - HEPES (N-2-hydroxyethylpiparizine-N-2-ethanesulphonic acid) - HL high light (500 mol m^–2 s^–1) - LL low light (35 mol m^–2 s^–1) - ₇₃₀ fluorescence yield (emission at 730 nm) - PCP peridinin-Chl a-binding protein - PMSF phenyl-methyl-sulfonyl-fluoride - PS I Photosystem I - PS II Photosystem II     

PNA-binding glycans are expressed at high levels on horse mature and immature T lymphocytes and a subpopulation of B lymphocytes

In mammals, the binding of peanut agglutinin (PNA) on the plasma membrane defines subpopulations among lymphocytes from peripheral blood and lymphoid organs. PNA binds Gal 1,3GalNAc residues provided that they are not sialylated. Here, we studied the expression of PNA-binding glycans on healthy horse peripheral blood, thymus, lymph node and spleen lymphocytes. We first demonstrated the binding specificity of PNA for galactose residues by competition experiments and the inhibitory role of sialic acids in PNA binding by sialidase digestion. Unlike human and murine lymphocytes, all equine lymphocytes were found positive by flow cytometry analysis. Double-staining analyses showed that lymphocytes expressing high levels of PNA-binding glycans (PNA^high lymphocytes) were made up of the great majority of CD5⁺, CD4⁺ and CD8⁺ cells, and of 30 and 50% of sIg-bearing lymphocytes in peripheral blood and in lymph nodes or spleen, respectively. Lectin histochemistry suggested that lymph node germinal centres contained PNA^high B cells. Contrary to what is found in humans and mice, PNA staining intensity on CD5⁺, CD4⁺ and CD8⁺ cells did not differentiate immature from mature T lymphocytes in the equine thymus. The functional consequences of these differences are discussed. Published in 2005.     

Membrane receptors for estrogen,progesterone, and testosterone in the rat brain: Fantasy or reality

Summary 1. There are numerous circumstantial evidence supporting the concept that steroid hormones control cellular function by means other than the nuclear receptor steroid binding mechanism. It is the intent of this report to present evidence indicating that steroids bind to specific sites in neuronal membranes.2. Some of the criteria to define steroid membrane receptors using steroid-BSA conjugates that can be radioiodinated to desired specific activity have been fulfilled for each of the three sex steroids using crude synaptosomal membrane preparations (P2 fractions) from the CNS of female and male rats. Ligand binding for each of the three steroids indicate high-affinity and high-capacity sites with distinct brain selectivity and stereospecificity. For example, 17-E-6-[¹²⁵I]BSA binds hypothalamic P2 fractions (HYP-P2) with an estimatedK _d of about 3±0.7 nM (X ± SE;n=3), whereas the cerebellum P2 (CB-P2) fractions bind the ligand with aK _d of 34±7 nM and, aB _max of 3 and 42 pmol/mg protein, respectively. Estrogen and testosterone binding fit best a one-single site, while progesterone binding sites can be best represented by a two-binding site, one high-affinity (K _d=1–2 nM) and one low affinity (K _d=62 nM), in CB-P2 fractions from intact adult female rat brain. Kinetics studies for T-3-[¹²⁵I]BSA indicate that the estimatedK _d of 30±2 nM for the olfactory bulb P2 fractions (OB-P2) from male rats is in good agreement withK _d values computed from Scatchard-derived data using the LIGAND algorithm.3. 17-E-6-[¹²⁵I]BSA binding sites are stereospecific and appears to be present as early as 5 days of age in both the OB- and the CB-P2 fractions without changes during development. In contrast, P-6-[¹²⁵I]BSA binding sites are practically absent during days 5 and 12 and appear by day 22.4. Finally, membrane receptor molecules for estrogen and progesterone have been isolated and purified by affinity chromatography and characterized by PAGE and Western blot. Microsequencing of one of the membrane estrogen binding proteins indicates that the high-affinity site corresponds to the OSCP subunit of the proton ATP synthase.5. It remains to be determined if P and T also bind to this complex enzyme or if they bind to other subunits of the family of proton ATPases. Overall the data indicate that steroid hormones conjugated to BSA are important tools to study the reality of membrane steroid receptors.     

High-resolution radioautography of phlorizin-3H in rings of hamster intestine

Quantitative light microscope radioautographs of galactose-³H and phlorizin-³H were prepared from freeze-dried plastic-embedded hamster small intestine incubated in vitro. The usual uphill epithelial cell accumulation of galactose accompanied by a somewhat smaller lamina propria accumulation was observed in control tissue incubated 3 min in 1 mM galactose-³H. The addition of 5 x 10^-4 M phlorizin to the medium blocked uphill accumulation, but did not prevent galactose equilibration with the epithelial cells. The galactose content of the lamina propria was considerably less than the galactose content of the epithelial cell. Varying the phlorizin-³H content of the medium from 0.6 to 60 µM revealed a brush border binding of phlorizin which followed a Langmuir adsorption isotherm with a half-saturation constant of 13 µM and a maximum binding of 84 µmoles of phlorizin/liter of microvilli or 2.6 x 10⁶ sites/epithelial cell. The phlorizin content of the epithelial cell compartment, excluding microvilli, never exceeded 10% that of the medium after 20 min of incubation. These findings directly support the view that phlorizin is a nontransported inhibitor which binds glucose-galactose carriers at the surface of epithelial cell microvilli.     

Characterization of lactoferrin binding to the MAC-T bovine mammary epithelial cell line using a biotin-avidin technique

• 1.1. A non-radioisotopic method utilizing a biotin-avidin approach was used to characterize lactoferrin binding to the clonal MAC-T bovine mammary epithelial cell line.
• 2.2. Binding of lactoferrin to MAC-T cells and isolated membranes was specific and saturable.
• 3.3. Unlabeled lactoferrin competed for and displaced biotin-labeled lactoferrin from binding sites on mammary epithelial cells. In contrast, unlabeled transferrin did not compete.
• 4.4. Scatchard analysis of lactoferrin binding to MAC-T cell crude membranes was nonlinear, revealing two classes of binding sites with association constants (K_a) of 2.36 × 10⁷ and 3.36 × 10⁶M⁻¹.
• 5.5. Binding of lactoferrin to MAC-T cells may be associated with the initial events which result in decreased MAC-T cell proliferation.

     

Effect of Synthetic β-Endorphin-Like Peptide Immunorphin on Human T Lymphocytes

-Endorphin and the synthetic -endorphin-like decapeptide Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr (referred to as immunorphin), corresponding to the sequence 364-373 of the CH3 domain of human immunoglobulin G heavy chain, were shown to stimulate concanavalin A-induced proliferation of T lymphocytes from the blood of healthy donors. [Met⁵]Enkephalin and the antagonist of opioid receptors naloxone examined in parallel were inactive. The stimulating effect of -endorphin and immunorphin on T lymphocyte proliferation is not inhibited by naloxone. Studies on receptor binding of ¹²⁵I-labeled immunorphin to T lymphocytes revealed that it binds with high affinity to naloxone-insensitive receptors (K _d = 7.0 ± 0.3 nM)). Unlabeled immunorphin completely inhibits ¹²⁵I-labeled -endorphin specific binding to naloxone insensitive receptors on T lymphocytes (K _i = 0.6 ± 0.1 nM)). Thus, -endorphin and immunorphin interact with common naloxone insensitive receptors on T lymphocytes.