Biomedical subjects

N Ling

Publications and source records attributed to N Ling.

At least 55 records · Page 3Linked to original sources

PubMed1996

Differential activation of human autoreactive T cell clones by altered peptide ligands derived from myelin basic protein peptide (87-99).

We have examined the functional consequences induced by interaction of DR2a-restricted myelin basic protein (MBP) (87-99)-specific T cell clones (TCC) with altered peptide ligands (APL) derived from MBP peptide (87-99). The immunodominant MBP peptide (87-99) has been implicated as a candidate antigen in multiple sclerosis (MS) by several lines of evidence. In the present study, we have defined the T cell receptor (TCR) contact residues for DR2a-restricted, (87-99)-specific T helper type 1 T cells to design APL suitable to modify the functions of such T cells potentially relevant for the pathogenesis of MS. We show that neutral (L-alanine substitutions) or conservative exchanges of the primary and secondary TCR contact residues lead to various alterations of T cell function, ranging from differences in interleukin-2 receptor up-regulation to anergy induction and TCR antagonism. The potential usefulness of APL as an immunomodulating therapy for DR2+ MS patients is discussed.

Amino Acid Sequence↗

PubMed1996

Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle.

The orientation of the light-chain region of myosin heads in relaxed, rigor, and isometrically contracting fibers from rabbit psoas muscle was studied by fluorescence polarization. Cysteine 108 of chicken gizzard myosin regulatory light chain (cgRLC) was covalently modified with iodoacetamidotetramethylrhodamine (iodo-ATR). Native RLC of single glycerinated muscle fibers was exchanged for labeled cgRLC in a low [Mg2+] rigor solution at 30 degrees C. Troponin and troponin C removed in this procedure were replaced. RLC exchange had little effect on active force production. X-ray diffraction showed normal structure in rigor after RLC exchange, but loss of axial and helical order in relaxation. In isolated myofibrils labeled cgRLC was confined to the regions of the sarcomere containing myosin heads. The ATR dipoles showed a preference for orientations perpendicular to the fiber axis, combined with limited nanosecond rotational motion, in all conditions studied. The perpendicular orientation preference was more marked in rigor than in either relaxation or active contraction. Stretching relaxed fibers to sarcomere length 4 microns to eliminate overlap between actin- and myosin-containing filaments had little effect on the orientation preference. There was no change in orientation preference when fibers were put into rigor at sarcomere length 4.0 microns. Qualitatively similar results were obtained with ATR-labeled rabbit skeletal RLC.

Animals↗

PubMed1996

Orientation changes in myosin regulatory light chains following photorelease of ATP in skinned muscle fibers.

The orientation of the light-chain region of myosin heads in muscle fibers was followed by polarized fluorescence from an extrinsic probe during tension transients elicited by photolysis of caged ATP. Regulatory light chain from chicken gizzard myosin was covalently modified with iodoacetamidotetramethylrhodamine and exchanged into skinned fibers from rabbit psoas muscle without significant effect of the tension transients. Fluorescence polarization ratios Q parallel = (parallel I parallel-perpendicular I parallel)/ (parallel I parallel+perpendicular I parallel) and Q perpendicular = perpendicular I perpendicular - parallel I perpendicular)/ (perpendicular I perpendicular + parallel I perpendicular), where mIn denote fluorescence intensities for excitation (pre-subscript) and emission (post-subscript) parallel or perpendicular to the fiber axis, were simultaneously measured at 0.5 ms time resolution. Q perpendicular decreased and Q parallel increased promptly after ATP release in the presence or absence of CA2+, indicating changes in orientation of the light-chain region associated with ATP binding or cross-bridge detachment. Little further change in the Q signals accompanied either active tension development (+Ca2+) or the final relaxation (-Ca2+). The Q and tension transients slowed when liberated ATP concentration was reduced. Assuming that ATP is released at 118 s-1 (20 degrees C), the apparent second-order rate constants were 3-10 x 10(5) M-1 s-1 for Q parallel, 1-5 x 10(5) M-1 s-1 for Q perpendicular, and 0.5-2 x 10(5) M-1 s-1 for the convergence of tension traces starting from different rigor values. Fitting of model orientation distributions to the Q signals indicated that the angular disorder increases after ATP binding. This orientation change is specific to ATP because photo release of ADP caused much smaller changes in the Q signals.

Adenosine Diphosphate↗

PubMed1996

Measurement of corticotropin-releasing factor (CRF), CRF-binding protein (CRF-BP), and CRF/CRF-BP complex in human plasma by two-site enzyme-linked immunoabsorbant assay.

The actions of human corticotropin-releasing factor (hCRF) in brain, pituitary, and plasma are modulated by a 37-kDa protein [CRF-binding protein (CRF-BP)] that binds to hCRF and neutralizes the peptide's biological activity, suggesting that only the free unbound peptide is biologically active. To accurately predict the biological consequences resulting from changes in total hCRF levels, we have developed two-site enzyme-linked immunosorbent assays (ELISAs) for hCRF-BP, free hCRF, and the hCRF-BP/hCRF complex. The assays were validated by measuring each factor in 1) maternal plasma at times when CRF and hCRF-BP levels are altered, and 2) plasma from normal elderly human subjects who have undergone a hCRF stimulation test. The hCRF-BP ELISA has a sensitivity of 2.7 fmol and a range of detection from 2.7-8000 fmol. Both the hCRF and hCRF-BP/ hCRF assays have a sensitivity of 0.4 fmol, with a useful range of detection from 0.4-40 fmol. Maternal plasma hCRF-BP levels remained unaltered between the 16-21 and 34-39 month gestational age groups. However, levels rose from 0.88 +/- 0.069 nmol/L in the 16-21 month gestational age group to 1.01 +/- 0.09 nmol/L in the 28-33 month gestational age group. Bound hCRF levels dramatically rose from undetectable at 16-21 months of gestation to 200 +/- 69 and 442 +/- 106 pmol/L in the 28-33 and 34-39 month gestational age groups, respectively. In comparison, free hCRF levels remained low throughout gestation, but dramatically rose to 318 +/- 120 pmol/L from 34-39 months of gestation. Binding site occupancy on the hCRF-BP decreased when bound and free hCRF levels were elevated. After treating the third trimester plasma sample with the high affinity hCRF-BP ligand, alpha-helical CRF-(9-41), all of the bound hCRF was displaced from the binding protein, and free hCRF levels rose from 87 to 284 pmol/L. The plasma hCRF-BP level was 0.9 +/- 0.08 nmol/L in normal human volunteers (10 men and 9 women; mean +/- SD age, 74.2 +/- 7.7 yr), decreased to 60% of basal levels 15 min after a bolus injection of 1 microgram/kg synthetic hCRF, and gradually returned to preinjection levels after 120 min. Conversely, bound and free hCRF levels increased from undetectable levels before hCRF injection to 0.58 +/- 0.03 nmol/L at 15 min and then rapidly decreased to undetectable levels at 120 min. These data validate the ELISAs in combination with high affinity hCRF-BP ligands for measuring bound and free hCRF in human plasma and suggest the utility of these assays for further determining alterations in peripheral CRF in conditions such as pregnancy.

Aged↗

PubMed1996

Circulating levels of follistatin from puberty to menopause.

OBJECTIVE: To determine the changes in circulating levels of follistatin, a binding protein for activin and inhibin, through the reproductive life cycle in women. DESIGN: An open, prospective descriptive study. SETTING: An academic endocrine research unit. PATIENTS: Prepubertal (n = 10), midpubertal (n = 7), and postpubertal (n = 25) (early adolescent) girls, normal cycling adult women (n = 8), postmenopausal women (n = 17), and men (n = 13) were studied. INTERVENTIONS: Normal cycling women were given Nal-Glu GnRH antagonist for 3 days in the follicular phase of the cycle. MAIN OUTCOME MEASURE: Serum concentrations of follistatin determined in a heterologous RIA. RESULTS: Mean follistatin levels did not change during puberty but were higher in adult and postmenopausal women. Levels of immunoreactive follistatin in men were lower than levels found in normal cycling women and postmenopausal women. Daily immunoreactive follistatin levels during the menstrual cycle remained constant and did not change significantly after ovarian suppression with GnRH antagonist. CONCLUSION: Because dynamic changes of serum immunoreactive follistatin do not occur during ovarian activation (puberty), suppression, and age-related ovarian failure, the increase in immunoreactive follistatin levels in adult and postmenopausal women may implicate sources of follistatin other than the ovary.

Adolescent↗

PubMed1996

125I-Tyro-sauvagine: a novel high affinity radioligand for the pharmacological and biochemical study of human corticotropin-releasing factor 2 alpha receptors.

Corticotropin-releasing factor (CRF) receptors encoded by two distinct genes have recently been identified and termed CRF1 and CRF2. CRF and the non-mammalian-related peptide sauvagine bind to and activate CRF1 receptors with high affinity and equal potency. Although CRF is significantly weaker at the CRF2 receptor, sauvagine retains its high affinity interactions with this receptor subtype. We expressed the human CRF1 and CRF2 receptor subtypes in stable cell lines and characterized 125I-Tyr0-sauvagine, a high affinity radiolabel suitable for the pharmacological and functional profiles of these proteins. 125I-Tyr0-sauvagine has high affinity (200-400 PM) for CRF1 receptors and demonstrates a pharmacological profile identical to that of 125I-Tyr0-ovine CRF-labeled CRF1 receptors. 125I-Tyr0-sauvagine binding to human CRF2 alpha receptors is saturable and of high affinity (KD = 100-300 PM) and demonstrates guanine nucleotide sensitivity typical of agonist binding to receptors. The pharmacological profile of 125I-Tyr0-sauvagine binding to CRF2 alpha receptors with respect to inhibition by CRF-related analogs is similar to the agonist profile of potencies obtained by measurements of cAMP production stimulated by these analogs in CRF2 alpha expressing cell lines and distinct from the profile of the CRF1 receptor subtype. Thus, the related nonmammalian peptides sauvagine and urotensin have high affinity and rat/ human CRF and ovine CRF have lower affinity for CRF2 receptors labeled with 125I-Tyr0-sauvagine. Because the distribution of CRF1 and CRF2 alpha receptors has been demonstrated to be distinct, suggesting selective functional roles for each receptor subtype, the ability to label CRF2 alpha receptors with 125I-Tyr0-sauvagine in vitro represents a unique opportunity for the discovery of subtype-selective nonpeptide ligands, which would presumably target different aspects of CRF-mediated disorders. We have thus identified and characterized a novel high affinity radioligand for the labeling of CRF2 receptors.

Amphibian Proteins↗

PubMed1995

Displacement of corticotropin releasing factor from its binding protein as a possible treatment for Alzheimer's disease.

In Alzheimer's disease (AD) there are dramatic reductions in the content of corticotropin releasing factor (CRF), reciprocal increases in CRF receptors, and morphological abnormalities in CRF neurons in affected brain areas. Cognitive impairment in AD patients is associated with a lower cerebrospinal fluid concentration of CRF, which is known to induce increases in learning and memory in rodents. This suggests that CRF deficits contribute to cognitive impairment. The identification in post-mortem brain of CRF-binding protein (CRF-BP), a high-affinity binding protein that inactivates CRF, and the differential distribution of CRF-BP and CRF receptors, provides the potential for improving learning and memory without stress effects of CRF receptor agonists. Here we show that ligands that dissociate CRF from CRF-BP increase brain levels of 'free CRF' in AD to control levels and show cognition-enhancing properties in models of learning and memory in animals without the characteristic stress effects of CRF receptor agonists.

Adult↗

PubMed1995

The heparin binding site of follistatin is involved in its interaction with activin.

Whether the heparin-binding site of follistatin would interact with activin has been examined. When a mixture of recombinant human follistatin-288 (rhFS-288) and -315 (rhFS-315) was applied to an activin-coupled affinity column, followed by stepwise elution of the column using 4M urea, 8M urea, 1M guanidine-HCl and 2M guanidine-HCl, rhFS-315 was eluted with 4M urea, while rhFS-288 was eluted with 2M guanidine-HCl. This finding implies that the carboxylterminal 27 amino acid extension of rhFS-315, which is not present in rhFS-288, affects the binding of follistatin with activin. Addition of heparin (50 micrograms/ml) to the elution solvent caused rhFS-288 to elute with 4M urea, whereas rhFS-315 was not affected. These data suggest for the first time that these two structurally related follistatin molecules interact with activin by different modes of binding and, in the presence of heparin, the interaction of rhFS-288 with activin is indistinguishable from that of rhFS-315. Two analogs of rhFS-288 mutated at the heparin binding site were eluted with 8M urea or 1M guanidine-HCl, distinct from the elution profile of the intact rhFS-288. These results indicated that mutation at the heparin binding site alters the activin binding affinity. In addition, bioassay of the two mutants showed that they were less potent than the rhFS-288. These findings suggest that the heparin binding site of follistatin also contributes to its binding for activin, and heparin may play an important role in the bioactivity of follistatin.

Activins↗

PubMed1995

Follistatin concentrations in follicular fluid of normal and polycystic ovaries.

Follistatin (FS) is an activin/inhibin binding protein which is believed to act in an autocrine/paracrine manner to regulate growth and differentiation. Although FS has been identified in human follicular fluid, it remains unclear how its concentration changes during selection and atresia, and what the concentrations of FS are in follicles of women with polycystic ovary syndrome (PCOS). Towards this goal, we have measured by radioimmunoassay the concentrations of FS in follicular fluid obtained from dominant and atretic cohort follicles of normal cycling women, preovulatory follicles of in-vitro fertilization (IVF) patients, and small Graafian follicles of patients with PCOS. In all cases, the follicular fluid concentration of FS was much higher (approximately 100-fold) than that reported in serum. The FS concentrations (ng/ml) were 203 +/- 42 (normal dominant), 185 +/- 17 (atretic cohort), 185 +/- 5 (IVF), and 250 +/- 14 (PCOS). There was no statistical difference between these mean values of FS. Further, there were no significant correlations between the follicular fluid concentrations of FS and the concentrations of oestradiol, progesterone, or androstenedione. These results indicate that human Graafian follicles, regardless of whether they are healthy or atretic, normal or PCOS, contain high steady-state concentrations of FS in the micro-environment. Collectively, these data fit with the hypothesis that major increases and decreases in the concentration of FS in the micro-environment may not play a key role in the mechanisms of selection, atresia, and PCOS in women. The possibility of regulation of intrinsic activin and inhibin activity through FS binding is discussed.

Case-Control Studies↗

PubMed1995

Interleukin-1 induces insulin-like growth factor binding protein-3 gene expression and protein production by Leydig cells.

Interleukin-1 (IL-1) is a potent inhibitor of Leydig cell function. IL-1 blocks human CG-induced cAMP and testosterone formation, as well as cytochrome P450 side-chain cleavage messenger RNA (mRNA) expression. IL-1 also decreases insulin-like growth factor-I (IGF-I) mRNA levels in Leydig cells. The effects of IGF-I are modified by IGF binding proteins (IGFBPs). In the present study, we evaluated the effects of IL-1 on IGFBP expression. Purified Leydig cells from adult rats were cultured with 0.1% heat-inactivated fetal bovine serum in Dulbecco's modified Eagles' medium/F12. Culture medium was changed to serum-free Dulbecco's modified Eagles' medium/F12 after 24 h and IL-1 beta (0.1-10 ng/ml) was added. Treatment of Leydig cells with IL-1 beta (10 ng/ml) for 2, 4, and 6 h resulted in a progressive induction of IGFBP-3 expression without affecting IGFBP-2 or IGFBP-4 mRNA levels. IL-1 beta in concentrations of 0.1, 1, and 10 ng/ml caused a 1.5-, 4-, and 6.5-fold induction of IGFBP-3 expression, respectively, whereas IGF-I mRNA levels were decreased in a dose-dependent manner. IL-1 beta increased the average transcription rate of IGFBP-3 by 3.3-fold. The t1/2 for IGFBP-3 mRNA was 2.07 h and was not affected by the treatment with IL-1 beta (2.21 h). The immunoblot of cell-conditioned media showed that the basal level of IGFBP-3 protein was low and IL-1 beta caused a dose-dependent increase in the production of IGFBP-3. These results indicate that IL-1 beta increases IGFBP-3 levels by increasing the rate of transcription rather than by changing the stability of IGFBP-3 mRNA. The addition of cycloheximide markedly inhibited IL-1 beta-induced IGFBP-3 mRNA levels. However, IL-1 beta was able to induce IGFBP-3 mRNA levels even in the presence of cycloheximide. This suggests that de novo protein synthesis may not be required for induction of IGFBP-3 mRNA by IL-1 beta. In conclusion, IL-1 beta inhibits IGF-I but increases IGFBP-3 expression in Leydig cells, and this may contribute to the inhibitory effects of IL-1 beta on Leydig cell steroidogenesis.

Animals↗

PubMed1995

Transients of fluorescence polarization in skeletal muscle fibers labeled with rhodamine on the regulatory light chain.

Structural changes of the myosin heads were correlated with mechanical events in the cross-bridge cycle by measuring fluorescence polarization signals at high time resolution from rhodamine probes bound to myosin regulatory light chains in skeletal muscle fibers. Motions of the cross-bridges were partially synchronized either by applying quick length changes to the fibers during active contractions or by activating the fibers from rigor by photolysis of caged ATP in the presence of Ca2+. With fibers in rigor, the fluorescence polarization values indicate that the probe dipoles are quite well ordered and are directed away from the muscle fiber axis. After photorelease of ATP from caged ATP, changes in polarization signals are consistent with broadening of the distribution of probe orientations. The signal deflections occur when ATP binds to actomyosin or when the cross-bridges detach, but the orientational distribution changes surprisingly little during active force development. In contrast, when staircases of quick releases are applied to labeled fibers during active contractions, the fluorescence polarization signals suggest a concerted rotation of the probes. The results indicate that the light chain region of myosin tilts during the quick release and/or during the tension recovery phase within the next few ms.

Adenosine Triphosphate↗

PubMed1994

Reversal of experimental autoimmune encephalomyelitis by a soluble peptide variant of a myelin basic protein epitope: T cell receptor antagonism and reduction of interferon gamma and tumor necrosis factor alpha production.

An immunodominant epitope of myelin basic protein (MBP), VHFFKNIVTPRTP (p87-99), is a major target of T cells in lesions of multiple sclerosis (MS) and in experimental allergic encephalomyelitis (EAE). T cells found in EAE lesions bear the same amino acids in the third complementary determining region of the T cell receptor (TCR) as those found in MS lesions. We analyzed the trimolecular interactions between MBP p87-99, class II major histocompatibility complex (MHC), and TCR, and designed soluble inhibitors for therapy. F, N, I, and V at positions 90, 92, 93, and 94 interact with MHC, whereas K, T, and P at positions 91, 95, and 96 interact with TCR. The peptides, p87-99[95T > A] and p87-99[96P > A] could compete more effectively with p87-99 for binding to MHC and could antagonize the in vitro response to T cells to p87-99 more effectively than p87-99[91K > A]. However, only p87-99[91K > A] prevented and reversed EAE, indicating that the extent of MHC or TCR competition does not predict success in treating EAE. To elucidate the mechanism of inhibition of EAE, draining lymph node cells from rats immunized with the native peptide alone or together with each of the three TCR antagonists were challenged in vitro with p87-99. Administration of p87-99[91K > A], but not p87-99 [95T > A] or p87-99[96P > A], reduced the production of tumor necrosis factor (TNF)- alpha and interferon (IFN) gamma. IFN-gamma and TNF-alpha are two cytokines that are critical in the pathogenesis of EAE and MS.

Amino Acid Sequence↗

PubMed1994

Identification of soluble forms of the fibroblast growth factor receptor in blood.

We have purified three acidic (FGF-1) and basic (FGF-2) fibroblast growth factor binding proteins (FGF-BP1, FGF-BP2, and FGF-BP3) from human plasma and calf serum and demonstrate the presence of these circulating FGF-BPs in blood. Each are truncated forms of the high-affinity FGF receptor (FGFR-1). FGF-BP1 and FGF-BP2 have estimated molecular masses of 70-85 kDa and 55-60 kDa, respectively, and are detected by using 125I-labeled FGF-2 ligand blotting. Immunoblotting with four distinct antibodies to FGFR-1 reveals that FGF-BP1 and FGF-BP2 are immunologically and biochemically related to the extracellular domain of FGFR-1. Reverse-phase HPLC chromatography resolves FGF-BP2 into two proteins with estimated molecular masses of 55 kDa and 60 kDa. Protein sequencing of the amino terminus of FGF-BP2 and FGF-BP3 reveals identity with the extracellular domain of the two-IgG-loop form of human FGFR-1. The FGF-BPs do not require heparin to bind FGF-2 on affinity columns, but heparin does enhance their recovery from blood. These FGF-BPs may play an important physiological role in regulating the biological activity of FGF and the other members of the FGF family of growth factors.

Amino Acid Sequence↗

PubMed1994

Hormonal regulation of insulin-like growth factor II and insulin-like growth factor binding protein expression by breast cancer cells in vivo: evidence for stromal epithelial interactions.

Insulin-like growth factors (IGFs) I and II are potent mitogens for breast cancer cells. Their proliferative activity is likely to be influenced by their binding proteins (IGFBPs), a family of newly identified proteins. We report here on the in vivo hormonal regulation of mRNAs for IGF-II and IGFBPs in the N-nitrosomethylurea-induced rat mammary tumor, a well-established model of hormone-responsive mammary cancer. IGF-II mRNA levels tended to decrease in regressing tumors following ovariectomy, and they markedly increased upon reactivation of tumor growth with hormone repletion. Ovariectomy induced a drastic increase in IG-FBP-6 mRNA which was reversible with hormone repletion. Similar but more modest changes were observed with IGFBP-2 mRNA. In contrast, IGFBP-3 and IGFBP-4 mRNAs tended to decrease with ovariectomy and increase with hormone repletion. These latter effects, however, were modest, variable, and not statistically significant. In situ hybridization analysis revealed that IGF-II, IGFBP-5, and IGFBP-6 mRNAs were localized in the stromal component of the tumor, whereas IGFBP-2 mRNA was expressed by epithelial cells. We conclude that hormonal regulation of IGFBP expression is heterogeneous, thus suggesting divergent biological functions for these peptides. Our data also emphasize the importance of potential stromal-epithelial interactions in the control of breast cancer cell proliferation by IGFs.

Animals↗

PubMed1994

Restraint changes pentobarbital-induced sleeping time in rats: evidence that arousal is modulated by brain corticotropin-releasing hormone and opioid in stress.

The effect of restraint of different duration on sodium pentobarbital (PbNa)-induced sleeping time was examined in rats. 1 h-restraint significantly shortened PbNa (50 mg/kg b.wt., administered i.p. immediately after restraint)-induced sleeping time as reported previously, whereas 2 h-restraint significantly prolonged the sleeping time. Naloxone (1 mg/kg b.wt.) administered i.p. 15 min before the start of restraint further exaggerated the 1 h-restraint-caused shortening of PbNa-induced sleeping time, and it blocked the 2 h-restraint-caused prolongation of the sleeping time. SDZ202-250 (0.1 pmol and 0.5 pmol), a selective mu agonist, but not [D-Pen2-D-Pen5]enkephalin (0.1 pmol-1.0 nmol), a selective delta agonist, or U50488H (0.1 pmol-1.0 nmol), a selective kappa agonist, administered i.c.v. 15 min before the i.p. injection of PbNa significantly prolonged PbNa-induced sleeping time; its prolongation was blocked by naloxone. These results suggest that a mu receptor-binding opioid prolongs PbNa-induced sleeping time in stress. The 2 h-restraint-caused prolongation of PbNa-induced sleeping time was also blocked by alpha-helical CRH(9-41) (26 nmol), a corticotropin-releasing hormone (CRH) receptor antagonist, administered i.c.v. 15 min before the start of restraint. In conjunction with our previous findings that the i.c.v. administration of CRH shortens PbNa-induced sleeping time and the 1 h restraint-caused shortening of PbNa-induced sleeping time is blocked by the CRH receptor antagonist, the present results suggest that CRH may stimulate an opioid-specific sedative mechanism, thus causing the prolongation of PbNa-induced sleeping time in 2 h-restraint.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1994

Expression of a nuclear envelope protein recognized by the monoclonal antibody BU31 in lung tumours: relationship to Ki-67 antigen expression.

The production of the murine monoclonal antibody BU31 is described. This antibody identifies a nuclear envelope protein which is expressed in some but not all cells, and which resembles statin, a protein reported to be expressed by non-proliferating cells. BU31 was applied onto frozen sections of a series of 78 lung tumours and the staining patterns were compared with those obtained with Ki-67. There was an inverse correlation between the proportion of tumour nuclei labelled with the two reagents (r = -0.61, 95 per cent confidence intervals -0.73 to -0.45). However, the four neuroendocrine neoplasms were BU31-negative. Squamous cell carcinomas often showed a peripheral distribution of the cells stained positively with Ki-67, whereas BU31 tended to label centrally situated cells. These observations are consistent with the concept that the antigen recognized by BU31 is expressed by non-proliferating cells in these tumours.

Adult↗

PubMed1994

Porcine ovarian granulosa cells secrete insulin-like growth factor-binding proteins-4 and -5 and express their messenger ribonucleic acids: regulation by follicle-stimulating hormone and insulin-like growth factor-1.

Using ligand blotting, Western immunoblotting, and Northern analysis, we have characterized the insulin-like growth factor (IGF)-binding proteins (IGFBPs) produced by cultures of porcine granulosa cells. Ligand blot analysis of conditioned medium from untreated cultures of moderately differentiated granulosa cells (MDGCs; from 4-6-mm follicles) revealed mainly IGF-binding activity associated with IGFBP-2 (34 kDa) and IGFBP-3 (40/44-kDa doublet), which have previously been identified and characterized. In addition, these cultures secreted 30- and 22-kDa forms under some circumstances. The identification and regulation of these IGFBPs of lower molecular mass were the focus of the current studies. Treatment of these MDGCs with IGF-I dramatically stimulated the production (to a detectable level) of a 30-kDa IGFBP that was identified by immunoblotting with antiserum to IGFBP-5 but not antisera to IGFBP-1, -2, -3, -4, or -6. Production of IGFBP-5 was attenuated by concurrent treatment with FSH. IGFBP-5 mRNA in these cultures was correspondingly stimulated by IGF-I but unaffected by FSH. FSH increased the level of a minor 22-kDa IGFBP. Messenger RNAs for IGFBP-1, -4, and -6 were also examined but only IGFBP-4 mRNA was detectable, suggesting that the 22-kDa band was IGFBP-4. These results were compared to those in cultures of immature granulosa cells from 1-3-mm follicles, in which 22- and 30-kDa IGFBPs were readily detectable. An antiserum to IGFBP-4 precipitated the 22- and 30-kDa bands whereas deglycosylation shifted the 30-kDa IGFBP to 22 kDa, suggesting that both these bands represent glycosylation variants of IGFBP-4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Insulin-like growth factor binding proteins show distinct patterns of expression in the rat uterus.

An intrinsic insulin-like growth factor (IGF) system, complete with IGF ligands, receptors, and biological responses, is present in the rat uterus, where it is thought to regulate uterine homeostasis by autocrine/paracrine mechanisms. It is known that IGF binding proteins (IGFBP) modulate IGF-I and IGF-II action, but very little information is available concerning their cellular localization in the uterus. Therefore, we have employed in situ hybridization to localize IGFBP-1, -2, -3, -4, -5, and -6 mRNAs in the adult rat uterus during the estrous cycle. IGFBP-1 was undetectable in all uteri examined. IGFBP-2 mRNA was localized only in the luminal epithelium of the endometrium. It was abundant during proestrus (P1000 h, P2000 h) and early estrus (E0200 h), but was relatively low at other stages of the cycle. IGFBP-3 mRNA was localized to the stroma cells juxtaposed to the endometrium. A weak signal was detected on estrus morning (E0200 h, E1000 h), but high levels of IGFBP-3 mRNA were observed in the stroma cells on Day 12 of pregnancy. IGFBP-4 mRNA was localized only in the luminal epithelium of the endometrium. It was moderately abundant at diestrus I and II, but the signal was very low or absent at other times in the cycle. IGFBP-5 mRNA was localized in the circular and longitudinal muscle layers of the myometrium. The IGFBP-5 hybridization signal was maximal at diestrus, weak on proestrus, and moderate during estrus. IGFBP-6 mRNA was also expressed in the myometrium. The signal was strong on estrus morning (E0200 h and E1000 h) and low or absent at other times in the cycle. These results provide the first direct evidence that the genes encoding the six IGFBP are expressed in a tissue-specific manner in the adult rat uterus. Equally important, the levels of the mRNA for each IGFBP appear to change throughout the estrous cycle, but not in a parallel fashion. These results support the hypothesis that inducible and tissue-specific expression of IGFBP-2 to -6 may be involved in modulating the activity of the IGF ligands during the proliferative and secretory phases of the uterine cycle.

Animals↗