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K C Flanders

Publications and source records attributed to K C Flanders.

117 records · Page 7Linked to original sources
PubMed1986

Transforming growth factor-beta. A very potent inhibitor of myoblast differentiation, identical to the differentiation inhibitor secreted by Buffalo rat liver cells.

Transforming growth factor-beta (TGF-beta) is now known to have a number of actions in addition to the induction of phenotypic transformation in fibroblastic cells. In this paper, we characterize its inhibition of differentiation in rat myoblasts of Yaffe's L6 strain and demonstrate its identity or very close similarity to the differentiation inhibitor (DI) secreted by Buffalo rat liver cells cultured in serum-free medium. At concentrations as low as 60 pg/ml, TGF-beta gave detectable inhibition of differentiation measured as myoblast fusion and creatine kinase elevation; maximal inhibition was observed at and above 0.5 ng/ml (20 pM). The inhibition persisted as long as fresh TGF-beta was added at 48-h intervals, but it was reversed upon removal of the factor. By itself or in the presence of mitogens, TGF-beta had no mitogenic activity in the L6 cells. Concentration dependencies of human TGF-beta and the rat DI were closely parallel in three assays: inhibition of myoblast differentiation, stimulation of normal rat kidney cell growth in soft agar, and competition for displacement of labeled TGF-beta from binding sites on A549 human lung carcinoma cells. We conclude that most if not all of the DI activity found in medium conditioned by Buffalo rat liver cells can be attributed to the presence of TGF-beta or a very similar molecule. These observations also offer a potentially useful approach to study the control of myogenesis; the process(es) can be blocked in cloned L6 myoblasts by incubation with very small quantities of a pure protein in fully defined serum-free medium.

Animals↗
PubMed1986

Biosynthesis of an asparagine-linked oligosaccharide-containing calcitonin by a rat medullary thyroid carcinoma cell line.

Calcitonin contains an amino acid sequence that provides a potential site for glycosylation of the peptide at the asparagine at position 3. Preliminary evidence has suggested that there are glycosylated forms of calcitonin and its precursor, procalcitonin. The CA-77 rat medullary thyroid carcinoma cell line, recently developed to study calcitonin biosynthesis, was used to demonstrate the synthesis of glycosylated forms of this hormone by intact cells. Cultures were incubated in medium containing either [3H]mannose or [35S]methionine. Two species incorporating both labels were specifically immunoprecipitated when cell extracts were treated with calcitonin antibodies. Gel filtration chromatography in 6 M guanidine hydrochloride indicated that one peptide had a molecular weight of 5500, approximately 2000 daltons larger than calcitonin, while the second peptide had a molecular weight of 14 400, the approximate size of procalcitonin. Treatment of the [3H]mannose-labeled cell extract with endo-beta-N-acetylglucosaminidase H before immunoprecipitation removed the labeled sugar from the calcitonin species. Microsequence analysis of the radiolabeled immunoreactive 5500-dalton calcitonin species showed methionine at cycle 8 and mannose at cycle 3, suggesting that this peptide is calcitonin containing an N-linked oligosaccharide at Asn-3. These results suggest that in this cell line a minor but significant biosynthetic pathway exists for the production of glycosylated calcitonin from glycosylated procalcitonin.

Amino Acid Sequence↗
PubMed1984

Semisynthetic derivatives of glucagon. The contribution of histidine-1 to hormone conformation and activity.

Semisynthetic N epsilon- acetimidoglucagon was prepared from the [des- His1 ]analogue by coupling the N-hydroxysuccinimide ester of N alpha- tBoc - Nimidazole -DNP-L-histidine to the peptide in dimethylformamide in the presence of 1-hydroxybenzotriazole. The deprotected, purified product was chemically identical to N epsilon- acetimidoglucagon and equipotent to N epsilon- acetimidoglucagon and native glucagon in its ability to activate adenylate cyclase and displace [125I] iodoglucagon from rat liver plasma membranes. Semisynthetic [ Phe1 ]-, [ Ala1 ]-, and [des- His1 ] glucagons prepared similarly achieved 85, 55, and 35% of the maximal activity and 22, 2, and 6% of the binding potency of N epsilon- acetimidoglucagon . The biological assays indicate that the amino group is involved to a greater extent in transduction than in binding, but the aromatic nature and hydrogen bonding capability of the imidazole ring of histidine-1 are important for both binding and transduction. In circular dichroism studies, all derivatives exhibited increased helicity in 2-chloroethanol. The [ Phe1 ] analogue although less soluble behaved similarly to native glucagon, while the [ Ala1 ] and [des- His1 ] derivatives exhibited an increased helical content in 0.01 N HCl as a result of an increased propensity of these derivatives to self-associate in the absence of 2-chloroethanol. The unexpected conformational changes throughout the molecule may have relevance for the functional activity.

Adenylyl Cyclases↗
PubMed1983

Noncooperative receptor interactions of glucagon and eleven analogues: inhibition of adenylate cyclase.

Glucagon and 11 glucagon derivatives were characterized and compared with respect to the cooperativity of their receptor interactions and their ability to elicit a biphasic (activation-inhibition) response from the adenylate cyclase system of rat liver plasma membranes. Slope factors were evaluated from two sets of experimental data, binding to hepatocyte receptors and activation of adenylate cyclase. The results are consistent with noncooperative binding to a single affinity state of the glucagon receptor for all derivatives, irrespective of the modification and the agonist properties of the derivatives. High-dose inhibition of adenylate cyclase activity was observed for native glucagon and all of the derivatives which were examined at high concentrations (greater than 10(-5) M). Partial agonism of some low-affinity glucagon derivatives is not caused by high-dose inhibition. Several mechanisms which might give rise to high-dose inhibition such as receptor cross-linking or multivalent receptor binding are discussed in relationship to the glucagon-receptor interaction. These phenomena indicate that significant differences exist between the glucagon system and the beta-adrenergic system.

Adenylyl Cyclase Inhibitors↗
PubMed1982

Semisynthetic derivatives of glucagon: (des-His1)N epsilon-acetimidoglucagon and N alpha-Biotinyl-N epsilon-acetimidoglucagon.

N epsilon-Acetimidoglucagon to be used for semisynthesis was prepared by reacting glucagon with methyl acetimidate hydrochloride at pH 10.2, favoring acetimidation of the sole epsilon-amino group. N epsilon-Acetimidoglucagon was isolated from the crude acetimidoglucagon mixture by anion-exchange chromatography at pH 9.4, producing a derivative which was identical with native glucagon on isoelectric focusing and which by amino acid analysis had greater than 98% of the lysine blocked. The yield was greater than that obtained when tetrahydrophthalic anhydride was used as a chromatographic handle to remove peptides with unreacted amino groups. N epsilon-Acetimidoglucagon closely resembled native glucagon in its biological activity and binding affinity, eliminating the need for deprotection. Semisynthetic N alpha-biotinyl-N epsilon-acetimidoglucagon, prepared by reacting (N-hydroxysuccinimido)biotin with N epsilon-acetimidoglucagon and purified by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 5.2) and exhibited 1.2% of the binding affinity, 2.4% of the biological potency, and 30% of the maximum activity of the native hormone. Preliminary fluorescence microscopy demonstrated binding of N alpha-biotinyl-N epsilon-acetimidoglucagon to glucagon specific receptors following exposure to fluorescein-labeled avidin. Capping of labeled receptors could be visualized with time. (Des-His1)N epsilon-acetimidoglucagon, prepared via a manual Edman degradation of N epsilon-acetimidoglucagon and isolated by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 5.2). The second residue, serine, has also been removed. Semisynthetic coupling of alternative residues to such derivatives will provide insight into the role of the amino-terminal residues in mediating the biological actions of the hormone.

Adenylyl Cyclases↗
PubMed1982

Glucagon carboxyl-terminal derivatives: preparation, purification, and characterization.

Chemical and enzymatic methods have been used to prepare the following series of seven glucagon derivatives modified in the carboxyl-terminal region important for hormone-receptor binding: [des-Asn28,Thr29](homoserine lactone27)glucagon, [des-Asn28,Thr29](homoserine27)glucagon, (S-methyl-Met27)glucagon, [des-Thr29](S-methyl-Met27)-glucagon, [des-Thr29]glucagon,[des-Asn28,Thr29](S-methyl-Met27)glucagon, and [des-Asn28,Thr29]glucagon. The derivatives were isolated in high yield, extensively purified, and chemically characterized. All were found to be full agonists of native glucagon. Binding affinity was evaluated by displacement of mono[125I]iodoglucagon prepared by new methods. Binding and biological activities closely correlated, indicating that most modifications affected the relative binding affinity and relative biological potency of glucagon to a comparable extent. Circular dichroism measured in dilute acid solution resembled that of native glucagon except for [des-Asn28,Thr29]glucagon which displayed increased alpha helicity (25%). All derivatives formed helical structures in 2-chloro-ethanol, although the amount of helicity induced was not closely correlated with biological activity. Binding and biological activities were not affected by removal of Thr-29, though both were reduced 20-fold when Asn-28 was also removed, irrespective of whether homoserine or native methionine remained at the carboxyl terminus. Lactone formation was associated with a further 5-fold reduction in binding affinity but not in activity. Methylation of Met-27 had essentially the same effect as removing the two carboxyl-terminal residues, although the combined effect of both modifications was greater than 100-fold reduction in binding and activity. These findings provide additional insight concerning glucagon structure-function relationships.

Adenylyl Cyclases↗
PubMedDate not supplied

Analogous tamoxifen and estrogen effects on transforming growth factor-betas 1 and 2 in the rat uterus.

Estrogenic stimulation is a potent risk factor for the development of uterine cancer. More recently, analysis of patients in prospective breast cancer trials have established that tamoxifen also increases uterine cancer risk. In this report, uteri of oophorectomized rats were examined to ascertain the effects of estrogen and tamoxifen on the uterine induction of two isoforms of transforming growth factor-beta (TGF-beta). In contrast to studies of cells derived from breast epithelium, our studies reveal that both estrogen and tamoxifen increase immunoreactive TGF-beta. These changes were particularly pronounced in the endometrial stroma. Effects of progesterone also were examined and found to be distinct and relatively restricted to the glandular epithelium. These studies indicate that, in the uteri of oophorectomized rats, tamoxifen exerts estrogen-like effects on a peptide previously implicated in the control of cellular growth and differentiation. We hypothesize that induction of TGF-beta isoforms may be an important mediator of both estrogen- and tamoxifen-induced proliferative disorders in the uterus.

Animals↗
PubMedDate not supplied

TGF-beta receptors-I and -II immunoexpression in Alzheimer's disease: a comparison with aging and progressive supranuclear palsy.

The transforming growth factor-betas (TGF-betas) influence cell survival, and TGF-beta2 shows increased immunoexpression in neurofibrillary tangle-bearing neurons and reactive glia in Alzheimer's disease (AD) and progressive supranuclear palsy (PSP). We compared immunohistochemical expression of TGF-beta type I (RI) and type II (RII) receptors in eight patients with AD, eight controls and three cases of progressive supranuclear palsy. Mild intraneuronal immunoreactivity for the RI receptor was observed in all cases. Intraneuronal TGF-beta RII receptor immunoexpression was more common in all groups, and its frequency did not differ between groups. We observed increased immunoreactivity for both RI and RII subtypes in reactive glia in the AD frontal cortex (RI: U = 0.5, p = 0.002; and RII: U = 9.000, p = 0.006) and parahippocampal gyrus (RI: U = 9.500, p = 0.013; RII: U = 14.5, p = 0.05) compared to control cases. We conclude that TGF-beta RI and II immunoreactivity is increased in reactive glia in AD and progressive supranuclear palsy, and RI immunoreactivity may occasionally be increased in neurons in cases with advanced AD.

Activin Receptors, Type I↗