FMS receptor for M-CSF (CSF-1) is sensitive to the kinase inhibitor imatinib and mutation of Asp-802 to Val confers resistance.

The kinase inhibitor imatinib is used in the treatment of chronic myeloid leukaemia, where it targets the intracellular Bcr-Abl tyrosine kinase, and gastrointestinal stromal tumours, where it targets either the KIT or PDGF tyrosine kinase receptors. Here, we report that imatinib is also an effective inhibitor of the closely related FMS receptor for macrophage colony stimulating factor and that mutation of Asp 802 of FMS to Val confers imatinib resistance. Imatinib readily reverted the transformed phenotype of haemopoietic and fibroblast cell lines that express the oncogene v-fms and also inhibited the growth of the Bacl.2F5 macrophage cell line. The cellular IC50 value of imatinib for FMS was similar to those for Bcr-Abl and KIT. Consequently, imatinib may also prove effective for the treatment of diseases whose progression is dependent upon macrophage-colony stimulating factor, this includes certain aspects of cancer and inflammation.

A family of phosphoinositide 3-kinases in Drosophila identifies a new mediator of signal transduction.

BACKGROUND: Mammalian phosphoinositide 3-kinases (PI 3-kinases) are involved in receptor-mediated signal transduction and have been implicated in processes such as transformation and mitogenesis through their role in elevating cellular phosphatidylinositol (3,4,5)-trisphosphate. Additionally, a PI 3-kinase activity which generates phosphatidylinositol 3-phosphate has been shown to be required for protein trafficking in yeast. RESULTS: We have identified a family of three distinct PI 3-kinases in Drosophila, using an approach based on the polymerase chain reaction to amplify a region corresponding to the conserved catalytic domain of PI 3-kinases. One of these family members, PI3K_92D, is closely related to the prototypical PI 3-kinase, p110 alpha; PI3K_59F is homologous to Vps34p, whereas the third, PI3K_68D, is a novel PI 3-kinase which is widely expressed throughout the Drosophila life cycle. The PI3K_68D cDNA encodes a protein of 210 kDa, which lacks sequences implicated in linking p110 PI 3-kinases to p85 adaptor proteins, but contains an amino-terminal proline-rich sequence, which could bind to SH3 domains, and a carboxy-terminal C2 domain. Biochemical analyses demonstrate that PI3K_68D has a novel substrate specificity in vitro, restricted to phosphatidylinositol and phosphatidylinositol 4-phosphate, and is unable to phosphorylate phosphatidylinositol (4,5)-bisphosphate, the implied in vivo substrate for p110. CONCLUSIONS: A family of PI 3-kinases in Drosophila, including a novel class represented by PI3K_68D, is described. PI3K_68D has the potential to bind to signalling molecules containing SH3 domains, lacks p85-adaptor-binding sequences, has a Ca(2+)-independent phospholipid-binding domain and displays a restricted in vitro substrate specificity, so it could define a novel signal transduction pathway.

Recruitment of the class II phosphoinositide 3-kinase C2beta to the epidermal growth factor receptor: role of Grb2.

Previously we demonstrated that the class II phosphoinositide 3-kinase C2beta (PI3K-C2beta) is rapidly recruited to a phosphotyrosine signaling complex containing the activated receptor for epidermal growth factor (EGF). Although this association was shown to be dependent upon specific phosphotyrosine residues present on the EGF receptor, the underlying mechanism remained unclear. In this study the interaction between PI3K-C2beta and the EGF receptor is competitively attenuated by synthetic peptides derived from each of three proline-rich motifs present within the N-terminal region of the PI3K. Further, a series of N-terminal PI3K-C2beta fragments, truncated prior to each proline-rich region, bound the receptor with decreased efficiency. A single proline-rich region was unable to mediate receptor association. Finally, an equivalent N-terminal fragment of PI3K-C2alpha that lacks similar proline-rich motifs was unable to affinity purify the activated EGF receptor from cell lysates. Since these findings revealed that the interaction between the EGF receptor and PI3K-C2beta is indirect, we sought to identify an adaptor molecule that could mediate their association. In addition to the EGF receptor, PI3K-C2beta(2-298) also isolated both Shc and Grb2 from A431 cell lysates. Recombinant Grb2 directly bound PI3K-C2beta in vitro, and this effect was reproduced using either SH3 domain expressed as a glutathione S-transferase (GST) fusion. Interaction with Grb2 dramatically increased the catalytic activity of this PI3K. The relevance of this association was confirmed when PI3K-C2beta was isolated by coimmunoprecipitation with anti-Grb2 antibody from numerous cell lines. Using immobilized, phosphorylated EGF receptor, recombinant PI3K-C2beta was only purified in the presence of Grb2. We conclude that proline-rich motifs within the N terminus of PI3K-C2beta mediate the association of this enzyme with activated EGF receptor and that this interaction involves the Grb2 adaptor.

Class II phosphoinositide 3-kinases are downstream targets of activated polypeptide growth factor receptors.

The class II phosphoinositide 3-kinases (PI3K) PI3K-C2alpha and PI3K-C2beta are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2alpha and PI3K-C2beta represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2alpha and PI3K-C2beta immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca(2+) as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2alpha is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2beta was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2beta with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2beta occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2beta was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3' phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.

The regulation of neuropeptide expression in rat anterior pituitary following chronic manipulation of estrogen status: a comparison between substance P, neuropeptide Y, neurotensin, and vasoactive intestinal peptide.

The neuropeptides substance P, neuropeptide Y, neurotensin, and vasoactive intestinal peptide are present in normal rat anterior pituitary gland and hypothalamus and can influence the secretion of classical pituitary hormones. We investigated the effects of estrogen manipulation on pituitary and hypothalamic expression of these four peptides by specific RIAs and cDNA probe analysis. Surgical ovariectomy produced a significant rise in the pituitary content of substance P immunoreactivity (IR) (130.2 +/- 4.8 fmol/gland vs. control 29.1 +/- 2.2, P less than 0.001), neuropeptide Y IR (34.9 +/- 3.9 vs. 19.6 +/- 2.0, P less than 0.05) and neurotensin IR (85.1 +/- 8.2 vs. 62.4 +/- 7.2, P less than 0.05), while vasoactive intestinal peptide IR showed a fall (201.2 +/- 18.8 vs. 285.4 +/- 25.9, P less than 0.05). These patterns were reversed with estrogen replacement or high dose estrogen treatment. Changes in peptide content were accompanied by parallel changes in the mRNA for each peptide. Treatment with an antiestrogen (tamoxifen) resulted in no change in substance P, neuropeptide Y, and neurotensin expression, while vasoactive intestinal peptide IR content decreased to below the assay detection limit. Hypothalamic expression of these peptides did not change with any of the treatments. These results indicate that: 1) the control of the pituitary expression of the four peptides under the influence of estrogen occurs predominantly at the level of gene transcription and 2) normalization of the castration induced changes by exogenous estrogen replacement suggests the changes to be mediated by the absence of this steroid. The regulation of the pituitary expression of these peptides by estrogen support the possibility of their having an autocrine or paracrine role.

Developmental patterns of glucagon-like peptide-1-(7-36) amide and peptide-YY in rat pancreas and gut.

Glucagon-like peptide-(17-36) amide [GLP-1-(7-36) amide] and peptide tyrosine tyrosine (PYY) are both products of the enteroglucagon cell. To examine the changes occurring during development, we analyzed by RIA the pancreatic concentrations of these two peptides in fetal and neonatal rats. The levels obtained were compared with those of the classical islet hormones, insulin, somatostatin, and glucagon. The total hormone content of the pancreas increased with age for insulin, glucagon, and somatostatin. The amounts of GLP-1-(7-36) amide immunoreactivity (IR) and PYY, however, remained approximately constant in the 3-, 8-, and 30-day-old and adult pancreas. GLP-1-(7-36) amide IR showed only a single peak by gel chromatography, but further analysis by anion exchange chromatography showed that during the fetal period (-18 days) and 3 days postpartum GLP-1-(7-36) amide was the main product, whereas 30 days postpartum and in adult pancreas mainly GLP-1 and an intermediate form were found. Similar analyses of gut extracts revealed that only GLP-1-(7-36) amide is produced during intestinal development. The gut content of GLP-1-(7-36) amide IR and PYY IR increased approximately 100 times between the fetus and the 30-day-old rat. This study reveals a complex and specific regulation of posttranslational processing during maturation for these two peptides.

Localization of 7B2, neuromedin B, and neuromedin U in specific cell types of rat, mouse, and human pituitary, in rat hypothalamus, and in 30 human pituitary and extrapituitary tumors.

The distributions of three novel peptides, 7B2, neuromedin B, and neuromedin U, in rat, mouse, and human pituitaries, rat hypothalamus, and 30 human pituitary tumors were investigated with immunocytochemistry. Immunoreactivity for 7B2 was present in rat, mouse, and human gonadotropes, in intermediate lobe cells and posterior lobe nerve fibers in rats and mice, in rat hypothalamus (particularly in the median eminence), and in eight human pituitary gonadotropinomas. In gonadectomized rats, larger, more numerous LH beta- and 7B2-immunoreactive gonadotropes were seen than in controls. Extractable 7B2-like immunoreactivity was elevated but not significantly so in gonadectomized rat pituitaries [males: castrated, 37.4 +/- 4.3 (mean +/- SE); controls, 26.9 +/- 4.3; females: ovariectomized, 27.2 +/- 2.7; controls, 19.1 +/- 2.2 pmol/gland]. Neuromedin B immunoreactivity was found in normal rat and mouse thyrotropes and weakly in "thyroidectomy" cells in hypothyroid rats, in which extractable pituitary neuromedin B was significantly depleted (thyroidectomized, 87.0 +/- 14.0; methimazole-treated, 82.0 +/- 11.4; control, 230.7 +/- 25.6 fmol/gland). Hyperthyroid rat pituitaries showed increased TSH beta and neuromedin B immunoreactivities and neuromedin B content (TRH-treated, 385.2 +/- 30.2; T4-treated, 352.6 +/- 20.2; control, 230.7 +/- 25.6 fmol/gland). Neuromedin U immunoreactivity occurred in corticotropes of all species, in rat and mouse intermediate lobe, and throughout the rat hypothalamus, with immunoreactive cell bodies in the arcuate nucleus. Neuromedin U-immunoreactive cells were present in six of six human pituitary and five of six human extrapituitary corticotropinomas. In adrenalectomized rats, corticotropes were larger and more numerous than in controls, but extractable anterior pituitary neuromedin U-like immunoreactivity was not raised (adrenalectomized, 3.30 +/- 0.45; control, 3.32 +/- 0.27 pmol/gland). Our findings suggest that 7B2, neuromedin B, and neuromedin U may be involved in pituitary function.

Is the C-terminal flanking peptide of rat cholecystokinin double sulphated?

A specific radioimmunoassay was developed to the predicted nine amino acid C-terminal flanking peptide of cholecystokinin (peptide serine serine, PSS). In aqueous extracts of rat brain, PSS was undetectable unless the extracts were first treated with arylsulphatase, which also resulted in desulphation of cholecystokinin. The reverse-phase HPLC analysis of partially desulphated extracts showed the presence of two peaks intermediate to the naturally occurring and the completely desulphated forms. It is therefore proposed that the CCK-flanking peptide PSS has both tyrosine residues sulphated.

Occurrence and developmental pattern of neuromedin U-immunoreactive nerves in the gastrointestinal tract and brain of the rat.

Neuromedin U is a newly described regulatory peptide, found by radioimmunoassay in significant concentrations in both the brain and gut of the rat. The aim of the present study was to localize this peptide immunoreactivity to discrete structures of the gut and brain and to map its distribution using immunocytochemistry. In the gut, neuromedin U was confined to nerve fibres mainly in the myenteric and submucous plexuses and the mucosa of all areas except stomach. Immunoreactive ganglion cells were seen in both ganglionated plexuses and their number did not increase following colchicine administration. This observation and the finding that the population of neuromedin U-immunoreactive nerves in the ileum was not affected by complete extrinsic denervation indicated that the nerves are mostly intrinsic in origin. Colocalization studies revealed neuromedin U and calcitonin gene-related peptide were present in the same myenteric and submucosal ganglion cells. Transection experiments showed that, like calcitonin gene-related peptide-immunoreactive nerves, fibres containing neuromedin U project for very short distances in both an oral and anal direction. At the electron microscopic level, neuromedin U immunoreactivity, demonstrated using the immunogold technique, was localized to large granular vesicles. In the central nervous system, neuromedin U immunoreactivity was localized to fibres which were widespread throughout the brain, except in the cerebellum. The presence of neuromedin U-immunoreactive cell bodies was restricted to the rostrocaudal part of the arcuate nucleus. Colocalization studies showed that a proportion of the neuromedin U-immunoreactive cell bodies in the arcuate nucleus also contained pro-opiomelanocortin. Neuromedin U-immunoreactive fibres were first detected in the rat intestinal mucosa at day 1 after birth. In the brain, the arcuate nucleus showed neuromedin U-immunoreactive neuronal cell bodies at E16 but not at E14. In conclusion, neuromedin U is a new member of the group of molecules known as brain-gut peptides.

The influence of adrenal hormone status on neuroendocrine peptides in the rat anterior pituitary gland.

Neuropeptide Y (NPY), neurotensin (NT), substance P (SP) and vasoactive intestinal peptide (VIP) are four structurally unrelated neuroendocrine peptides which affect anterior pituitary function. All four peptides appear to be locally synthesized in the anterior pituitary gland and have been shown to be regulated by thyroid and/or sex hormone status. We show here that NT, SP and VIP but not NPY are influenced by adrenal hormone status in the male rat pituitary gland. Adrenalectomy increased the content of VIP (35.4 +/- 4.0 (S.E.M.) vs control 11.9 +/- 1.1 pmol/g wet weight) but decreased that of SP (18.8 +/- 2.3 vs control 36.7 +/- 3.5 pmol/g wet weight). Adrenalectomy combined with castration decreased the content of SP (14.6 +/- 3.5 vs control 36.7 +/- 3.9 pmol/g wet weight) but had no effect on VIP content. Treatment with dexamethasone produced significant decreases in NT, SP and VIP contents (17.8 +/- 2.3 vs control 32.6 +/- 3.4 pmol/g wet weight, 5.5 +/- 0.9 vs control 36.7 +/- 3.9 pmol/g wet weight and 4.2 +/- 0.6 vs control 11.9 +/- 1.1 pmol/g wet weight respectively). The changes in pituitary peptide contents occurred in parallel with changes in mRNA levels, suggesting that alterations in glucocorticoid hormone status can alter the synthesis of these peptides. These results, together with the known effects of these neuroendocrine peptides suggest possible functions for locally produced SP and VIP in regulating the secretion of adrenocorticotrophin and/or other pro-opiomelanocortin-derived peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

The anterior pituitary content of neuromedin U-like immunoreactivity is altered by thyrotrophin-releasing hormone and thyroid hormone status in the rat.

In this study we have examined the effects of TRH, thyroid hormones and dopamine on the rat anterior pituitary content of neuromedin U-like immunoreactivity. Oral administration of TRH (20 mg/100 g per day) to euthyroid animals evoked a fivefold increase in peptide content after 12 days of treatment. This effect was found to be dependent upon circulating levels of thyroid hormone, since administration of TRH to thyroidectomized animals failed to show a similar effect without simultaneous treatment with tri-iodothyronine. The possibility that the TRH-induced increase in anterior lobe neuromedin U content reflected alterations in prolactin secretion or synthetic rate was also examined. Treatment of euthyroid animals with a dopamine agonist and antagonist was, however, without effect. These results demonstrate a unique relationship between TRH and thyroid hormone levels in increasing the anterior lobe content of neuromedin U immunoreactivity.

Chronic administration of the somatostatin analogue SMS 201-995 does not lead to endogenous antibody formation.

Plasma samples of seven patients with gut and pancreatic endocrine tumours who have been on long-term treatment with a long-acting somatostatin analogue (SMS 201-995) were investigated for endogenous antibodies to the peptide by incubation with radiolabelled SMS 201-995. The duration of treatment with the somatostatin analogue was between 9 and 26 months and the dose from 100 to 300 micrograms day-1. In none of the patients could antibodies to SMS be detected. The effect of this somatostatin analogue is unlikely to be impaired by formation of endogenous antibodies, even after long-term treatment.

Neuromedin U--a study of its distribution in the rat.

The distribution of neuromedin U, a novel peptide originally isolated from porcine spinal cord, was investigated in the rat using a recently developed radioimmunoassay. High concentrations of neuromedin U-like immunoreactivity were found in the pituitary gland and gastrointestinal tract. Significant concentrations of immunoreactivity were also found in several regions of the rat brain, spinal cord and both male and female genitourinary tracts. In the small intestine, neuromedin U-like immunoreactivity was restricted to the submucosal muscular layers, suggesting localization in neurones rather than in epithelial cells. Chromatographic analysis of pituitary, spinal cord and gut revealed a single peak of immunoreactivity which did not co-elute with either synthetic porcine neuromedin U-25 nor neuromedin U-8, indicating inter-species molecular heterogeneity.

The purification and sequence analysis of an avian neuromedin U.

In this study we have purified an avian homologue of neuromedin U from the chicken. Each step of the purification process was followed by a specific radioimmunoassay developed using porcine neuromedin U. Microsequence analysis characterised the peptide to be 25 amino acid residues long with the following sequence: Y-K-V-D-E-D-L-Q-G-A-G-G-I-Q-S-R-G-Y-F-F-F-R-P-R-N. Chicken neuromedin U has marked sequence similarity with the porcine peptide at its bioactive C-terminal region. Our findings demonstrate that the amino acid sequence of neuromedin U is markedly conserved in species which have diverged millions of years ago in evolutionary terms.

A case of somatostatinoma: responses to food and SMS 201-995 administration.

Plasma somatostatin response to food and the administration of the long-acting somatostatin analogue SMS 201-995, as well as pituitary responses to insulin-induced hypoglycemia, growth hormone-releasing hormone (GHRH) and thyrotropin-releasing hormone (TRH), were assessed in a 60-year-old woman with biopsy-proven metastatic somatostatinoma. SMS 201-995 alone did not change plasma somatostatin-like immunoreactivity (SRIF), but levels of low-molecular-weight forms (SRIF-14 and SRIF-28) more than doubled in response to a standard meal. This postprandial response was not affected by pretreatment with SMS 201-995. Although a growth hormone response to insulin-induced hypoglycemia and supramaximal GHRH was absent, the patient's thyroid-stimulating hormone response to TRH was normal. These results suggest that somatostatin analogues may not influence tumor autonomy and that a SRIF response to food may contribute to early satiety in patients with somatostatinoma. In addition, the long-term use of somatostatin analogues to suppress hormone production by other peptide-secreting tumors may not have predictable results.

The motor effect of neuromedin U on rat stomach in vitro.

A series of neuromedin U (NmU)-like peptides were found to evoke concentration-response contraction of rat fundic circular muscle in vitro. Isometric contraction of this tissue was induced by rat NmU, porcine NmU-8 and NmU-25, and frog NmU. Rat NmU was significantly more potent than frog NmU. The contractile action of rat NmU upon rat fundic strips was not affected by either atropine or tetrodotoxin indicating a direct effect. Maximal NmU-induced contractions were 10% of the maximal contraction induced by carbachol and ranged between those of the bradykinin and angiotensin II. None of the NmU peptides were active on the circular smooth muscle of the frog stomach or on the small and large intestinal longitudinal smooth muscle of either rat or frog. The results of this study demonstrate that members of the NmU peptide family all induce in vitro contraction of rat gastric circular smooth muscle independent of cholinergic or other neuronal mechanisms. Their activity, however, appears to be both tissue and species specific.

Comparison of neuromedin-N and neurotensin on net fluid flux across rat small intestine.

Neuromedin-N, a hexapeptide recently isolated and purified from porcine spinal cord, has close sequence homology with the C-terminal region of the tridecapeptide neurotensin. Both peptides have a remarkably similar peripheral distribution. Little is known of the biological activity of neuromedin-N. Neurotensin and peptide histidine methionine are known to stimulate net fluid secretion into rat small intestine. We have therefore tested the effect of neuromedin-N and the hexapeptide neurotensin-(8-13), the smallest fully active analogue of neurotensin in this system, compared with that of neurotensin and peptide histidine methionine. All four peptides reduced net absorption in low doses and caused net secretion in larger doses. However, whereas peptide histidine methionine was active in all areas of the small intestine, neurotensin, neurotensin-(8-13) and neuromedin-N were inactive in the duodenum. In the post-duodenal areas neurotensin was approximately 7 times more active than peptide histidine methionine, 21 times more potent than neuromedin-N and 33 times more potent than neurotensin-(8-13).

The generation of valosin-like peptides from a precursor protein in vitro as an extraction artifact.

Valosin is a 25 amino acid peptide recently isolated from the porcine gastrointestinal tract. The molecular forms of valosin-like immunoreactivity (VLIR) were examined following different tissue extraction procedures. Fractionation of tissue extracted with cold 0.1 M sodium hydroxide by Sephadex G50 gel permeation chromatography revealed a large form of VLIR (Kav = 0). Smaller forms of VLIR, Kav = 0.36 and 0.57 were obtained in tissue extracted by boiling in 0.5 M acetic acid. Acidification and boiling of the 0.1 M sodium hydroxide tissue extracts also generated smaller forms of VLIR of Kav = 0.36 and 0.57. Partially purified preparations of the large forms of VLIR extracted with sodium hydroxide could be disrupted into a smaller form of Kav = 0.57 by acidification and boiling. This smaller molecular form co-eluted with the synthetic 25 amino acid valosin standard. We conclude that valosin does not occur naturally but is an artifact generated by cleavage of a larger protein precursor upon acid extraction of tissues. Workers should be aware of the need to verify their extraction procedures when characterising novel peptides to avoid potential pitfalls such as acid/thermal cleavage of proteins.

Peptide histidine valine-42 stimulates gastric acid secretion in man.

The effect of peptide histidine valine-42 (PHV-42) on gastric acid secretion was studied in man. PHV-42 was infused into 5 healthy volunteers at a dose of 10 pmol/kg/min. This dose caused a significant stimulation of basal gastric acid and potassium output. There were no significant changes in circulating gastrin throughout the infusion. In 2 subjects with a background of submaximal pentagastrin stimulation, PHV-42 infusion at the same dose did not alter acid secretion in either subject. The previous observation that PHV-42 is found particularly in the stomach and the new finding that it stimulates basal gastric secretion suggest the possibility that PHV-42 could have a role in local control of acid secretion.

[The century-long trends of the French hospital system: an analysis in terms of regulation]. / La dynamique séculaire du système hospitalier français: une analyse en termes de régulation.

This article is an inquiry about growth of hospitals expenses. The analysis is founded on the long term construction of monetary series. These series show evidence of three characteristics: growth of hospitals expenses between 1815 and 1993, development trough a succession of stages, existence of long term cyclic fluctuations contrary to the Kondratieff's movements.