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Segmental identity of structures located in the hindbrain and branchial arches of vertebrates are determined, at least in part, through the action of Hox paralog group 2 (PG2) genes. Despite an overall similarity in their function during development, Hox PG2 genes from evolutionarily divergent lineages have distinctly different spatio-temporal expression patterns, particularly in comparisons between actinopterygians and sarcopterygians. Results from our laboratory, including the cloning and expression analysis of an unprecedented three Hox PG2 genes (Hoxa2a, a2b, and b2a) from Morone saxatilis (striped bass), show that the trans-orthologs Hoxa2a and a2b are differentially expressed in both rhombomeres and branchial arch tissue whereas their paralogs expression (Hoxb2a) is restricted solely to the rhombomeres. The pattern of Hox PG2 expression documented for M. saxatilis contrasts sharply with that of Danio rerio (zebrafish) and mammals but appears to be similar to that of Gallus gallus (chicken). Our results indicate that while the Hox PG2 gene complement in actinopterygians is greater than that of sarcopterygians, expression patterns in these two major vertebrate lineages are similarly divergent, indicative that an increased number of genes does not lead to concordant increases in expression pattern divergence. Our results also reveal a considerable plasticity in the Hox gene-related evolutionary developmental specification of vertebrate hindbrain and branchial arch structures, at least some of which may underlie the variation in morphology observed in these divergent organisms.

The well conserved family of Hox genes code for transcription factors that are active regulators of segmental identity along the anterior-posterior axis in developing embryos. This activity is reportedly seen in the AP axis of the developing digestive tract in chick and mouse where Hox genes exhibit patterns of expression specific to the region of the gut. In the hindgut, the expression of Abd-B-like genes (paralogs 913) seems to be restricted in the mesoderm during differentiation where conducive signaling directs the epithelial transition of the endoderm. Here we investigate the expression of the Abd-B-like genes in the human colon adenocarcinoma cell line Caco-2, which is a well defined model for enterocyte differentiation. RT-PCR and Northern Blot results show differential expression of HoxD10, HoxC12, HoxB13 and HoxD13 when evaluated before and after differentiation. Transcripts for HoxD13 are present during exponential growth but not after differentiation, while HoxC12, HoxD10 and HoxB13 show an increase in transcripts post differentiation. Functional studies will further clarify these genes as potential regulators of proliferation and/or differentiation within this cell model. This work was supported by the American Cancer Society, Institutional Research Grant ECU 5-89812.

We developed a computational method for the identification of putative cis-regulatory elements, which incorporates a direct measure of the effects of selection as a determinant in non-protein coding sequence evolution. As such, the method provides a biologically relevant framework for comparisons of non-protein coding sequence evolution. Comparisons of orthologous HoxA, Aa and Ab cluster paralogous group 2 intergenic and intron-related sequences from Mus muscu/us(mouse), Homo sapiens (humans), Heterodontus francisci (horn shark), Polypterus senegalus (bichir), Danio rerio (zebrafish), Oreochromis niloticus (Nile tilapia), Morone saxatilis (striped bass), and Takifugu rubripes (Japanese pufferfish) revealed three sequence domains that are under purifying selection in all the species and on both clusters, indicative that these three regions likely represent fundamental regulatory control elements of HoxA cluster paralogous group 2 genes in their common gnathostome ancestor. Detailed analysis of one of these three domains using both Mus musculus and Morone saxatilis HoxA paralogous group 2 regions as the index for comparisons demonstrated the presence of four general gnathostome elements, one general actinopterygian element, two general teleost elements and a host of mammalian and perciform-specific elements. The results also showed the partitioning of cis-regulatory elements between the actinopterygian HoxAa and Ab clusters relative to the inferred ancestral gnathostome HoxA cluster. Overall, our results indicate that the evolution of Hox paralogous group 2 cis-regulatory regions is dominated by the evolution of novel lineage-specific elements superimposed on a framework of shared common ancestral elements.

Trophic changes of the exocrine pancreas after in vivo gastrin (G)/CCK treatment are well documented but up to now the study of the mechanisms involved is restricted by the lack of a suitable in vitro model. Nevertheless the in vivo trophic effect induced by gastrin/CCK peptides has been associated with an increase of ornithine decarboxylase (ODC) activity. In the present work, using the AR42J cell line in which CCK receptors and stimulation of amylase release by CCK peptides has already been demonstrated, we investigated the presence of gastrin binding sites and the possible modulation of proliferation by an inhibitor of ODC activity. 125I-BH-G17ns binding is saturable, reversible and specific. Potencies of the different analogues tested are G17ns greater than CCK8 greater than CCK8ns greater than or equal to G6s greater than G/CCK4. Furthermore dBt cGMP, a non-peptide antagonist for CCK receptors, does not compete for gastrin binding. This indicates the existence of a subclass of gastrin binding sites. Difluoromethyl ornithine (DFMO) (1 mM), an irreversible inhibitor of ODC, inhibits cell growth from day 3 up to day 7. This growth inhibition is dose dependent and closely related to an intracellular polyamine modulation. Putrescine and spermidine levels fell under detectable values while spermine levels increased. All these data suggest that this cell line could be a useful in vitro model to study the mechanisms of gastrin induced growth control. [ABSTRACT FROM PUBLISHER]

Purpose: We investigated the combination of tivantinib, a c-MET tyrosine kinase inhibitor (TKI), and bevacizumab, an anti-VEGF-A antibody., Methods: Patients with advanced solid tumors received bevacizumab (10 mg/kg intravenously every 2 weeks) and escalating doses of tivantinib (120-360 mg orally twice daily). In addition to safety and preliminary efficacy, we evaluated pharmacokinetics of tivantinib and its metabolites, as well as pharmacodynamic biomarkers in peripheral blood and skin., Results: Eleven patients received the combination treatment, which was generally well tolerated. The main dose-limiting toxicity was grade 3 hypertension, which was observed in four patients. Other toxicities included lymphopenia and electrolyte disturbances. No exposure-toxicity relationship was observed for tivantinib or metabolites. No clinical responses were observed. Mean levels of the serum cytokine bFGF increased (p = 0.008) after the bevacizumab-only lead-in and decreased back to baseline (p = 0.047) after addition of tivantinib. Tivantinib reduced levels of both phospho-MET (7/11 patients) and tubulin (4/11 patients) in skin., Conclusions: The combination of tivantinib and bevacizumab produced toxicities that were largely consistent with the safety profiles of the individual drugs. The study was terminated prior to establishment of the recommended phase II dose (RP2D) due to concerns regarding the mechanism of tivantinib, as well as lack of clinical efficacy seen in this and other studies. Tivantinib reversed the upregulation of bFGF caused by bevacizumab, which has been considered a potential mechanism of resistance to therapies targeting the VEGF pathway. The findings from this study suggest that the mechanism of action of tivantinib in humans may involve inhibition of both c-MET and tubulin expression., Trial Registration: NCT01749384 (First posted 12/13/2012)., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: To address multidrug resistance, we developed engineered Cationic Antimicrobial Peptides (eCAPs). Lead eCAP WLBU2 displays potent activity against drug-resistant bacteria and effectively treats lethal bacterial infections in mice, reducing bacterial loads to undetectable levels in diverse organs., Objective: To support the development of WLBU2, we conducted a mass balance study., Methods: CD1 mice were administered 10, 15, 20 and 30 mg/kg of QDx5 WLBU2 or a single dose of [14C]-WLBU2 at 15 mg/kg IV. Tolerability, tissue distribution and excretion were evaluated with liquid scintillation and HPLC-radiochromatography., Results: The maximum tolerated dose of WLBU2 is 20 mg/kg IV. We could account for greater than >96% of the radioactivity distributed within mouse tissues at 5 and 15 min. By 24h, only ~40-50% of radioactivity remained in the mice. The greatest % of the dose was present in liver, accounting for ~35% of radioactivity at 5 and 15 min, and ~ 8% of radioactivity remained at 24h. High radioactivity was also present in kidneys, plasma, red blood cells and lungs, while less than 0.2% of radioactivity was present in brain, fat, or skeletal muscle. Urinary and fecal excretion accounted for 12.5 and 2.2% of radioactivity at 24h., Conclusion: WLBU2 distributes widely to mouse tissues and is rapidly cleared with a terminal radioactivity half-life of 22 h, a clearance of 27.4 mL/h/kg, and a distribution volume of 0.94 L/kg. At 2-100 μg-eq/g, the concentrations of 14C-WLBU2 appear high enough in the tissues to account for the inhibition of microbial growth., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Background: Operating room (OR) and post-anesthesia care unit (PACU) activity are closely linked since the number, type, and sequence of surgeries influence subsequent PACU activity. We aimed to explore the relationship between duration-of-surgery (DOS) and PACU length-of-stay (LOS), the PACU-LOS:DOS ratio, since it is among the determinants of the number of PACU beds and nurse staffing required to insure efficient egress of patients from the OR., Methods: PACU-LOS:DOS ratio was examined using retrospective data from a tertiary medical center's surgical information system (Phase 1) and prospectively collected data from a convenience sample of post-operative patients (Phase 2)., Results: Phase 1 included 17 047 patients, the majority (73%) with PACU-LOS:DOS ratios >1.0, indicating PACU-LOS longer than DOS. Median PACU-LOS was 117 minutes, median DOS was 80 minutes, and median PACU-LOS/DOS ratio was 1.5. PACU-LOS showed greater variability than DOS because of extended PACU stays. Phase 2 (n = 2054) confirmed Phase 1 results (median PACU-LOS/DOS ratio - 1.8). In both phases at a DOS of >130 minutes PACU-LOS/DOS ratio became <1.0. In 24% of Phase 2 patients PACU-LOS was prolonged because of administrative issues. Post-operative, more than pre- and intra-operative, measurements influenced PACU-LOS., Conclusions: The PACU-LOS/DOS ratio proved useful for demonstrating interactions between 2 central components of the surgical system. The many patients with PACU-LOS:DOS ratios >1.0 provides objective evidence for the number of PACU beds exceeding the number of ORs., (© 2019 The Acta Anaesthesiologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

DNA damaging chemotherapy and radiation are widely used standard-of-care modalities for the treatment of cancer. Nevertheless, the outcome for many patients remains poor and this may be attributed, at least in part, to highly effective DNA repair mechanisms. Ataxia-telangiectasia mutated and Rad3-related (ATR) is a key regulator of the DNA-damage response (DDR) that orchestrates the repair of damaged replication forks. ATR is a serine/threonine protein kinase and ATR kinase inhibitors potentiate chemotherapy and radiation. The ATR kinase inhibitor VX-970 (NSC 780162) is in clinical development in combination with primary cytotoxic agents and as a monotherapy for tumors harboring specific mutations. We have developed and validated an LC-MS/MS assay for the sensitive, accurate and precise quantitation of VX-970 in human plasma. A dilute-and-shoot method was used to precipitate proteins followed by chromatographic separation with a Phenomenex Polar-RP 80Å (4μm, 50×2mm) column and a gradient acetonitrile-water mobile phase containing 0.1% formic acid from a 50μL sample volume. Detection was achieved using an API 4000 mass spectrometer using electrospray positive ionization mode. The assay was linear from 3 to 5,000ng/mL, proved to be accurate (94.6-104.2%) and precise (<8.4% CV), and fulfilled criteria from the FDA guidance for bioanalytical method validation. This LC-MS/MS assay will be a crucial tool in defining the clinical pharmacokinetics and pharmacology of VX-970 as it progresses through clinical development., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Pneumocystis jiroveci pneumonia is a classic opportunist infection affecting AIDS patients. However it is less frequent since systematic prophylaxis and antiretroviral therapies. Treatment resistance is rare in France. We report the case of a severe Pneumocystis jiroveci pneumonia with treatment resistance to standard treatment and fatal outcome. The different causes of treatment resistance, notably the role of CMV co-infection, were reviewed and discussed.

We investigated the role of the cAMP link to the signal transduction mechanism coupled with adenosine A(2A) and A(2B) receptors in cultured human coronary artery endothelial cells (HCAEC) and porcine coronary artery endothelial cells (PCAEC). 2-[4-[2-¿2-[(4-aminophenyl)methylcarbonylamino]ethylaminocarbon yl¿eth yl]phenyl]ethylamino-5'- ethylcarboxamidoadenosine ((125)I-PAPA-APEC) (PAPA-APEC) was used to demonstrate the specific binding in PCAEC membranes. The specific binding was saturable and reversible with a maximal number of binding sites (B(max)) of 240 fmol/mg protein, and scatchard analysis revealed a single class of binding site with an equilibrium dissociation constant (K(d)) of 1. 17 +/- 0.035 nM. In competition experiments, adenosine receptor agonists showed the following order of potency (based on IC(50)): 5'-(N-ethylcarboxamido)adenosine (NECA) >/= CGS-21680 > 2-chloroadenosine. This order appears to be consistent with the A(2) adenosine receptor classification. We also studied the effects of adenosine agonists on the accumulation of cAMP as an indirect approach to show the presence of functional A(2) receptors. Similarly, the same adenosine agonists (10(-7)-10(-4) M) elicited the production of cAMP in intact endothelial cells in a dose-dependent manner, exhibiting consistently with the A(2) adenosine receptor classification. A selective A(2A) adenosine receptor antagonist (ZM-241385, 10(-8) M) significantly inhibited the effect of CGS-21680 on cAMP but only partly inhibited the effect of NECA, suggesting the presence of both A(2A) and A(2B) receptors. Western blot analysis further showed the immunoreactivity of A(2A) and A(2B) receptor at 45 and 36 kDa, respectively, in both HCAEC and PCAEC. Direct evidence for the presence of A(2A) and A(2B) receptors in cultured HCAEC and PCAEC by reverse transcription-polymerase chain reaction (RT-PCR), revealed expected PCR product sizes (205 and 173 bp) for A(2A) and A(2B) receptors in HCAEC and PCAEC, respectively. The data show that adenylate cyclase-coupled adenosine A(2A) and A(2B) receptors are present in coronary endothelial cells.

The physical mapping of Hox gene clusters from a limited number of vertebrates has shown an overall conservation in gene organization in which major evolutionary changes appear to be primarily restricted to the deletion of one or more genes, with the exception of the amplification of additional clusters as postulated from zebrafish. We have sequenced a 31 kb region of the HoxA cluster from the teleost Morone saxatilis (striped bass), both to provide a detailed physical map of this region and to better understand the nature of Hox cluster evolution among vertebrate taxa. We identified five linked Hox genes: Hoxa4, Hoxa5, Hoxa7, Hoxa9, and Hoxa10, which are organized similarly to those of other vertebrates. Furthermore, we have documented the absence of the Hoxa6 and Hoxa8 genes within the 31 kb contig. Comparison of our results to those published for other vertebrates suggests that the absence of Hoxa6 is a common characteristic of teleosts, whereas the absence of Hoxa8 is common to vertebrates in general, with the possible exception of zebrafish. Further comparisons between the HoxA genes from Morone with those from the pufferfish, Fugu rubripes, revealed the likely presence of a previously unreported Hoxa7 gene, or gene fragment, in the Fugu genome, which suggests that the Hoxa7 gene, unlike Hoxa6 or Hoxa8, is present in teleosts. In addition to these differences in vertebrate Hox cluster structure, we also observed a marked reduction in the length of the Hoxa4--a10 region between vertebrate lineages representative of teleosts and mammals. Comparative analysis of HoxA cluster organization among teleosts and mammals suggests that cluster length reduction and lineage-specific gene loss events are hallmarks of Hox cluster evolution.

Although the major target organ of paraquat toxicity is the lung, the primary route of absorption of the herbicide is across the gastrointestinal epithelium. Thus, uptake of paraquat was investigated in the rat duodenal crypt cell line, IEC-6. The herbicide was toxic; as measured by cell number, the LD50 was 75 microM. Using 2 microM [14C]paraquat as tracer, uptake of the herbicide was found to be slow but linear over 24 hr at 37 degrees C. No accumulation was observed at 4 degrees C. Inhibition studies showed paraquat inhibited both putrescine and spermidine uptake after only a 15-min incubation. The Km for putrescine was increased when incubated in the presence of paraquat whereas Vmax was not changed, suggesting a competitive mode of inhibition. In the reverse situation, putrescine also inhibited paraquat uptake (IC50 10 microM). Paraquat uptake was reduced in a dose-dependent manner by the putative calmodulin antagonist W-7, but was not affected by KN-62, a Ca2+/calmodulin kinase II specific inhibitor. These results provide evidence that paraquat uptake occurs through the polyamine transport system in IEC-6 cells. Furthermore, this process is modulated by intracellular events involving a major signaling protein, Ca2+/calmodulin.

In the present work the effects of the novel neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) on both AR4-2J cell growth and the modulation of ornithine decarboxylase activity were investigated. Both PACAP38 and the amidated form PACAP27 caused a concentration-dependent stimulation of AR4-2J cell growth; the maximal increase was seen at 1 nmol/L (30% above control, P less than 0.01) with a half-maximal effect at 0.01 nmol/L. Ornithine decarboxylase activity was also increased by PACAP in a dose-dependent manner, reaching half-maximal stimulation at 0.5 nmol/L. The addition of 1 nmol/L of somatostatin analog SMS 201-995 totally suppressed PACAP-stimulated AR4-2J cell growth. Vasoactive intestinal polypeptide (3 mumol/L) and 8-bromo-cyclic adenosine monophosphate (1 mmol/L) had no effect on cell proliferation. Treatment of cells by pertussis toxin (25 ng.mL-1.day-1) suppressed PACAP-stimulated AR4-2J cell growth but enhanced PACAP-induced stimulation of adenylate cyclase activity. It was concluded that PACAP stimulates AR4-2J cell proliferation by a mechanism that seems independent of cyclic adenosine monophosphate production. The mitogenic effect of PACAP depends on a pertussis toxin-sensitive G protein and is associated with an increase of ornithine decarboxylase activity.

Polyamines are essential for cell growth and differentiation. Their specific uptake contributes to the regulation of intracellular polyamine levels. In this study, we describe the modulation of this transport mechanism in a rat tumoral pancreatic acinar cell line (AR4-2J) and analyze the transport system characteristics of the normal rat pancreatic acini. Normal acini had a common carrier for spermidine and spermine, like AR4-2J cells, but not a specific putrescine carrier. Intracellular polyamine deprivation enhanced putrescine and spermidine uptake of AR4-2J cells with no modification of polyamine carrier affinity. Uptake was modulated during growth and decreased for both polymaines at confluence. AR4-2J cell differentiation with dexamethasone prevented cell proliferation and diminished uptake of both putrescine and spermidine without affecting their respective carrier affinities. Our data show, first, that the polyamine transport system could be modulated by polyamine metabolism with no change in its affinity characteristics. Second, in rat pancreatic acinar cells, neoplastic transformation was partly characterized by induction of a high-affinity putrescine carrier. This phenotype was not reversed by dexamethasone-induced cell differentiation.

Many reports emphasized the role of gastrin as growth factor on normal gastrointestinal mucosa and pancreas. In the present study, we analyzed the proliferative effects of cholecystokinin (CCK) and gastrin peptides on a rat tumoral pancreatic cell line, AR42J, which possesses both CCKA and CCKB receptor subtypes. The results showed a good correlation between the binding of gastrin to CCKB receptor [Kd 1.125 +/- 0.3 (SD) nM] and its ability to either induce ornithine decarboxylase activity [50% effective concentration, 0.6 +/- 0.3 nM] and [3H]-thymidine incorporation [50% effective concentration, 2 +/- 0.4 nM]. Furthermore, the ability of different cholecystokinin and gastrin antagonists such as proglumide and asperlicin derivatives (respectively, CR1409, CR1505, and L364,718) were tested. We found that all antagonists displaced 125I-labeled gastrin binding, with the following order of potencies: L364,718 greater than CR1409 greater than CR1505 greater than proglumide. Furthermore, the 50% inhibitory concentration of CR1409 and CR1505 to inhibit gastrin stimulated ornithine decarboxylase activity (an early event involved in cell proliferation) and [3H]thymidine incorporation were in agreement with their constants of inhibition (Ki) on gastrin binding. The L364,718 compound, at a concentration which fully occupied the CCKA without affecting the CCKB, had no effect on gastrin stimulated ornithine decarboxylase activity and [3H]thymidine incorporation. In addition, this compound appeared to be a full agonist on CCKB receptor. These results confirm the implication of the CCKB receptor in the proliferative response of AR42J cells to gastrin.

Doppler echocardiography was performed in 112 patients with normal aortic valve prostheses and 13 patients with dysfunction (3 obstructions, 10 regurgitations) confirmed at catheterisation and/or surgery. The maximum and mean transprosthetic pressure gradients were measured in all patients by continuous wave Doppler. The prosthetic valve surface area was calculated by applying the continuity equation in the last 67 patients and compared with the effective surface area deduced from hemodynamic studies in the literature: --There was a great variability in the values of mean pressure gradient and prosthetic valve area within each group and with each size of prosthesis in patients with normal valves. No significant difference was observed between the gradients of different prostheses. However, for a given size, the St Jude Medical prosthesis had larger calculated surface areas than the other prostheses. The Doppler valve surface area did not differ significantly from the area measured invasively for the different categories of prostheses, and it increased with the size of the prosthesis. Mild aortic regurgitation was observed in 16 per cent of bioprostheses and 30 per cent of mechanical prostheses. The three cases of obstruction were characterised by a high mean pressure gradient (59 +/- 16 mmHg, p less than 0.01 vs normal prostheses) and a reduced Doppler surface area (0.7 +/- 0.2 cm2, p less than 0.005 vs normal prostheses) and were correctly diagnosed by Doppler echocardiography. Eight of the ten prosthetic regurgitations were correctly quantified by Doppler. Cardiac Doppler coupled with echocardiography is a very valuable method of non-invasive assessment of aortic valve prostheses.(ABSTRACT TRUNCATED AT 250 WORDS)

Dipyridamole thallium scintigraphy (tomo SMTD) (fig. 2) and a bicycle stress-testing ECG were performed in a prospective study of 102 patients (Tabl. VIII) referred for aortic reconstruction surgery with aortic cross-clamping (fig. 1), carried out without knowledge of tomo SMTD results. A clinical coronary artery disease (CAD) was suspected in 45 patients (44%). The exercise test (tabl. IV) was inadequate in 78%, normal in 11% and abnormal in 11% of patients. Tomo SMTD (tabl. VI et X) was normal in 60 patients (59%), abnormal with persistent defect in 21 (20.5%) and abnormal with transient defect in 21 (20.5%). Post-operative cardiac events (tabl. I) occurred in 4 patients (3.9%): 2 deaths, 1 non-fatal myocardial infarction and 1 rest angina; but only 2 of these had documented CAD. All 4 had abnormal tomo SMTD (3 with transient, 1 with persistent defect). It is concludes that abnormal tomo SMTD had a significant pronostic value (p less than 0.03) in predicting post-perative cardiac events (tabl. XI, fig. 3) after abdominal aortic surgery with cross-clamping. This test facilitates selection of patients for coronary angiography.

Recent studies have demonstrated the presence of both CCKA and CCKB receptors on dog and guinea pig pancreas. Although CCKA receptors are implicated in enzymatic secretion, biological effects of CCKB receptors are still unknown. We have previously found that a rat acinar pancreatic cell line (AR4-2J) possesses both receptor subtypes. In this work we report the ability of various CCK/gastrin agonists and antagonists to bind with these receptors. We found that gastrin, pentagastrin and Gastrin/CCK4 induce ornithine decarboxylase activity, an early event involved in cell proliferation, as well as 3H-thymidine incorporation. Furthermore, these effects occur at doses at which these peptides interact only with the CCKB receptor subtype. In view of these data we propose that modulation of AR4-2J cell growth by gastrin agonists specifically involve occupation of the CCKB receptor.

Basic fibroblast growth factor (bFGF) is a potent mitogen for various cell types. We report here the first study of the effects of bFGF on a digestive tract-derived cell line. The effect of bFGF on the proliferation of AR4-2J cells, tumor cells of acinar pancreatic origin, was investigated together with modulation of ornithine decarboxylase (ODC) activity, an intracellular event involved in cell proliferation. bFGF caused a concentration-dependent stimulation of AR4-2J cell growth, with a half maximal effect (EC50) at 22 +/- 2 pM. ODC activity, assayed by the CO2-trapping method, was also increased by bFGF in a dose-dependent manner, reaching half-maximal stimulation at 20 pM. We conclude that bFGF is a very potent growth promoting factor for cells of pancreatic origin, already effective at picomolar concentrations. The parallelism between the growth assay and the ODC activity assay implicates the involvement of ODC activity in the pathway of the mitogenic effect of bFGF. The stimulation of ODC activity therefore seems to be a reliable early marker for cell proliferation in this model.

In order to characterize the CCK receptor in guinea-pig pancreas, iodinated CCK-39 was bound to pancreatic membranes and the reversible complex was solubilized using various non-denaturing detergents. In term of recovery of ligand stabilized receptors, the relative potencies were Zwittergent 3-14 greater than CHAPS = CHAPSO greater than digitonin greater than MEGA 10 greater than octyl beta-D-glucopyranoside. The stability of receptor complexes was increased by glycerol. Chromatographic analysis revealed that digitonin was the most efficient detergent for disaggregation of CCK receptor complex since it yielded a 76 kDa component in addition to the large components obtained after solubilization with CHAPS and Zwittergent. Furthermore, CCK receptors were covalently labelled using dissuccinimidyl suberate or UV irradiation of labelled membranes by photoactivable radioiodinated CCK-39 and subsequently solubilized by CHAPS + SDS or by SDS alone. A predominant molecule was characterized by chromatography (76 kDa) and SDS-PAGE (89 kDa). In addition to this component, other components having molecular masses of 130-150 kDa, 57 kDa and 40 kDa were detected by SDS-PAGE. They correspond to minor bands. These bands, except the 40 kDa band, were protected from covalent labelling by the presence of CCK-39 (10(-6) M) during initial incubation. Reduction under beta-mercaptoethanol mainly resulted in the decrease of high molecular weight aggregates (Mr greater than 200 kDa). We concluded that for a given detergent a specific molecular weight pattern of solubilized CCK receptor complex is achieved. The minimal component had a molecular mass of 71-84 kDa according to the method of biochemical analysis used.

(Thr28,Nle31)CCK(23-33) (CCK-9) and gastrin(1-17)I (gastrin) inhibited adenylate cyclase activity in membranes from the tumoral rat pancreatic acinar cell line AR 4-2J through a Bordetella pertussis toxin-sensitive mechanism. This contrasted with the stimulatory effect exerted by CCK-9 on adenylate cyclase activity in membranes from normal rat pancreas. The relative potency of CCK-9, gastrin, and related peptides in inhibiting adenylate cyclase, when confronted with previous evidence, suggests that 'non-selective CCK-gastrin CCK-B receptors' predominating over 'selective CCK-A receptors' in the AR 4-2J cell line, favored the coupling of the first receptors to adenylate cyclase through Gi, while CCK-A receptors capable of stimulating the enzyme through Gs were detected only after Bordetella pertussis toxin pretreatment.

It is known that the human exocrine pancreas responds to secretin stimulation more than does VIP, a structurally related peptide. We looked for the receptors for those polypeptides in a human pancreatic cancer cell line grown in culture and in nude mice. By analysing the cAMP responses and the 125I-VIP binding we found VIP receptors with a KD of 1.5 10(-9) M. Secretin stimulates the adenylate cyclase through the VIP receptor sites with a KD of 1.7. 10(-6) M. We noted also that during cell proliferation in culture there was about a 5 fold increase of the cAMP response to VIP.

The binding of bombesin to its receptors on normal human pancreatic membranes was investigated using high specific activity, radioiodinated bombesin ([125I]-Tyr4-bombesin), prepared by an oxidative method with chloramine-T. Binding was specific, temperature-dependent, saturable, reversible and linearly related to membranes protein concentration. After a 30 min period of incubation with membranes the degradation of the tracer has never been found superior to 20%. Scatchard analysis of binding data was compatible with a single class of binding sites with a high affinity (0.96 nM) and a Bmax of 753 fmol/mg protein. [125I]-Tyr4-bombesin binding to human pancreatic membranes was competitively inhibited by (1-Tyr4-)bombesin, GRP, the nonapeptide of bombesin and litorin but not by unrelated hormones such as somatostatin, CCK, human gastrin, etc. These results describe for the first time the presence of specific receptors for bombesin on human pancreatic membranes. The binding characteristics obtained are comparable with those found in other species.

Crude membranes (27,000 g pellets) from five normal human pancreases were prepared. In the presence of GTP, the peptides of the secretin family stimulated adenylate cyclase activity, their order of potency being: secretin greater than helodermin greater than peptide histidine isoleucinamide (PHI) greater than or equal to vasoactive intestinal peptide (VIP) greater than growth hormone releasing factor (GRF) (1-29)-NH2. In addition, helodermin and PHI were more efficient than secretin. Secretin (3-27) inhibited fully the secretin stimulation and partially only the helodermin and PHI stimulation of the enzyme. Secretin receptors were investigated by the ability of secretin and related peptides to inhibit tracer binding. [125I]Secretin binding was fully inhibited by secretin (Kd 0.8 nM), helodermin (Kd 200 nM), and PHI (Kd 250 nM). VIP and GRF(1-29)-NH2 induced partial (20%) inhibition at a high 10 microM concentration. The fragments secretin (2-27), (3-27), (4-27), and (7-27) showed the same low potency and efficacy based on their ability to stimulate adenylate cyclase and to occupy secretin receptors. The analogues [Val5]secretin and [Ala2]secretin had a higher potency than secretin. Based on this comparison of adenylate cyclase stimulation and [125I]secretin binding inhibition, it is tempting to conclude that the human pancreas: (a) possesses highly specific secretin receptors and (b) such receptors could not fully account for the whole pattern of adenylate cyclase activation by related peptides, so that the presence of an added type of "helodermin-PHI-preferring" receptors is suggested.

Crude membranes (27,000 x g pellets) from three normal human pancreata were prepared. Muscarinic receptors were investigated by the ability of three antagonists (atropine, pirenzepine, and AF-DX 116) and three agonists (carbamylcholine, oxotremorine, and pilocarpine) to inhibit [3H]NMS binding. These receptors showed for pirenzepine and AF-DX 116 a M2 beta specificity, typical of secretory glands and smooth muscle, that was comparable to that of rat pancreatic membranes, i.e., a low affinity for the two antagonists (Ki of 0.4 and 0.2 microM, respectively). In addition, these receptors were predominantly in a low affinity state for the agonist carbamylcholine (Ki of 100 microM).

VIP and secretin control the secretory function of the normal pancreas. We analysed their regulatory functions in a human pancreatic cancer cell line: Capan-1. Saturation binding experiments with 125I-VIP showed the existence of one class of binding sites of very high affinity: KD 6.4 +/- 3.0 X 10(-11) M and a low Bmax: 12 fmoles/10(6) cells, in both intact cells and membrane preparations. This site has not yet been described in normal or tumorous digestive cells. Competition binding experiments let us characterize two more binding sites, KD: 2.1 +/- 0.7 X 10(-9) M and 5.0 +/- 0.6 X 10(-8) M and the corresponding Bmax: 120 and 500 fmoles/10(6) cells. These sites are similar to those found on cells of the digestive tract. Competition binding experiments gave the following IC50: 3.0 +/- 0.9 X 10(-9) M for VIP; 2 +/- 0.6 X 10(-6) M for PHI; and 1 +/- 0.7 X 10(-5) M for secretin. VIP elicited a cAMP rise, the half maximal response being obtained at 1.2 X 10(-10) M. Secretin induced a cAMP response but only for concentrations higher than 10(-8) M. VIP receptors were found to be modulated by two factors: cell ageing and cell density. Cells chronically treated with VIP showed a slight decrease of their proliferation; insulin exerted an opposite effect. It is concluded that at the difference of normal pancreatic cells, the present cell line lacks secretin-preferring receptors and acquires some of the properties of intestinal cells.

A technique for preparing a suspension of dispersed functional acini from human pancreas has been developed. The changes in pancreatic enzyme secretion and accumulation of cellular cyclic AMP caused by various secretagogues have been studied. Ca2+-mobilizing agents stimulated amylase release from human pancreatic acini. The relative potencies with which secretagogues increased amylase release were as follows: gastrin-releasing peptide's potency (Ec50, 0.1 +/- 0.01 nM) was greater than bombesin 14's (Ec50, 0.2 +/- 0.01 nM), which was greater than litorin's (Ec50, 0.6 +/- 0.18 nM), which was greater than bombesin 9's (Ec50, 6 +/- 0.1 nM). For CCK-peptides, the relative potencies were as follows: CCK-39's potency (Ec50, 0.28 +/- 0.15 microM) was equal to cerulein's (Ec50, 0.3 +/- 0.07 microM). Both of these potencies were greater than CCK-8's (Ec50, 1.6 +/- 0.1 microM), which was greater than that of CCK-4. Carbamyl choline was poorly potent (Ec50 greater than 1 mM). The 12-O-tetradecanoylphorbol-13-acetate (TPA) was active from 0.1 nM to 0.1 microM. Neither secretin nor VIP increased amylase release from human pancreatic acini but they did cause an accumulation of cellular cyclic AMP, secretin (Ec50, 0.5 +/- 0.2 nM) being more potent than VIP.