Ions-free electrochemically synthetized in aqueous media flake-like CuO nanostructures as SERS reproducible substrates for the detection of neurotransmitters.

The process of catalytic destruction of tumor cells can be strengthened by introducing copper(II) oxide nanostructures (CuONSs) with receptor's agonists/antagonists immobilized on their surface. Here we show a simple and reliable electrochemical method for the fabrication ions-free flake-like CuO nanostructures in a surfactant/ions free aqueous environment. For the determination of the metal surface plasmon, size, rheology, and structure of the fabricated nanostructures ultraviolet-visible (UV-Vis), Fourier-transform infrared (FT-IR), Raman, and X-ray photoelectron (XPS) spectroscopies as well as scanning electron microscope (SEM), high-resolution transmission electron microscopy with energy dispersive X-ray (HDTEM-EDS), X-ray powder diffraction (XRD), and dynamic light scattering (DLS) analysis were used. The fabricated nanostructures were used as highly sensitive, uniform, and reproducible sensors of a natural ligand (bombesin) of some types of metabotropic seven transmembrane G protein-coupled superfamily receptors (GPCRs), which are over-express on the surface of many malignant tumors. Surface-enhanced Raman scattering (SERS) was used to monitor the geometry of adsorbate, separate, enrich, and detect various bombesin C-terminal fragments. It has been shown that the type of used substrate, surface development, and ions present in the solution have little effect on the mode of adsorption.

(18)F-AmBF3-MJ9: a novel radiofluorinated bombesin derivative for prostate cancer imaging.

A novel radiofluorinated derivative of bombesin, (18)F-AmBF3-MJ9, was synthesized and evaluated for its potential to image prostate cancer by targeting the gastrin releasing peptide receptor (GRPR). AmBF3-MJ9 was prepared from an ammoniomethyl-trifluoroborate (AmBF3) conjugated alkyne 2 and azidoacetyl-MJ9 via a copper-catalyzed click reaction, and had good binding affinity for GRPR (Ki=0.5±0.1nM). The (18)F-labeling was performed via a facile one-step (18)F-(19)F isotope exchange reaction, and (18)F-AmBF3-MJ9 was obtained in 23±5% (n=3) radiochemical yield in 25min with >99% radiochemical purity and 100±32GBq/µmol specific activity. (18)F-AmBF3-MJ9 was stable in mouse plasma, and was partially (22-30%) internalized after binding to GRPR. Positron emission tomography (PET) imaging and biodistribution studies in mice showed fast renal excretion and good uptake of (18)F-AmBF3-MJ9 by GRPR-expressing pancreas and PC-3 prostate cancer xenografts. Tumor uptake was 1.37±0.25%ID/g at 1h, and 2.20±0.13%ID/g at 2h post-injection (p.i.) with low background uptake and excellent tumor visualization (tumor-to-muscle ratios of 75.4±5.5). These data suggest that (18)F-AmBF3-MJ9 is a promising PET tracer for imaging GRPR-expressing prostate cancers.

Bombesin staining in neuroendocrine cell hyperplasia of infancy (NEHI) and other childhood interstitial lung diseases (chILD).

AIMS: We have analysed levels of bombesin-positive neuroendocrine cells (NECs) in neuroendocrine cell hyperplasia of infancy (NEHI) and other childhood interstitial lung diseases (chILDs) in order to validate proposed histological criteria for NEHI and investigate its aetiology. METHODS AND RESULTS: The extent of bombesin-positive cells within airway epithelium was analysed in lung biopsies from seven patients diagnosed with NEHI, including two classified previously as non-diagnostic, and other chILDs (n = 64) with age ranges of 1 month-18 years. NECs were counted and calculated as a percentage of airways containing NECs, average percentage of NECs per airway, percentage of airways with >10% NECs and number of neuroendocrine bodies (NEBs). Correlation with age and gender was also undertaken. Patients with NEHI had the highest average percentage of bombesin-positive NECs per airway compared to other chILDs. However, NEH was also seen in many other chILDs, and appears to be most prominent in disorders associated with lung immaturity such as histological patterns associated with surfactant protein-related disorders and pulmonary interstitial glycogenosis. CONCLUSIONS: NEH may, to a degree, be a marker of airway immaturity rather than the direct cause of NEHI. This possibility is supported by the fact that the number of bombesin-positive NECs decreased with age in this cohort, independent of disease type. The average percentage of bombesin-positive NECs per airway appears to be the best histological criterion for assessing the extent of NECs in the context of NEHI.

DMAP-BODIPY alkynes: a convenient tool for labeling biomolecules for bimodal PET-optical imaging.

Several new boron dipyrromethene/N,N-dimethylaminopyridine (BODIPY-DMAP) assemblies were synthesized as precursors for bimodal imaging probes (optical imaging, OI/positron emission tomography, PET). The photophysical properties of the new compounds were also studied. The first proof-of-concept was obtained with the preparation of several new BODIPY-labeled bombesins and evaluation of the affinity for bombesin receptors by using a competition binding assay. Fluorination reactions were investigated on DMAP-BODIPY precursors as well as on DMAP-BODIPY-labeled bombesins. Chemical modifications on the BODIPY core were also performed to obtain luminescent dyes emitting in the therapeutic window (650-900 nm), suitable for in vivo imaging, making these compounds promising precursors for PET/optical dual-modality imaging agents.

LC/MS evaluation of metabolism and membrane transport of bombesin peptides.

Two bombsin peptides, GRPR agonist [Aca-QWAVGHLM-NH(2)] and antagonist [fQWAVGHL-NHEthyl] were evaluated. We employed the highly sensitive Waters Q-Tof Premier MS coupled with a UPLC system to identify the metabolites produced by rat hepatocytes or PC-3 human prostate cancer cells; and we utilized the AB/MDS 4000 Q-Trap LC/MS/MS system with highly sensitive quantitative and qualitative performance, to quantitatively analyze the internalization of GRPR agonist and antagonist in PC-3 cells. The major metabolites of both GRPR agonist and antagonist were the result of peptide bond hydrolysis between W and A which was demonstrated by observation of the N-terminal fragment m/z 446 (Aca-QW-OH) for agonist and m/z 480 (fQW-OH) for antagonist. Both peptides were also hydrolyzed between A and V which formed peaks m/z 517 [Aca-QWA-OH] and m/z 555 (VGHLM-NH2) for the agonist and m/z 551 [fQWA-OH] and m/z 452 (VGHL-NHEthyl) for the antagonist. The peptide agonist also formed a unique metabolite that resulted from hydrolysis of the C-terminal amide. The antagonist showed significantly slower metabolism as compared to the agonist in both rat hepatocytes and PC-3 cells. The antagonist also showed significantly lower PC-3 cell internalization rate than that of the agonist. In conclusion, the metabolism profiles of both GRPR agonist and antagonist peptides were identified by LC/MS. The antagonist peptide was more stable than the agonist peptide in rat hepatocyte incubation. One major factor could be the hydrolysis-resistant C-terminal L-NHEthyl group compared with the unsubstituted amide of the agonist. Another factor could be different amino acid sequences of the agonist and antagonist that may also influence the enzymatic hydrolysis. The antagonist ligand is potentially more useful for receptor-targeted imaging due primarily to its higher metabolic stability.

Potential-dependent characterization of bombesin adsorbed states on roughened Ag, Au, and Cu electrode surfaces at physiological pH.

This paper reports the direct surface-enhanced Raman spectroscopic (SERS) and generalized two-dimensional correlation analysis observations of the different orientations of the neurotransmitter bombesin (BN) chemisorbed on electrochemically roughened Ag, Au, and Cu electrode surfaces at different applied electrode potentials and at physiological pH. The presence of the indole ring of Trp(8) and the amide bond between Gln(7) and Trp(8) of BN on these surfaces generates a specific SERS profile of BN adsorbed on the roughened Ag and Au electrodes that is affected by the electrode potential. Furthermore, for BN on Au, slight changes are observed in the band enhancement in comparison to what is observed for this neurotransmitter immobilized on Ag. In addition, there are larger changes in the spectra triggered by the substitution of Ag with Au electrodes and Ag with Cu electrodes than by substitution of Au with Cu electrodes.

Selected pathological, immunohistochemical and ultrastructural changes associated with an infection by Diphyllobothrium dendriticum (Nitzsch, 1824) (Cestoda) plerocercoids in Coregonus lavaretus (L.) (Coregonidae).

The pathological changes induced by an infection of Diphyllobothrium dendriticum (Nitzsch, 1824) plerocercoids in powan, Coregonus lavaretus (L.), from Loch Lomond, Scotland, were assessed using immunohistochemical and ultrastructural techniques. In a sample of 26 powan, the occurrence of encysted plerocercoids of D. dendriticum on the outer surface of the stomach was 38.5% (n = 10) with the number of cysts ranging from 4 to 15 and measuring 4.2 +/- 1.0 mm x 3.4 +/- 0.9 mm (mean +/- SD). Histological examination of intestinal samples also revealed plerocercoids (2-21) encapsulated within a proliferation of mesenteric fibrous tissues of the gastric wall and, occasionally, by the gut lamina propria-submucosa and lamina muscularis. In section, cysts were tri-layered and were formed from a series of concentric whorls of fibroblast and collagen fibre-based connective elements. The extent of necrosis within each muscle layer and the serosa of the stomach differed, notably within the latter that was marked by a chronic inflammatory reaction and fibrosis. Within the cyst and around it, a large number of degranulating mast cell/eosinophilic granule cells were seen, in addition to melano-macrophage centres. Immunohistochemical staining of sections of infected stomach revealed a high density of elements, in close proximity to plerocercoids, staining positive for serotonin, bombesin, substance P and galanin. Uninfected material did not present the same levels of activity. Sections through both infected and uninfected tissue were also tested for elements containing vasoactive intestinal peptide, met-enkephalin, calcitonin gene-related peptide, neuropeptide Y and nitric oxide synthase, but these were absent.

In vitro and in vivo evaluation of Alexa Fluor 680-bombesin[7-14]NH2 peptide conjugate, a high-affinity fluorescent probe with high selectivity for the gastrin-releasing peptide receptor.

Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14-amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H(2)N-glycylglycylglycine-BBN[7-14]NH(2) peptide with the following general sequence: H(2)N-G-G-G-Q-W-A-V-G-H-L-M-(NH(2)). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H(2)N-G-G-G-BBN[7-14]NH(2) in dimethylformamide (DMF). In vitro competitive binding assays, using (125)I-Tyr(4)-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 +/- 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.

Molecular imaging of gastrin-releasing peptide receptor-positive tumors in mice using 64Cu- and 86Y-DOTA-(Pro1,Tyr4)-bombesin(1-14).

Bombesin is a tetradecapeptide neurohormone that binds to gastrin-releasing peptide receptors (GRPR). GRPRs have been found in a variety of cancers including invasive breast and prostate tumors. The peptide MP2346 (DOTA-(Pro(1),Tyr(4))-bombesin(1-14)) was designed to bind to these GRP receptors. This study was undertaken to evaluate radiolabeled MP2346 as a positron emission tomography (PET) imaging agent. MP2346 was radiolabeled, in high radiochemical purity, with the positron-emitting nuclides (64)Cu (t(1/2) = 12.7 h, beta+ = 19.3%, E(avg) = 278 keV) and (86)Y (t(1/2) = 14.7 h, beta+ = 33%, E(avg) = 664 keV). (64)Cu-MP2346 and (86)Y-MP2346 were studied in vitro for cellular internalization by GRPR-expressing PC-3 (human prostate adenocarcinoma) cells. Both (64)Cu- and (86)Y-MP2346 were studied in vivo for tissue distribution in nude mice with PC-3 tumors. Biodistribution in PC3 tumor-bearing mice demonstrated higher tumor uptake, but lower liver retention, in animals injected with (86)Y-MP2346 compared to (64)Cu-MP2346. Receptor-mediated uptake was confirmed by a significant reduction in uptake in the PC-3 tumor and other receptor-rich tissues by coinjection of a blockade. Small animal PET/CT imaging was carried out in mice bearing PC-3 tumors and rats bearing AR42J tumors. It was possible to delineate PC-3 tumors in vivo with (64)Cu-MP2346, but superior (86)Y-MP2346-PET images were obtained due to lower uptake in clearance organs and lower background activity. The (86)Y analogue demonstrated excellent PET image quality in models of prostate cancer for the delineation of the GRPR-rich tumors and warrants further investigation.

Comprehensive expression analysis of L-dopa decarboxylase and established neuroendocrine markers in neoadjuvant hormone-treated versus varying Gleason grade prostate tumors.

Current hormone withdrawal therapies used for treatment of advanced prostate cancer lead to androgen-independent tumor growth. Increased prostatic neuroendocrine (NE) cell density has been implicated in promoting progression of prostate cancer, but the process by which this occurs remains unclear. The aim of this study was to determine whether there is an association of increased NE differentiation with neoadjuvant hormone therapy and Gleason grade. Using adjacently sectioned tissue microarrays, the expression profile of novel and known NE markers were monitored. L-Dopa decarboxylase (DDC), a catecholamine synthesis enzyme and androgen receptor (AR) coregulator protein, was identified as an additional NE marker of prostate cancer. Immunohistochemical analysis of DDC with the established NE markers, chromogranin A and bombesin, revealed a significant increase in NE differentiation after 6 months of hormone therapy and after progression to androgen independence but no apparent correlation with Gleason grade. In addition, dual immunofluorescence analysis revealed that approximately 55% of the mixed population of DDC- and chromogranin A-expressing NE cells continue to express AR. Taken together, these results suggest that the increase of NE differentiation in prostate cancers depends specifically on duration of hormone therapy. This increase may be due to the transdifferentiation of AR-expressing epithelial-derived adenocarcinoma cells into an NE cell phenotype.

A matrix-assisted laser desorption/ionization compatible reagent for tagging tryptophan residues.

Chemical tagging of amino acids is an important tool in proteomics analysis, and has been used to introduce isotope labels and mass defect labels into proteolytic peptides by derivatization of cysteine or lysine residues. Here, we present a new reagent with chemical specificity for tryptophan residues. Previously, 2-nitrobenzenesulfenyl chloride has been used as a highly specific reagent for labeling tryptophan residues. We show that this tag undergoes UV dissociation during matrix assisted laser desorption/ionization (MALDI). The multiplicity of photofragments increases the difficulty of characterizing the derivatization products. To overcome this problem, we have synthesized a new reagent, 2-(trifluoromethyl)benzenesulfenyl chloride, which is shown to react quantitatively with tryptophan in peptides and proteins. Most significantly, it exhibits high photostability in MALDI-Fourier transform mass spectrometry analyses.

Expression of matrix metalloproteinase-9 and bombesin/gastrin-releasing peptide in human prostate cancers and their lymph node metastases.

Neuroendocrine differentiation and subsequent excretion of neuropeptides have been demonstrated to be associated with progression of human prostate cancer. Among neuropeptides found to exist in the prostate, bombesin/gastrin-releasing peptide has been shown to upregulate matrix metalloproteinase-9 (MMP-9) in human prostate cancer cell lines. Expression levels of bombesin, MMP-9, and neuron-specific enolase were examined by immunohistochemistry in 41 cases of clinically organ-confined prostate cancers including 9 with microscopic lymph node metastases. Twenty-seven (64%) of the 41 radical prostatectomy specimens were positive for both MMP-9 and bombesin. Expression of these molecules was observed in almost the same population of the cancer cells. The remaining 14 cases were negative for both MMP-9 and bombesin. High-grade tumors (Gleason sum > or = 7) were more likely to express MMP-9 and bombesin (21/24:88%) than low-grade tumors (Gleason sum > or = 6) (7/17:41%). In eight of the nine cases with pathological lymph node metastases, expression of MMP-9 and bombesin was also noted in metastatic sites. Neuron-specific enolase was positive in 16 cases (39%) and not always associated with the expression of bombesin. Expression of bombesin and expression of MMP-9 are common in human prostate cancers and may be related to an aggressive phenotype.

Analysis of peptides using partial (no discharge) atmospheric pressure chemical ionization conditions with ion trap mass spectrometry.

A novel approach, based on the use of atmospheric pressure chemical ionization ion trap mass spectrometry (APCI-ITMS) conditions, but without using corona discharge, was used to analyze peptides. The proposed method was applied to three standard peptides (bombesin, trityrosine and tyrosine-glycine-glycine) as well as peptides obtained through enzymatic digestion of two standard proteins (horse cytochrome c and horse myoglobin).

Immunocytochemical and ultrastructural characterization of endocrine cells in the larval stomach of the frog Rana temporaria tadpoles: a comparison with adult specimens.

According to immunostaining and ultrastructural patterns, Rana temporaria tadpole stomach displays a well-differentiated endocrine population comprising, at least, six cellular types: ECL, EC [serotonin], D [somatostatin] - all three of them abundant -, P [bombesin] - less numerous -, CCK-8 [cholecystokinin/gastrin] and A [glucagon/glicentin] - both very scarce. Larval endocrine cells are mainly located in the surface epithelium and show open or closed morphologies. Cellular diversity is similar in tadpoles and frogs, with the exception of immunoreactivity for gastrin-17, found in adults in numerous cells. Larval cells display mature ultrastructural traits, although with smaller secretory granules. The different distribution of endocrine cells, which in adults are preferentially located in the glands, probably refers to different functional requirements. However, the rich vascular plexus present in larval mucosa may be an efficient transport medium of surface hormones to-gastric targets. The enhancement in adults of endocrine population and correlative increase in hormonal secretion indicates a more active functional role, probably related to the shift from herbivorous to carnivorous habits. In summary, the tadpole gastric endocrine population, although not as numerous as that of adult frogs, displays histological traits that indicate a relevant (immunoreactive and ultrastructural properties, cellular diversity) and specific (surface location, relative abundance of open-type cells) role of local regulatory factors in amphibian larval gastric function.

Comparative study of serotonin and bombesin in adenocarcinomas and neuroendocrine tumors of the colon.

The aim of this study was to investigate serotonin and bombesin expression in colorectal adenocarcinomas and neuroendocrine colorectal tumors to clarify their role in the progression of colon cancer. The investigation was carried out by electron microscope immunocytochemistry. The ultrastructural study revealed that some cases of colorectal adenocarcinomas were characterized by the presence of amphicrine cells containing endocrine granules and mucus granules. Poorly differentiated adenocarcinomas and liver metastases were poorly granulated compared with highly differentiated tumors. Neuroendocrine tumors nevertheless were characterized by the presence of numerous malignant neuroendocrine cells filled with secretory granules and mucus granules. Bombesin appeared to be located in enterochromaffin-like endocrine cells, which are primarily responsible for the production of serotonin. In colorectal adenocarcinomas there was an inverse correlation between serotonin levels and the degree of differentiation. High serotonin levels characterized colorectal adenocarcinomas with composite phenotype and colorectal neuroendocrine tumors. Increased bombesin expression was correlated with colorectal adenocarcinomas exhibiting poor histological grade and their liver metastases. In conclusion, the findings suggest that high serotonin levels may be an indicator of neuroendocrine differentiation, and bombesin may be a useful marker for colorectal adenocarcinomas with aggressive behavior,

Increased content of bombesin/GRP in human synovial fluid in early arthritis: different pattern compared with substance P.

OBJECTIVE: Bombesin (BN) and the mammalian homologue gastrin-releasing peptide (GRP) are known trophic factors, neurotransmitters and paracrine hormones. BN/GRP has not previously been demonstrated in synovial fluid. In this study, the amounts of BN/GRP and substance P (SP) present in synovial fluid from the knee joints of patients with rheumatoid arthritis (RA) and of healthy controls were measured. METHODS: Synovial fluid from the knee joint was collected from patients with either longstanding RA (n = 32) or early arthritis (symptoms for < 12 months; n = 9) and from control subjects, i.e., individuals without known joint disease (n = 10). These samples were analyzed using radioimmunoassays. RESULTS: Levels of BN/GRP-like peptide were below the assay detection limits in synovial fluid from controls. Detectable levels of immunoreactive BN/GRP were present in the majority of patients with either longstanding RA or early arthritis. The levels were significantly higher in the synovial fluid from patients classified as having early arthritis compared with those with longstanding RA (p < 0.05). There was a strong correlation between BN/GRP levels and the number of leukocytes in the synovial fluid in the patients with early arthritis. The levels of SP-like peptide in the patients, whether with early arthritis or longstanding RA, were significantly elevated compared with controls. However, there was no difference in the levels between these two patient groups. CONCLUSIONS: These observations show that BN/GRP-like peptide is present in the synovial fluid of joints affected by arthritis and that the pattern of BN/GRP increase differs from that of SP. It appears as if the presence of BN/GRP is particularly related to the early processes of joint involvement. These observations are of interest because BN/GRP has well-known trophic and paracrine effects and chondrocytes have recently been shown to produce neuropeptides such as BN/GRP.

An immunohistochemical study of endocrine cells in the alimentary tract of the red-bellied frog, Bombina orientalis.

The regional distribution and relative frequency of endocrine cells was studied immunohistochemically (PAP method) in the alimentary tract of the red-bellied frog, Bombina orientalis, using antisera against serotonin, somatostatin, chromogranin (CG), cholecystokinin (CCK)-8, bombesin, secretin, glucagon and pancreatic polypeptide (PP). Eight kinds of endocrine cells were identified in this study. These immunoreactive cells were located in the gastric glands of the stomach regions and in the intestinal or esophageal epithelium with variable frequencies. They were spherical or spindle-shaped. Serotonin- and somatostatin-immunoreactive cells were demonstrated in the whole alimentary tract including esophagus. CG-immunoreactive cells were restricted to the stomach. CCK-8-immunoreactive cells were observed from the antrum to the ileum. Bombesin-immunoreactive cells were restricted to the stomach. Secretin-immunoreactive cells were demonstrated in the pylorus, duodenum and ileum. Glucagon-immunoreactive cells were found in the antrum and duodenum. PP-immunoreactive cells were detected from the antrum to the rectum. In conclusion, throughout the alimentary tract of the red-bellied frog, the different regional distribution and relative frequency of endocrine cells were demonstrated. The regional distributions and relative frequencies of the endocrine cells in the alimentary tract of the red-bellied frog were resembled to those of the other anuran species except for esophagus.

Neuroendocrine differentiation in non-small cell lung carcinomas.

The immunohistochemical expression of ten neuroendocrine and neural differentiation-related antigens in 200 specimens from patients with surgically resected non-small cell lung carcinomas were evaluated. Poorly differentiated adenocarcinomas and undifferentiated large cell carcinomas showed the highest percentage of positive samples (30%-60%) for the markers evaluated with diffuse and intense immunostaining. Poorly differentiated squamous carcinomas bronchioalveolar adenocarcinomas, and giant cell carcinomas showed a lower percentage of positive samples (20%) with moderate immunostaining. Well differentiated tumors were very rarely positive for the neuroendocrine markers. It is concluded that neoplasms positive for the neuroendocrine markers can be considered as non-small cell carcinoma with neuroendocrine features, even if they do not have the histological appearance of neuroendocrine lung neoplasms.

The ciliated hepatic foregut cyst, an unusual bronchiolar foregut malformation: a histological, histochemical, and immunohistochemical study of 7 cases.

The ciliated hepatic foregut cyst is an unusual solitary cystic lesion of the liver. In a series of 7 cases of hepatic ciliated cysts, we performed a histological, histochemical, and immunohistochemical study to better define the histogenesis of this rare entity. The patients were 4 women and 3 men, aged 39 to 75 years. Four patients presented with abdominal pain. In 3 cases the cyst was discovered incidentally on ultrasonography. The cysts measured from 1 to 4 cm in diameter. Microscopically, the lining of the columnar epithelium was composed of ciliated cells and mucin secreting goblet cells. The wall was composed of bands of smooth-muscle fibers surrounded by an outer fibrous capsule. The goblet cells stained with PAS, alcian blue, and high-iron diamine. The immunohistochemical study showed that endocrine cells were present within the cyst epithelium, positive for chromogranin, synaptophysin, bombesin, and calcitonin, and negative for serotonin, somatostatin, glucagon, insulin, gastrin, and pancreatic polypeptide. In all the cases, immunoreactivity of some cells for CC10 strongly suggested the presence of Clara cells. Our study shows that the epithelium lining ciliated hepatic foregut cysts has histological, histochemical, and immunohistochemical features similar to those observed in the bronchiolar epithelium. This lesion is a developmental ventral foregut abnormality that could arise from a bronchiolar bud of the tracheobronchial diverticulum.

Granin proteins (chromogranin A and secretogranin II C23-3 and C26-3) in the endocrine pancreas of amphibians.

The occurrence and cellular distribution of chromogranin A (CgA) and of two synthetic secretogranin II (SgII)-fragments (termed C23-3 and C26-3) has been investigated immunohistochemically in the endocrine pancreas of five amphibian species. Immunoreactivity for CgA was detected only in specimens of the genus Rana, whereas for SgII it was found in all the urodeles and anurans studied. Either CgA or the SgII-fragment displayed its own cellular distribution patterns in the endocrine pancreas of a given species. Moreover, immunoreactivity for both regions (C23-3 and C26-3) of the SgII-molecule exhibited by the same endocrine cell population have been encountered in newt and frog organs. Besides the interspecific heterogeneous distribution of CgA and of the two SgII-fragments in relation to the insular cell types, a striking heterogeneity of their immunostaining density among the endocrine cells of the same type was also revealed. The above findings entirely support the concept of a good conservation of granins during phylogeny; they do not support, however, the previously ascribed usefulness of these anionic glycoproteins as markers for all neuro-endocrine cells.