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Publications and source records attributed to J Lainé.
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We describe here, in Golgi-impregnated rat cerebellar cortex, a new group of large granular layer neurons. These cells have a globular soma located at variable depths in the granular layer, and three to four long radiating dendrites coursing through the three layers of the cortex. The axon projects more or less directly into the molecular layer, where it expands in a local plexus of oblique and tortuous thick collaterals ascending through the major part of the layer. Interestingly, the axons of several of these cells give off a collateral that courses for a long distance in the transverse direction, just above the Purkinje cell somata, parallel to the parallel fibers. While the granular layer location and the polymorphous somato-dendritic pattern of these cells is reminiscent of that of Golgi cells, their axonal pattern is clearly of the same type as that of another large granular layer interneuron, the Lugaro cell. Moreover, double anti-calretinin and anti-calbindin immunolabellings show that Lugaro cells as well as some globular somata dispersed in the granular layer are both calretinin-positive and in close apposition with numerous calbindin-positive varicosities of Purkinje cell axon recurrent collaterals. These latter are known from previous ultrastructural studies to be pre-synaptic to Lugaro cells. The common granular layer location and calretinin labelling, the striking similarity in axonal projection pattern, and the important common recurrent afferentation by Purkinje cell axons strongly argue in favor of the classification of these globular interneurons as a subgroup of a widened Lugaro cell type.
Large increases in cAMP concentration inside the cell are generally growth inhibitory for most cell lines of mesenchymal and epithelial origin. Moreover, recent data suggest a role of cAMP in survival of different cell types. Herein, the ability of forskolin (an adenylyl cyclase activator) and IBMX (3-isobutyl-1-methylxanthine) (a phosphodiesterase inhibitor) to modulate cell cycle progression and survival of human pancreatic cancer cells was evaluated. We showed that forskolin + IBMX inhibited serum-induced ERK activities, Rb hyperphosphorylation, Cdk2 activity, and p27(Kip1) downregulation and caused G1 arrest in MIA PaCa-2 cells. Furthermore, forskolin + IBMX protected pancreatic cells against apoptosis induced by prolonged inhibition of ERK activities by preventing Bcl-X(L) downregulation, activation of caspases 3, 6, 8, and 9, and PARP cleavage and by inducing Bad phosphorylation (ser112). Taken together, our data demonstrate for the first time that cAMP is an inhibitor of cell cycle progression and apoptosis in human pancreatic cancer cells.
Normal cellular prion protein, a necessary protagonist in fatal neurodegenerative prion diseases, was mapped in rodent cerebellum to establish its cellular and ultrastuctural localization. Existing morphological data about native prion protein distribution in brain tissues remain, indeed, contradictory and do not fit with biochemical and cell biological results. Using ultrastructural preembedding immunocytochemistry and a monoclonal anti-mouse prion protein antibody, this report shows that cellular prion protein is present in all cortico-cerebellar and deep nuclei neuronal cell types, as well as in all glial cell types. The heaviest expression appears on parallel fibres and astrocytic processes. The protein is exclusively located on the outer cell membrane and in Golgi and endosomal intracytoplasmic organelles, with no cytoplasmic or synaptic vesicle labelling. Most important, and in contrast with previous ultrastructural data, cellular prion protein is shown to be distributed on all portions of neurons, without any preferential synaptic targeting. The present morphological report shows, for the first time in vivo, that the cellular prion protein is present on the entire cell surface membrane of all neuronal and glial cell types of the rat cerebellum. This ubiquitous presence supports the notion that prion protein has a generalized cellular function in brain tissue rather than a specialized role restricted to synaptic transmission.
BACKGROUND AND AIMS: Growth factors are well known for their participation in the regulation of cell proliferation and survival. However, the intracellular signaling pathways by which growth factors promote survival are still poorly understood. In the present study, using the MIA PaCa-2 cell line, a well-established model of pancreatic cancer cells, we analyzed the roles of ERK1/2 activities in the regulation of cell survival and investigated some of the mechanisms involved. METHODS: The ability of the MEK inhibitor PD98059 to modulate survival of the MIA PaCa-2 cells was evaluated, and the responses were correlated with expression of Bcl-2 homologs and caspases 1, 3, 6, 8, and 9 activities. RESULTS: Herein, we showed that inhibition of ERK1/2 activities caused (1) a G1 arrest; (2) a down-regulation of the expression levels of the anti-apoptotic homologs Bcl-2, Mcl-1, and Bcl-X(L) without affecting the pro-apoptotic levels of Bax and Bak; (3) a promotion of caspases 3, 6, 8, and 9 activities; (4) a stimulation of PARP cleavage; and (5) a programmed cell death by apoptosis. CONCLUSION: Our data suggest that activation of the ERK pathway functions to protect pancreatic tumor cells from apoptosis as well as to regulate their progression in the cell cycle.
This study was undertaken to characterize the biochemical properties of rat pancreatic phospholipase D (PLD). Based on Western blot analysis of pancreas subcellular fractions, PLD1 was detected as a protein of 120 kDa associated with the microsomal fraction, whereas PLD2 appeared as a 105-kDa protein enriched in the microvesicular fraction. In these fractions, a low level of PLD activity was measured with an exogenous substrate containing phosphatidylinositol-4,5-bisphosphate (PIP2), unresponsive to guanosine triphosphate (GTP)gammaS and adenosine diphosphate (ADP)-ribosylation factor (ARF). Addition of unsaturated but not saturated fatty acids stimulated an oleate-dependent PLD activity that colocalized with the PLD1 enzyme in the crude plasma membrane and microsomal fractions. The transphosphatidylation reaction was maximal with either 200-400 mM (1.2-2.3%) ethanol or 25 mM (0.23%) 1-butanol, with an optimal pH between 6.5 and 7.2. Lipids extracted from the pancreatic membranes were potent inhibitors of the HL60 cell PLD activity when compared with those isolated from HL60 cells. Oleate-dependent PLD activity was less susceptible to these inhibitions. A phospholipase A1 (PLA1) activity hydrolyzing phosphatidylethanol also was found in the pancreatic membrane fractions and was nearly absent in the HL60 cells. This activity was completely inhibited by 400 nM tetrahydrolipstatin (THL), a lipase inhibitor. Pancreatic PLD1 and PLD2 activities could be measured after a chromatographic separation from microsomal membranes and high-speed supernatants, respectively. Activities of both enzymes were inhibited by oleate and required the presence of PIP2 in the substrate vesicles. ARF1 strongly activated PLD1 in a dose-dependent manner, and PLD2 was slightly responsive. Indirect immunofluorescence revealed that PLD2 is distributed throughout the pancreas, with a more intense staining in the islets. This study presents for the first time biochemical characteristics of the pancreatic PLD activities and shows the presence of oleate-dependent PLD1 and PLD2 activities, as well as PLD1 and PLD2 proteins in this gland.
Positive signs of pancreas regeneration were observed in rats after induced pancreatitis and partial pancreatectomy (1,2). Although the human pancreas did not regenerate after partial anatomic resection (3), the pig pancreas exhibited growth responses to bombesin after partial pancreatectomy (4). This study was undertaken to establish the time course of pancreatic inflammation, apoptosis, and hypertrophy and/or hyperplasia after partial pancreatectomy in pigs.
In rodents, cholecystokinin (CCK) induces pancreatic enzyme secretion and pancreas growth through its CCK(A) receptors. It is unknown whether occupation of the CCK(B) receptors present in pig and human pancreas can cause the same effects. This study evaluates CCK(B) receptor expression in rat, mouse, pig, and fetal human pancreata using Northern blot, Western blot, and immunofluorescence techniques. The reported 2.7-kb CCK(B) receptor mRNA transcript in the rat brain and gastric fundus is absent in pancreas; the message was, however, detected by RT-PCR and by a CCK(B) receptor antibody as an 80-kDa protein present uniquely in islet delta-cells. Proteins of 50 and 80 kDa appear in mouse pancreas, and proteins of 50 and 115 kDa appear in pig and human pancreas, respectively, all localized in islet delta-cells. Gastrin mRNAs are strongly present in fetal rat pancreas, and the hormone is localized in islets; both are repressed 10 days after birth. In conclusion, the CCK(B) receptors are present in pancreas of four species with exclusive location in islet delta-cells. In such a location, they could be indirectly involved in the control of enzyme secretion.
We have evaluated the presence and localization of the CCK(A) receptor in rat, mouse, pig and human fetal pancreas by Northern, Western blots and immunofluorescence techniques. In the rat, parallelism exists between development of the CCK(A) receptor mRNA and protein with maximal peaks of expression during the suckling period. In the course of pancreatitis induction, CCK(A) receptor mRNA were maximally expressed and sustained during the gland's regeneration. In the rat and mouse pancreas, the CCK(A) receptor protein is localized around the acinar cells and beta cells of the islets of Langerhans. In the adult pig and fetal human pancreas, the CCK(A) receptor proteins were detected by Western blot. By immunofluorescence, its detection was possible only in the islet of Langerhans of the pig pancreas. These new findings support the views that CCK plays important and various roles in specific physiological systems of the pancreas of different species.
Some regulated secretory proteins are thought to be targeted to secretory granules through an acidic-dependent aggregation in the trans-Golgi network. In this report we use pancreatic zymogens, a paradigm of regulated proteins, to test this hypothesis, because they qualitatively aggregate upon acidification in vitro. Pig zymogens were found to start to aggregate significantly at pH approximately 6.0, a pH slightly lower than that at which rat zymogens aggregate, but still compatible with the pH of the cell-sorting compartments. When pig zymogen granule membranes were mixed with the zymogens in the aggregation assay, membranes that normally floated on 1 M sucrose were observed to be pelleted by the aggregating zymogens. Rat membranes were pelleted by pig zymogens and vice versa. Igs, typical constitutively secreted proteins, which needed chemical cross-linking to serve as an aggregated protein control, pelleted membranes almost independently of pH. Corresponding cross-linked zymogen-binding ability and pH dependence was unaffected by the chemical modification. Membranes treated with sodium carbonate, pH 11, or with protease K, were still pelleted by zymogens, suggesting that the aggregated zymogens bound to membrane lipids. This hypothesis was confirmed by the efficient pelleting of unilamellar vesicles composed of granule membrane lipids. Vesicles composed of single classes of phospholipids were also pelleted, but with various efficacies. We conclude that pancreatic zymogen aggregates, formed under the acidic conditions of the secretory pathway sorting compartments, have the capacity to bind firmly to membranes through their phospholipid constituents.
The implication of MAP kinases in the proliferation control of pancreatic cancer cells is still unknown. This study was undertaken to examine the contribution of the p44/p42 and p38 MAP kinases in the mitogenic response to epidermal growth factor (EGF) and bombesin in human pancreatic cancer cells, MIA PaCa-2 and PANC-1. Data indicate that EGF and bombesin stimulated growth of both cell lines. In MIA PaCa-2 cells, EGF and bombesin stimulated the in gel activation of p38 while p44/p42 kinases exhibited high basal activity and no response to stimuli. Growth and p38 activation were inhibited by genistein, wortmannin, PD98059 and SB203580, specific inhibitors of tyrosine kinase, phosphatidylinositol 3-kinase, MEK-1 and p38 kinases, respectively. In PANC-1 cells, EGF and bombesin stimulated p42 in gel activation; p44 remained highly activated and unresponsive to stimuli and p38 did not respond. Stimulated growth and p42 activation were inhibited by genistein, wortmannin and PD98059. Estimation of MAPK activities with a specific anti-active MAP kinase antibody indicated, however, that EGF increased the intensity of the bands corresponding to p42 and p44 MAP kinases in both cell lines, indicating that the mitogenic factor can regulate MAP kinase activity. Data also pointed out that ATP is sufficient to increase MAP kinase activity within the in gel assay technique and may thus explain the discrepancies existing between the in gel assay data and those obtained with the anti-active MAP kinase antibody.
Rat fetal pancreas development and maturation were investigated in vitro and in vivo, and the informations available on their controls do not agree. Our main objective was to reinvestigate fetal pancreas growth in vivo through treatments of the dams during their entire pregnancy. Pregnant rats were thus implanted subcutaneously with Alzet minipumps and received cerulein (0.25 microg kg-1 h-1), gastrin-releasing peptide (GRP; 0.18 microg kg-1 h-1), GRP antagonist (12 microg kg-1 h-1), pentagastrin (2.38 microg kg-1 h-1), L-365,260, a cholecystokinin B (CCKB) receptor antagonist (120 microg kg-1 h-1), and hydrocortisone (417 or 833 microg kg-1 h-1). After sacrifice at the end of pregnancy, the pancreata of the dams and those of their fetuses were excised for weight, protein, RNA, DNA, and digestive enzyme determinations. In the fetus, pancreas growth defined as hyperplasia was observed only in response to hydrocortisone, while aplasia occurred in response to cerulein. Gastrin and the GRP antagonist were the most effective hypertrophic agents, and the effect of the CCKB receptor antagonist was atrophic. In conclusion, hydrocortisone caused proliferation of the fetal rat pancreas, whereas gastrin induced its differentiation and maturation probably through CCKB receptor occupation.
Lugaro cells are fusiform neurons located just beneath the Purkinje cell layer. Their axon projects profusely in the molecular layer and emits some collaterals in the granular layer. We have analysed the molecular layer postsynaptic targets of a Golgi-impregnated Lugaro cell, using the electronmicroscopic gold-toning procedure and post-embedding anti-GABA immunocytochemistry. All the gold-labeled neuritic and somatic profiles were GABA-positive, thus confirming that the Lugaro cell is an inhibitory interneuron. The axonal varicosities of this cell form multiple symmetrical synaptic junctions with basket and stellate cell somata and proximal dendrites, and, perhaps, with the molecular layer dendrites of Golgi interneurons. Lugaro cells thus seem to exert a feed-back inhibitory control on Purkinje cells.
The intracellular events involved in normal pancreatic growth have been extensively investigated in response to cholecystokinin. Recent data indicate that tyrosine kinase, phospholipase D, phosphatidylinositol 3-kinase, and p42/p44 MAPK are stimulated in rat pancreatic acinar cells. Although we begin to understand the intracellular signaling pathways activated in normal pancreas, such information is not yet available in pancreatic cancer cells. This study was undertaken to identify the growth factors and hormones involved in cell proliferation of two human pancreatic cancer cell lines of ductal origin, the MIA PaCa-2, and PANC-1 cells, and to establish the intracellular events involved in the control of their growth. We demonstrated that FGF-2, IGF-1, cerulein, and gastrin but not FGF-1, HGF, secretin, and PACAP, stimulated proliferation of MIA PaCa-2 and PANC-1 cells. Autocrine factors such as gastrin and IGF-1 were also responsible for their proliferation. In response to EGF, FGF-2, IGF-1, cerulein, gastrin and bombesin, tyrosine kinase, and tyrosine phosphatase activities were stimulated in both cell lines. The close relationship established between cell growth and tyrosine kinase activation results from the observation that maximal growth stimulation paralleled with maximal enzyme activation and that genistein, the tyrosine kinase inhibitor, blocked cell growth and enzyme activation. The implication of PLD in growth-stimulated processes is doubtful since all growth factors and hormones tested failed to stimulate an already very active PLD activity. We finally observed a constitutive activity of p44 MAPK in both cell lines and of p42 in MIA PaCa-2 cells. However, p38 and p42 were stimulated in MIA PaCa-2 and PANC-1 cells, respectively, by all growth factors and hormones.
Membrane dipeptidase (EC 3.4.13.19) enzyme activity that is inhibited by cilastatin has been detected in pancreatic zymogen granule membranes of human, porcine and rat origin. Immunoelectrophoretic blot analysis of human and porcine pancreatic zymogen granule membranes with polyclonal antisera raised against the corresponding kidney membrane dipeptidase revealed that the enzyme is a disulphide-linked homodimer of subunit mass 61 kDa in the human and 45 kDa in the pig. Although membrane dipeptidase was, along with glycoprotein-2, one of the only two major components of carbonate high pH-washed membranes, no enzyme activity or immunoreactivity was detected in the zymogen granule contents. Digestion with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), and subsequent recognition by antibodies specific for the cross-reacting determinant, revealed that membrane dipeptidase in human and porcine pancreatic zymogen granule membranes is glycosyl-phosphatidylinositol-anchored. Membrane dipeptidase was released from the pancreatic zymogen granule membranes by an endogenous hydrolase, and the released form migrated as a disulphide-linked dimer on SDS/PAGE under non-reducing conditions. Under reducing conditions it migrated with the same apparent molecular mass as the membrane-bound form, and was still a substrate for bacterial PI-PLC. Treatment of kidney microvillar membranes with phospholipase A2 resulted in the release of membrane dipeptidase in a form that demonstrated electrophoretic and cilastatin-Sepharose binding properties identical to those of the endogenously released form of the enzyme from zymogen granule membranes. These results indicate that the glycosyl-phosphatidylinositol anchor on the pancreatic membrane dipeptidase is cleaved by an endogenous hydrolase, probably a phospholipase A, and that this cleavage may promote the release of the protein from the membrane.
We present a detailed description of the somatodendritic and axonal features of the Golgi-impregnated Lugaro cell in the rat cerebellar cortex. This neuron, present throughout the cerebellum, is characterized by a fusiform cell body located at the border between the granular and Purkinje cell layers, horizontal bipolar dendrites extending in the parasagittal plane and an axon projecting exclusively to the molecular and granular layers. A quantitative analysis of 77 Lugaro cells confirms the somatodendritic homogeneity of this cell type. Computer reconstructions showed that the bipolar dendrites spread like an horizontal X centered by the cell body, implying that Lugaro cells are arranged in a flat lattice just beneath the Purkinje cell layer. Fifty-seven Lugaro cells with sufficiently impregnated axons displayed a common pattern of organization with, nevertheless, a certain variability of the axonal trajectory. Some axons projected directly into the molecular layer with a profuse plexus spreading just above the parent cell. Other axons first traveled downwards into the granular layer, sometimes even reaching and passing into the white matter. They always hooked back to reascend through the granular layer and also finally terminate in the molecular layer, near and sometimes at a distance from the parent cell. In a few samples, one or two remote axon collaterals were seen to extend longitudinally in the lower molecular layer for a few hundred microns, in the same direction as the parallel fibers. In all cases a few collaterals projected into the granular layer. In view of its dense afferentation by Purkinje cell recurrent collaterals and its profuse inhibitory projection in the molecular layer, the Lugaro cell could act as a feedback interneuron on the corticocerebellar output.
To uncover the mechanisms involved in the biogenesis of secretory granules, we studied development of the exocrine pancreas in the pig from the fetus up to the mature animal by following the enzyme activities and expression (Northern blot) of five zymogens and GP-2, the major protein of the granule membrane. Fetal pancreas mainly contained chymotrypsinogen and barely detectable amounts of amylase, trypsin, lipase, and elastase. GP-2 was not notably expressed before the Day 21 of life. Ultrastructural examination of the fetal tissue embedded in Epon with osmium postfixation or in Lowicryl at -20 degrees C without postfixation showed dense granules with an irregular shape but also showed that most granules had uncondensed contents, with the aspect of immature granules, or had a dense core surrounded by light material. With immunogold cytochemistry, the concentration of chymotrypsinogen was directly associated with the acquisition of electron density by the granule matrix. These observations suggest that fetal granules have a slower rhythm of zymogen condensation and an irregular shape that could be due to the particular composition of the matrix and the absence of GP-2. We conclude that, in the exocrine pancreas, secretory granules can be formed under various conditions, even with a matrix containing a ratio of components very different from that of the normal mature animal.
A new cell type is described in silver-impregnated sections of the rat cerebellar cortex, uniformly distributed through all the cerebellar folia. The soma is rather small, roughly pyriform, vertically oriented, and squeezed, in a sandwich-like manner, between the Purkinje cell somata. One or two thick dendrites arise from the upper pole of the cell body and course through the entire molecular layer, dividing into a few, slightly oblique, branches that can reach the pia mater. These dendrites are covered with irregularly distributed spines. Some more slender dendrites emerge from the lower part of the cell body, or from the proximal trunk of a molecular dendrite, and spread tortuously for a short distance in the upper granular layer. A thick initial segment emerges directly from the soma or from the proximal portion of a dendrite, the axon winding then horizontally through or just above the Purkinje cell layer. During this horizontal course it gives off vertically oriented beaded branches ascending through the major part of the molecular layer. These branches, rather closely spaced, occupy different parasagittal planes, separated by about 10 to 30 microns. This axonal arborisation can thus be compared with a candelabrum. The peculiar three-dimensional spread of the axonal collaterals suggests a functional relationship between these branches and the dendritic trunks of neighbouring Purkinje cells. A comparative analysis of the morphological differences between this candelabrum interneuron and the other corticocerebellar interneurons found in the vicinity of the ganglionic layer confirms the specificity of this new cell class.