Reg I protein in healthy and seminoma human testis.

Regenerating gene (Reg), encodes a secretory protein with growth and differentiation stimulating effects mostly in digestive tissues. Overexpression of Reg proteins and specifically of Reg I, one member of the Reg family, is associated with several human diseases and cancers. In the present study we analyzed the expression of Reg I in normal rodent and human testes where germ cells normally proliferate and differentiate into spermatozoa, and in seminoma testis, the most common cancer of young men. Western blot analyses demonstrated the presence of a specific band at 19 kDa in human and rodent testis extracts. Immunofluorescence and deconvolution microscopy demonstrated that Reg I was present within the seminiferous tubules in both Sertoli and germ cells. By using a Sertoli cell line we demonstrated that Reg I was localized at the plasma membrane even in the absence of contact between neighboring cells and appeared before the tight junction associated protein ZO-1 was revealed at this location. Reg I was strongly expressed in human seminoma testis tissue and in a human tumor germ cell line where the immunoreactive signal was mainly detected at the plasma membrane level. These data showing for the first time the weak presence of Reg I in the normal testis and its strong expression in the testis cancer suggest a potential role of Reg I in normal and neoplastic germ cell proliferation.

Changes in cell proliferation and differentiation of adult rat small intestine epithelium after adrenalectomy: kinetic, biochemical, and morphological studies.

The effects of 10-day bilateral adrenalectomy on morphometry, proliferation, and apoptosis were determined in the small intestine of 3-month-old Sprague-Dawley rats. The activities of sucrase, lactase, and its respective mRNA, aminopeptidase N, and intestinal alkaline phosphatase were also evaluated. Adrenalectomy lead to partial atrophy and disorganization of the epithelium, with an increased number of goblet and Paneth cells and a reduction of crypt cell proliferation paralleled by a marked increase in villus apoptosis. Biochemical assays revealed that aminopeptidase N and intestinal alkaline phosphatase activities were significantly decreased, whereas disaccharidases were increased by adrenalectomy. The corresponding induction of lactase mRNA suggests an active response of the epithelium. In conclusion, adrenalectomy modified maturation and the differentiation processes of the small intestinal mucosa, especially in the proximal part of the small intestine. This result points to an important role of adrenals and glucocorticoids in the trophic status of the adult small intestinal mucosa.

Expression of REG protein during cell growth and differentiation of two human colon carcinoma cell lines.

We localized REG protein in Paneth cells and nonmature columnar cells of the human small intestinal crypts and speculated that this protein was associated with growth and/or differentiation. The aim of this study was to determine whether REG protein is present in two human colon cancer cell lines that exhibit enterocytic differentiation after confluence and to investigate changes in the level of its expression during growth and differentiation. Results were compared to those obtained on cells that remain undifferentiated. Western immunoblotting and immunofluorescence demonstrated the presence of REG protein in the three cell lines. With the antisera against human REG protein, the staining was diffusely spread throughout the cytoplasm at Day 2, and after Days 3-4 it appeared to have migrated to cell boundaries. After confluence, we observed only a punctate staining array along cell boundaries, which disappeared at Day 15. REG mRNA expression was demonstrated by RTPCR and REG mRNA hybridization until Day 13, but not after, in the three cell types. REG protein may be involved in cellular junctions. Its presence appears to be associated with the cell growth period and the protein must be downregulated when growth is achieved and differentiation is induced.

Cloning of a calcium channel alpha1 subunit from the reef-building coral, Stylophora pistillata.

While the mechanisms of cellular Ca2+ entry associated with cell activation are well characterized, the pathway of continuous uptake of the large amount of Ca2+ needed in the biomineralization process remains largely unknown. Scleractinian corals are one of the major calcifying groups of organisms. Recent studies have suggested that a voltage-dependent Ca2+ channel is involved in the transepithelial transport of Ca2+ used for coral calcification. We report here the cloning and sequencing of a cDNA coding a coral alpha1 subunit Ca2+ channel. This channel is closely related to the L-type family found in vertebrates and invertebrates. Immunohistochemical analysis shows that this channel is present within the calicoblastic ectoderm, the site involved in calcium carbonate precipitation. These data and previous results provide molecular evidence that voltage-dependent Ca2+ channels are involved in calcification. Cnidarians are the most primitive organisms in which a Ca2+ channel has been cloned up to now; evolutionary perspectives on Ca2+ channel diversity are discussed.

Pancreatic trypsinogen I expression during cell growth and differentiation of two human colon carcinoma cells.

Pancreatic trypsin has been found to induce tight junction or dome formation in some colon cancer cell lines (HT-29, Caco-2), and a tumor-associated trypsinogen, trypsinogen type II, has been isolated from another colon cancer cell line (COLO 205). We have tried to determine if trypsinogen is present and how its expression varies during cell culture in HT-29 Glc+/- and Caco-2 cells, which exhibit enterocytic differentiation, and in HT-29 Glc+ cells, which never differentiate. Trypsinogen mRNA presence and expression were demonstrated in these cells by mRNA hybridization, RT-PCR, cytoimmunofluorescence, Western immunoblot analysis, and gel filtration. Trypsinogen was found to be trypsinogen type I and was mainly in zymogen form in culture media. Differentiating cells exhibited variations in trypsinogen I expression, but cells that remained undifferentiated did not. In the differentiated cells, a high and transient peak in trypsinogen I expression was observed during the first steps of differentiation.

Diet supplemented with yoghurt or milk fermented by Lactobacillus casei DN-114 001 stimulates growth and brush-border enzyme activities in mouse small intestine.

The nutritional benefits of lactic acid bacteria in fermented dairy products have been well documented, especially in terms of weight gain and feed efficiency, but not in terms of small intestine adaptation. The effects of a diet supplemented (30% wt/wt) with milk fermented either by Lactobacillus casei DN-114 001 or yoghurt for 3 or 15 days were investigated in the small intestine of mice by morphometry, kinetic analysis and determination of brush-border enzyme activities. Results were compared with those obtained with standard or milk isocaloric diets. Cell proliferation and villous area were significantly increased in the proximal intestine of mice fed the fermented-milk-supplemented diets for 3 days and were associated with hypertrophy and hyperplasia of Paneth and goblet cells. Lactase-specific activity was increased by fermented-milk diets at days 3 and 15, whereas there was no variation in maltase-specific activity. Alkaline phosphatase-specific activity was increased after 3 days of the three tested diets in the whole intestine, and after 15 days in the proximal intestine. Aminopeptidase activity was increased in the distal part of the intestine after 3 days of the 3 diets. Our findings suggest that diets supplemented with fermented milks have a positive effect on the trophicity of the mucosa in the small intestine of mice.

Modulation of proliferation, second messenger levels, and morphotype expression of the rat intestinal epithelial cell line IEC-6 by fermented milk.

Trophic effects of milk fermented with Lactobacillus helveticus, Lactobacillus paracasei ssp. paracasei, Bifidobacterium sp., or the combination of Lactobacillus bulgaricus and Streptococcus thermophilus (yogurt) were studied on the IEC-6 intestinal epithelial cell line. Incorporation of [methyl-3H]thymidine, mitochondrial dehydrogenase activities, cyclic AMP production, and differentiation of levels of the IEC-6 strain were evaluated between the 15th and 30th passage in culture. All fermented and unfermented milks enhanced trophic responses of IEC-6 cells in a dose-dependent manner. Compared with the corresponding milks, supernatant fractions were more effective in stimulating mitochondrial dehydrogenase response. Fermented milk supernatants were also more effective than the corresponding unfermented fractions. Increases in DNA synthesis and cyclic AMP confirmed the activation observed with mitochondrial dehydrogenase. Yogurt induced the more trophic response with an increased number of the more differentiated cell morphotype. Fermentation with L. casei also demonstrated an important trophic adaptation of IEC-6 cells. Milk processing by lactic acid bacteria enhanced trophic and proliferation responses of intestinal epithelial cell line IEC-6. These results suggested that IEC-6 cells could represent an accurate and easy in vitro model for testing the trophic quality of various nutrients and for an optimization of physiological digestive functions.

Immunohistological localization of regenerating protein in ocular structures.

PURPOSE: To investigate the presence of pancreatic stone protein (PSP)/reg (regenerating) protein in eyes and extraocular structures of rabbits, monkeys and man, according to species and age. METHODS: Rabbit eyes, with normal or de-epithelialized corneas, were taken from albino and pigmented animals. Monkey eyes were taken from cynomolgus monkeys, 2 years old. Stillborn and adult human eyes were obtained after autopsy. They were studied by immunohistochemistry methods, using a monoclonal antibody raised against human PSP. RESULTS: On rabbit ocular structures, the anti-PSP monoclonal antibody showed a strong reactivity at the level of basement membranes and basal poles of the cells of corneal and conjunctival epithelia and on basement membranes of skin and palpebral conjunctiva in eyelids. On de-epithelialized rabbit eyes, the remaining basement membrane was labeled while, along the re-epithelialization, the anti-PSP reactivity appeared with the migrating cells which cover the denuded cornea. On young monkeys, the whole corneal epithelium was reactive. Similar results were obtained from stillborn eyes, whereas no reactivity was detected on autopsy specimens from aged persons. CONCLUSIONS: As in other tissues and organs, the reg protein, in the eye, is found in structures known for their continuous and rapid renewal. This protein seems not to exist (or persist) in eyes from aged donors while it is strongly expressed in young donor eyes (monkey, stillborn baby) as well as in regenerating corneal epithelium. These findings enforce the hypothesis about the involvement of reg protein in cell proliferation and differentiation phenomena and its probable correlation with aging.

Immunohistochemical study of secretory proteins in the developing human exocrine pancreas.

We have studied, by immunohistochemical methods using specific antisera, the development of three glycoproteins of human pancreatic secretion: lipase, carboxyl ester hydrolase (CEH) and the P19 protein (precursor of the non glycosylated protein X or "pancreatic thread/stone protein"). We have compared their development to that of trypsinogens (Tgs) and chymotrypsinogen A (ChTgA), as well as to that of FAP (feto acinar pancreatic protein), a glycoprotein associated with the differentiation of human pancreas. Our studies show the characteristic appearance and development of lipase, the immunoreactivity of which appears later (at the 21st week of pregnancy) than it does for Tgs and ChTg (at the 16th week of pregnancy). Moreover, the lipase labelling is first observed in a few acini dispersed in the pancreas and then spreads out progressively to be present in all the acini after the age of 15 days. By contrast, as soon as they appear, Tgs and ChTg are observed uniformly in all acinar cells. The intensities of the lipase, Tgs and ChTg labellings increase greatly at birth. The ontogenesis of CEH does not follow that of lipase but that of Tgs and ChTg. The ontogenesis of P19 is parallel to that of Tgs. As previously observed, FAP presents a maximal immunoreactivity at the 24th-27th weeks of pregnancy, which decreases slowly up until birth.

Immunocytochemical demonstration of a pancreatic secretory protein of unknown function in human duodenum.

We have previously isolated from human pancreatic juice a secretory glycoprotein of 19 KD (P19), devoid of known enzymatic activity. P19 gave by proteolysis a protein of 14 KD (P14), at first named protein X and also called pancreatic thread protein or pancreatic stone protein. Specific rabbit immunosera prepared against P19 and P14 were applied to localize these proteins in human small intestine. By comparison, antibodies directed against some human pancreatic enzymes (amylase, lipase, chymotrypsin, trypsinogen 1, trypsinogen 2, and trypsin 1) were also tested. Positive immunoreactivity was observed on Paneth cells with antisera directed against trypsinogens, trypsin 1, and P19-related proteins. In addition, antisera directed against P19-related proteins stained the columnar cells located in the crypts of Lieberkühn. These original findings are a further indication of the resemblance between Paneth and pancreatic acinar cells but show that their functional analogy is only partial. On the other hand, the presence of P19-related proteins on non-mature columnar cells suggests that this differential distribution is a consequence of differentiation.

Protective effect of misoprostol, a synthetic prostaglandin E1 analog, on cerulein-induced acute pancreatitis in rats.

This study was performed to assess the effects of misoprostol, a synthetic prostaglandin E1 analog, on cerulein-induced pancreatitis. Per group of 10 each, male Wistar rats received either cerulein (2.5 micrograms/kg/h subcutaneously), cerulein and misoprostol (500 micrograms/kg intraperitoneally at 0 and 4 h), or saline. Rats were killed 6 h after the first injection. Misoprostol treatment significantly reduced interstitial edema and acinar cell lesions induced by hyperstimulation. Pancreatic amylase and chymotrypsin contents were increased by cerulein and returned towards control levels in the misoprostol-treated group. The lysosomal volume density and the pancreatic beta-D-glucuronidase activity were significantly increased after hyperstimulation. The two parameters were significantly reduced by misoprostol. A protective effect of misoprostol against lesions induced by cerulein hyperstimulation would be a consequence of a lysosomal stabilizating effect.

Effect of long-term intraluminal perfusion of pentagastrin on rat small-intestinal mucosa.

Mucosal changes occurring during long-term intraluminal perfusion of pentagastrin in the duodenum of conscious adult rats (100 micrograms/24 h/kg, 6 days) were studied. Significant increases of labelling and of mitotic indices were noted in the whole small intestine, with an enlargement of the crypt epithelial proliferative compartment. Differential kinetic variations were observed between upper and lower parts of the small intestine when labelling indices were measured in accordance with the cell position in the crypts. Kinetic variations were correlated to the increases of villous and microvillous area. Alkaline phosphatase and leucine-aminopeptidase-specific activities were significantly increased, suggesting modifications of synthesis and/or maturation of these enzymes. The ileal Paneth cell number was also significantly increased in the ileum. Pharmacologic doses of pentagastrin intraluminally perfused have a trophic effect at all levels of the small-intestinal mucosa.

Protective effect of misoprostol, a synthetic prostaglandin E1 analog, on experimental pancreatitis induced by pancreatic duct ligation in rat.

This study was performed to assess the effects of misoprostol (M), a synthetic prostaglandin E1 analog, on experimental pancreatitis in rat. Pancreatitis was induced by ligation of the main pancreatic duct of 3-month-old male Wistar rats. Pancreatic lesions were observed at 6, 12, 24, 48, and 96 h after pancreatic duct ligation (PDL). A time of 48 h was chosen to evaluate M treatment. M was injected intraperitoneally (500 micrograms/kg every 4 h) between time 0 and 48 h after PDL. Stereological analysis was performed on light and electron microscopy. Total pancreatic amylase and chymotrypsin concentrations were determined. Four groups of five rats were studied: sham operated (SO), M without PDL (PG), duct ligation without M (DL), and duct ligation with M (DLPG). Edema, dedifferentiation of pancreatic acinar cells, and heterogeneous distribution of zymogen granule diameters observed after PDL were significantly decreased by M in the DLPG group. Enzyme concentrations were also decreased by M in the DLPG group. Enzyme concentrations were also decreased by M both in normal (PG) and duct ligated rats (DL). M has protective effects against pancreatic lesions induced by PDL. In this model, the protective effect of M may be due to a blockade of the autodigestive secretions of the pancreatic acinar cells.

Exocrine pancreatic secretion in the elderly.

In order to evaluate impairment of exocrine pancreatic function during aging, 27 subjects (mean age: 36 years +/- 7.8) and 28 subjects (mean age: 72 years +/- 3.2), with no clinical or radiological evidence of digestive disease, were selected. Duodenal aspirates over a 60 min period were obtained during continuous IV infusion of secretin (0.5 U/kg/h) and caerulein (75 ng/kg/h). Bicarbonate, lipase, chymotrypsin amylase concentrations and output were measured. Bicarbonate, lipase, chymotrypsin concentrations in the aged group were significantly reduced by 17%, 15% and 23% respectively (P less than 0.05) as compared with those in the young group. In addition, a significant reduction of approximately 45% in bicarbonate and enzyme output levels was observed. This study provides strong evidence for a marked functional involution of the exocrine pancreatic secretion during aging. The potential consequences of this phenomenon on the nutritional status in the elderly are discussed.

Silent human pancreatic glucagonoma and "A" nesidioblastosis.

Surgical fragments of healthy and tumor-bearing pancreas from a patient with pancreatic tumor were studied by electron or light microscopy, histochemistry, and immunocytochemistry (human insulin, glucagon, somatostatin, gastrin, and bovine pancreatic polypeptide). Histological results were compared to those obtained by radioimmunoassay, both in tumor and serum. The tumor was identified as a glucagonoma because reactions for Grimelius' silver impregnation and immunoreaction with an antiserum against glucagon were positive and because a very high level of glucagon in the tumor was observed. Insulin, somatostatin, and gastrin levels remained normal, both in tumor and serum, but the glucagon level was normal in serum. Associated with this silent glucagonoma, an uncommon nesidioblastosis was also diagnosed with many A cells irregularly mixed with acinar cells, isolated or clustered in small groups. Acinar "intermediate" cells of "A" type were also observed. Such associative histopathological processes evoked possible development of an endocrine tumor from nesidioblastic-like tissue. Its embryogenic origin remained uncertain.

Long-term comparative effect of cholecystokinin and gastrin on mouse stomach, antrum, intestine, and exocrine pancreas.

Mice were injected three times a day for 12 days with 300 micrograms/kg body weight of gastrin G17 or 37.5 Ivy dog U/kg body weight of CCK or saline. Other mice were also injected four times an hr for 1 hr with 7.5 micrograms/kg of gastrin, nine Ivy dog U/kg of CCK or saline; 1 hr before killing, they were injected with tritiated thymidine to evaluate the labelling indices in peptic, antral, duodenal, jejunal, and ileal mucosae. Four hours after the first injection of the two peptides, the peptic labelling indices increased while those of intestinal mucosa increased 8 hr after these injections. Long-term injections of CCK had a trophic effect on secretory cells of the digestive tract: the number of gastric zymogenic cells, Paneth cells, and the mucous cells of Brünner glands were hypertrophied. The pepsin, amylase, chymotrypsin, and lysozyme activities increased in stomach, exocrine pancreas, and intestine, respectively. Neither parietal cells nor intestinal enterocytes and hydrolase activities were affected. The trophic effect of long-term injections of gastrin is confirmed on parietal cells and exocrine pancreatic parenchyma and is demonstrated in Paneth cells. Confirming cytological results, pancreatic lipase and amylase activities and intestinal lysozyme activity were increased after gastrin. Although CCK and gastrin have a structural analogy, these two peptides did not affect the same cellular types. A specific action of CCK on the main secretory cells of the digestive mucosa is demonstrated.

Long-term effect of somatostatin 14 on mouse stomach, antrum, intestine and exocrine pancreas.

Mice were injected 3 times a day for 12 days with 8 micrograms/kg of somatostatin 14 which caused a hypoplasia of parietal and goblet cells, a hypotrophy and hypofunctionality of pancreatic acinar cells with a decrease in lipase and chymotrypsin activities, a decrease in the secretory fuction of the Brunner gland and in the number of dark granules of G cells. Neither villous and microvillous areas nor brush border hydrolase activities were affected. The number of peptic cells and Paneth cells increase as the level of pepsin and lysozyme. Mice were injected 4 times per hour with 2 micrograms/kg of somatostatin. 2 h after the first injection of somatostatin and 90 min after a single injection of tritiated thymidine, fundic, antral, jejunal and ileal labelling indexes strongly decrease (maximal effect in ileum). The inhibitory effect of somatostatin on the digestive epithelial cell proliferation compared to its long-term action only directed on specific cell types evokes probable compensatory mechanisms induced to maintain the equilibrium of the digestive epithelia.

Immunohistochemical localization of intestinal phospholipase A2 in rat paneth cells.

Using the peroxidase-anti-peroxidase (PAP) technique with a specific rabbit anti-swine intestinal-phospholipase-A2 serum, the immunoreactivity of this phospholipase A2 was localized in rat-intestinal Paneth cells. The specific rabbit anti-swine intestinal-phospholipase-A2 serum did not stain the rat-pancreatic acinar cells which were stained by a specific rabbit anti-swine pancreatic-phospholipase-A2 serum. Specific rabbit anti-swine pancreatic-phospholipase-A2 serum did not stain rat-intestinal Paneth cells. Therefore, there is no cross-immunoreactivity between pancreatic and intestinal phospholipases.

Histological variations of the duodenal mucosa in chronic human pancreatitis.

From 16 volunteers with chronic pancreatitis and 36 healthy subjects duodenal biopsies were taken 15--20 cm beyond the papilla of Vater. Several morphometric parameters were calculated. The main results show: a significant decrease of villous area and height but not of the number of intestinal villi; a significant increase of Paneth cells; and a slight decrease in the number of glandular mitoses. This study suggests in man, a possible relationship between exocrine pancreatic secretion and the intestinal mucosa and a trophic action of pancreatic juice on the proliferation and the differentiation of the intestinal mucosa.