Pdx-1-independent differentiation of mouse embryonic stem cells into insulin-expressing cells.

To investigate whether insulin-producing cells obtained from ES cells via the nestin-positive cell-mediated method are of the pancreatic lineage, we established a pdx-1 knockout ES cell line and analyzed its differentiation into insulin-producing cells. As a result, pdx-1 knockout ES cell expressed insulin 2 gene at the final differentiated cells. Thus, our study demonstrated that pdx-1 is not essential for insulin gene expression, at least in cells differentiated from this population of nestin-expression enriched ES cells, and suggested that the insulin-producing cells derived from ES cells may be different from the pancreatic beta cells in terms of their lineage.

Novel human and mouse genes encoding an acid phosphatase family member and its downregulation in W/W(V) mouse jejunum.

BACKGROUND AND AIMS: Interstitial cells of Cajal (ICC) are pacemakers and mediators of motor neurotransmission in gastrointestinal smooth muscles. ICC require cellular signalling via Kit, a receptor tyrosine kinase, for development and maintenance of phenotype. Much of the evidence demonstrating the functions of ICC comes from studies of W/W(V) mice, which have reduced Kit function and reductions in specific populations of ICC. The aim of the present study was to differentially examine gene expression in the small intestines of wild-type and W/W(V) mutant mice. METHODS AND RESULTS: RNA from the jejunums of wild-type and W/W(V) mutants was analysed using a differential gene display method. Eighteen queries were identified as novel genes that were differentially displayed in wild-type and W/W(V) mice. One candidate gene, encoding a novel acid phosphatase-like protein, was significantly suppressed in fed and starved W/W(V) mice. The full length clone of the murine gene and its human counterpart were designated acid phosphatase-like protein 1 (ACPL1). Human ACPL1 cDNA encodes a protein of 428 amino acids with homology to human prostatic acid phosphatase protein. This gene is located at 1q21. ACPL1 was abundantly expressed in the human small intestine and colon. Gene products were found to be cytoplasmic in transfected COS-7 cells. Reverse transcription-polymerase chain reaction analysis revealed expression of ACPL1 mRNA within single isolated ICCs. CONCLUSIONS: Gene analysis showed that ACPL1 was differentially expressed in the small intestines of normal and W/W(V) mice. ICC within the small intestine expressed mRNA for ACPL1. Specific downregulation of ACPL1 in the jejunums of W/W(V) mice and high expression in human intestinal tissue suggest that the ACPL1 gene could be associated with ICC function in mice and humans.

Ultrastructural study of mouse renal glomeruli under various hemodynamic conditions by an "in vivo cryotechnique".

Three-dimensional ultrastructure of mouse renal glomeruli under various hemodynamic conditions was studied by scanning electron microscopy with an "in vivo cryotechnique" followed by freeze-substitution. These results were also compared with those of conventionally fixed glomeruli at different perfusion pressures. Some kidneys of anesthetized mice were directly frozen in vivo under normal blood flow condition. Others were frozen in the same way after ligation of the lower abdominal aorta or their heart arrest. The frozen specimens were routinely freeze-substituted, freeze-dried and ion-sputtered for SEM. For another conventional fixation method, some kidneys were perfused with glutaraldehyde at pressures of 150 cm or 250 cm water, and other renal cortical tissues were routinely immersion-fixed with glutaraldehyde. In freeze-substituted specimens with the in vivo cryotechnique, interdigitating foot processes of podocytes exhibited smooth surface contours with less shrinkage and their surfaces were arranged more tightly than those seen by the conventional fixation method. In the anesthetized mice, filtration slits between the foot processes were found to be narrow after the heart arrest, but they were wide under the normal blood flow condition. After the aorta ligation, the filtration slits became wider and the foot processes were more elongated. The ultrastructure of capillary loops in functioning renal glomeruli were revealed to be dynamically changing in the living state, depending on various hemodynamic conditions.

Loss of interstitial cells of Cajal and development of electrical dysfunction in murine small bowel obstruction.

1. Partial obstruction of the murine ileum led to changes in the gross morphology and ultrastructure of the tunica muscularis. Populations of interstitial cells of Cajal (ICC) decreased oral, but not aboral, to the site of obstruction. Since ICC generate and propagate electrical slow waves in gastrointestinal muscles, we investigated whether the loss of ICC leads to loss of function in partial bowel obstruction. 2. Changes in ICC networks and electrical activity were monitored in the obstructed murine intestine using immunohistochemistry, electron microscopy and intracellular electrophysiological techniques. 3. Two weeks following the onset of a partial obstruction, the bowel increased in diameter and hypertrophy of the tunica muscularis was observed oral to the obstruction site. ICC networks were disrupted oral to the obstruction, and this disruption was accompanied by the loss of electrical slow waves and responses to enteric nerve stimulation. These defects were not observed aboral to the obstruction. 4. Ultrastructural analysis revealed no evidence of cell death in regions where the lesion in ICC networks was developing. Cells with a morphology intermediate between smooth muscle cells and fibroblasts were found in locations that are typically populated by ICC. These cells may have been the redifferentiated remnants of ICC networks. 5. Removal of the obstruction led to the redevelopment of ICC networks and recovery of slow wave activity within 30 days. Neural responses were partially restored in 30 days. 6. These data describe the plasticity of ICC networks in response to partial obstruction. After obstruction the ICC phenotype was lost, but these cells regenerated when the obstruction was removed. This model may be an important tool for evaluating the cellular/molecular factors responsible for the regulation and maintenance of the ICC phenotype.

Clathrin-dependent and clathrin-independent endocytosis are differentially sensitive to insertion of poly (ethylene glycol)-derivatized cholesterol in the plasma membrane.

We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin-coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin-independent fluid phase uptake and the internalization of fluorescent cholera-toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin-mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin-coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin-coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036-3047) and suggest here that the functional clathrin-lattice is much stiffer than typical phospholipid bilayers.

Immunoelectron-microscopic study of Kit-expressing cells in the jejunum of wildtype and Ws/Ws rats.

Interstitial cells of Cajal (ICC) are responsible for generating electrical slow waves in the gastrointestinal (GI) tract. Slow waves regulate the frequency of contractions of the tunica muscularis, and therefore ICC are critical for normal motility in the small intestine. ICC express Kit, the gene product of c-kit, a protooncogene that encodes a receptor tyrosine kinase. Physiological evidence demonstrating that ICC are pacemakers has come from experiments on W-mutant mice which have few Kit-positive cells at the level of the myenteric plexus (IC-MY) and also lack electrical slow waves. In the past identification of ICC required the use of electron microscopy, however the discovery that ICC express Kit has facilitated studies of the distribution of ICC in several species. Immunoelectron microscopy to relate ultrastructure to Kit expression has only been performed in a limited number of studies of mice. We examined the ultrastructure of Kit-expressing cells in the rat using immunoelectron microscopy and an anti-Kit antibody. We compared the presence and appearance of Kit-expressing ICC in wildtype and Ws/Ws rats, which carry a mutation in the white spotting locus and have a phenotype similar to W/Wv mutant mice. Kit-expressing cells could be detected in the myenteric plexus (MY) and deep muscular plexus (DMP) regions of the small intestine of wildtype animals. In Ws/Ws rats, Kit-expressing cells were not observed in the region of MY, but were observed in the DMP. The density of Kit-positive cells in the DMP of Ws/Ws rats was similar to those in wildtype rats. Electron microscopy showed that Kit-expressing cells at the level of the MY of the rat had similar ultrastructural features as IC-MY in wildtype mice. IC-DMP in the rat of both wildtype and Ws/Ws mutants were similar in structure to IC-DMP of the mouse. We conclude that wildtype rats have IC-MY and IC-DMP in the tunica muscularis of the jejunum. ICC express Kit-like immunoreactivity (Kit-LI) in the rat as in the mouse. IC-MY are absent in the small intestine of Ws/Ws rats, and this corresponds to the lack of Kit-labeling in this region. Ws/Ws rats, however, possess IC-DMP with normal ultrastructural features and Kit-LI. The absence of IC-MY of Ws/Ws rats is likely to account for the abnormal contractile activity of the GI tract observed in these mutants. The present study suggests that Ws/Ws rats could provide an interesting model to investigate the physiological significance of pacemaker activity because they manifest a defect in IC-MY.

Differential gene expression in the small intestines of wildtype and W/W(V) mice.

UNLABELLED: Much of the evidence demonstrating the role of interstitial cells of Cajal (ICC) in pacemaking and neurotransmission in the gastrointestinal tract comes from studies of W/W(V) mice. These animals have few pacemaker ICC in the small bowel due to reduced functional Kit protein. We examined gene expression in the small intestines of wildtype and W/W(V) mice. RNA expression in the jejunums of wildtype and W/W(V) mutants was studied using a differential gene expression METHOD: Seven known genes were differentially expressed in wildtype and W/W(V) mice. COX7B (cytochrome c oxidase, subunit VIIb) and SORCIN (encoding multidrug-resistance complex, class 4) were suppressed in both fed and fasted W/W(V) mice. Expression of another five genes was increased in W/W(V) mice: ADA (adenosine deaminase), MDH1 (malate dehydrogenase), RPL-8 (ribosomal protein L8), SPTB2 (spectrin, nonerythroid, beta subunit), and p6-5 (encoding phosphorylcholine [PC] T-cell suppressor factor [TsF]). Differential expression was the same in fasted and fed animals, suggesting that the differences were independent of the dietetic state. We conclude that several genes are differentially expressed in the small intestines of W/W(V) mice where the major lesion is loss of pacemaker ICC. Differential gene display may help develop a molecular profile of motility disorders in which ICC are lost.

Changes of in vivo gastrointestinal motor pattern in pacemaker-deficient (WsRC-Ws/Ws) rats.

In vitro studies on pacemaker-deficient W-mutants have revealed a disappearance of rhythmic contraction in their gastrointestinal tracts. Their contractile force has not been diminished, however. In contrast, W-mutants often present dysmoility-like symptoms with distension of the gastrointestinal tract in vivo. Gastrointestinal motility of W-mutant rats was examined in vivo by an extraluminal strain-gauge force transducer method. We examined a normal gastrointestinal motor pattern in the rats with two distinct motor phases, digestive and interdigestive. Moreover, we detected a failure to form an interdigestive contractile complex in pacemaker-deficient rats. The interdigestive motor activity of the gastrointestinal tract is important for cleaning gastrointestinal tract in preparation for the next meal. The impairment of the interdigestive contractile complex may be related to the dysmoility-like symptoms of W-mutant rats in vivo.

Remodeling of networks of interstitial cells of Cajal in a murine model of diabetic gastroparesis.

Patients with long-standing diabetes commonly suffer from gastric neuromuscular dysfunction (gastropathy) causing symptoms ranging from postprandial bloating to recurrent vomiting. Autonomic neuropathy is generally believed to be responsible for diabetic gastropathy and the underlying impairments in gastric emptying (gastroparesis) and receptive relaxation, but the specific mechanisms have not been elucidated. Recently, it has been recognized that interstitial cells of Cajal generate electrical pacemaker activity and mediate motor neurotransmission in the stomach. Loss or defects in interstitial cells could contribute to the development of diabetic gastroparesis. Gastric motility was characterized in spontaneously diabetic NOD/LtJ mice by measuring gastric emptying and by monitoring spontaneous and induced electrical activity in circular smooth muscle cells. Interstitial cells of Cajal were studied by Kit immunofluorescence and transmission electron microscopy. Diabetic mice developed delayed gastric emptying, impaired electrical pacemaking, and reduced motor neurotransmission. Interstitial cells of Cajal were greatly reduced in the distal stomach, and the normally close associations between these cells and enteric nerve terminals were infrequent. Our observations suggest that damage to interstitial cells of Cajal may play a key role in the pathogenesis of diabetic gastropathy.

Topographical difference of cytoskeletal organization in smooth muscle cells of rat duodenum revealed by quick-freezing and deep-etching method.

The sarcolemmal domain of rat duodenal smooth muscle cells includes caveolae and associated cytoskeletal or filamentous elements. We have used the quick-freezing, deep-etching method to examine the three dimensional relationships between these components. Replica membranes for separated strips of rat duodenal muscle layers were routinely prepared after extraction soluble proteins from cytoplasm and extracellular matrix. As results, 1) cytoskeletal elements in smooth muscle cells consisted mainly of striated thin filaments; 2) thin filaments were connected with some plasma membranes through filaments associated with the sarcolemma, which formed fine network structures beneath the sarcolemma; 3) many bridging structures between the filaments associated with the sarcolemma and the extracellular matrix were frequently detected in the plasma membrane; and 4) compact filaments associated with the sarcolemma almost disappeared near the caveolae, and only thin filaments were anchored to their neck parts. The special arrangement of the cytoskeletal components, which is probably necessary for the intestinal motility, characterizes the topographical difference of the smooth muscle sarcolemma.

Dynamic ultrastructure of mouse pulmonary alveoli revealed by an in vivo cryotechnique in combination with freeze-substitution.

A morphological approach to cell dynamics is usually difficult, since routine preparative techniques for electron microscopy always induce artifacts due to cessation of the blood supply into organs. An in vivo cryotechnique followed by the freeze-substitution method probably reduces such problems. It was applied for examining the pulmonary alveoli of BALB/c mice in vivo. The following ultrastructural features were revealed. (1) A surfactant layer provided a continuous covering to the alveolar epithelium. (2) Pleural epithelial cells, alveolar cells and endothelial cells contained many small vesicles and pits. In the alveolar epithelium, they were often localised near microtubules. (3) Typical lamellar structures in large alveolar epithelial cells were rarely detected. (4) Circulating erythrocytes with various shapes were observed in branching blood capillaries. (5) A close association between erythrocytes and the endothelium was seen at the peripheral alveolar septum. Such ultrastructural arrangements may be appropriate for the physiological functions of the pulmonary alveoli, such as exchanges of gases or materials in vivo.

Immunolocalization of dystrobrevin in the astrocytic endfeet and endothelial cells in the rat cerebellum.

Dystrobrevin is a newly discovered dystrophin-associated protein that is classified as alpha- and beta-dystrobrevin. Previous studies reported that dystrophin, utrophin, syntrophin and beta-dystroglycan were expressed in the cerebellum. In the present study, we examined cellular and subcellular localization of dystrobrevin in the adult rat cerebellum immunohistochemically. Confocal microscopy showed that dystrobrevin was expressed around blood vessels and under the pia mater as dystrophin, utrophin and beta-dystroglycan were. Immunoelectron microscopy demonstrated that dystrobrevin was localized not only in the astrocytic endfeet around blood vessels and under the pia mater, but also in endothelial cells. Considering the fact that dystrobrevin possesses multiple phosphotyrosine kinase residues, these data suggest that dystrobrevin plays a role in blood-brain barrier functions as a component of the dystrophin complex.

Ultrastructure of rat duodenal myenteric plexus revealed by quick-freezing and deep-etching method.

A quick-freezing and deep-etching (QF-DE) method was employed with whole-mount strips of rat duodenal muscle walls to exhibit the cytoskeletons of the myenteric plexus. Nerve fibers in the myenteric plexus, which contained fewer neurofilaments than other types of neurons examined, had many varicosed contours, and were bundled by enteroglial cells. Cytoskeleton arrays were rarely observed in the varicosed regions, where synaptic vesicles were often seen, although other nerve regions contained many neurofilaments running almost in parallel with the nerve fiber bundle. Enteroglial cells had short cytoskeletons predominantly across the cytoplasm, becoming thinner the around varicosed regions of the nerve bundles. Such enteroglial extruded areas were often in close association with neighboring nerve fibers, indicating intercommunications between the nerve fibers. In distal parts of enteric nerve processes, there were numerous synaptic vesicles, but few neurofilaments. Smooth muscle cells were closely associated with the enteric nerve processes. Fine network structures, responsible for the extracellular matrix, were present between the smooth muscle cells and the enteric nerve processes. These specific structures of the myenteric plexus could be important for signalling or for the transportation of neurotransmitters involved in gut motility.

"In vivo cryotechnique" in combination with replica immunoelectron microscopy for caveolin in smooth muscle cells.

A novel "in vivo cryotechnique" with replica immunoelectron microscopy was developed for detecting caveolin localization on replica membranes prepared directly from living smooth muscle cells. After quick-freezing mouse duodenal walls by our "in vivo cryotechnique", the specimens were prepared for freeze-fracture and deep-etch replica membranes. Then they were treated with 5% SDS and 0.5% collagenase to keep some antigens on the replica membranes. The immunogold method could be used to clarify the localization of the caveolin antigen in relation to three-dimensional ultrastructures of living smooth muscle cells. Our new cryotechnique can provide native organization of functional molecules in living cells.

[An investigation of the factors influencing serum pepsinogen levels--sex, age, smoking, drinking].

BACKGROUND: Until now, the influence of sex, age, smoking, drinking on serum pepsinogen levels has been assessed by single regression analysis. However, the influence of those factors on pepsinogen levels should be assessed exactly by multiple regression analyses. SUBJECTS AND METHODS: 891 subjects were collected from by questionnaire and serum tests. Analyses were done with serum pepsinogen I (PG I), pepsinogen II (PG II) and pepsinogen I/II ratio (PG I/II) as a criterion variable and as categorized explanatory variables, sex, age, current or past smoking habit, and current drinking habit. And analyses are done by Mann-Whitney U test, correlation coefficient, single regression method, multiple regression method. RESULTS: PG I level is significantly higher in men than in women by Mann-Whitney U test. But the effect of sex factor is not remarkable by multiple regression analyses. PG II level increased and PG I/II level decreased with progression of age by all analyses methods. Current or past smoking elevates PG I level by Mann-Whitney U test, but current smoking dose and past smoking amount showed no dose-dependent associations with PG I level. Current drinking elevates PG I level by Mann-Whitney U test, but current drinking dose showed no dose-dependent associations with PG I. However, the effects of current smoking and current drinking to serum PG levels is not so large by multiple regression analyses. CONCLUSION: The effects of sex, current smoking and current drinking to serum PG levels are not remarkable by multiple regression analyses. Significantly, PG II level increased and PG I/II level decreased with progression of age. Therefore it may not be necessary to consider the effects of sex, smoking habit and drinking habit when serum pepsinogen levels are used as markers for gastric cancer.

Dynamic structure of glomerular capillary loop as revealed by an in vivo cryotechnique.

Morphological studies using immersion or perfusion fixation methods do not reveal the ultrastructure of functioning kidneys with normal circulation. A simple apparatus was developed for freezing the kidneys in vivo without stopping the blood supply, and the ultrastructure of the glomerular capillary loops was examined under different haemodynamic conditions. Mouse kidneys were frozen under normal blood flow conditions; others were frozen in the same way after ligation of the abdominal aorta at a point caudal to the renal arteries. They were then processed for the freeze-substitution or deep-etching method. Good ultrastructural preservation was obtained within about 5 microM depth from the frozen tissue surface. Functioning glomeruli with normal blood flow possessed open capillary lumens, different shapes of foot processes and atypical basement membranes with low density. Moreover, heterogeneity in width between foot processes was identified on the replica membranes. Under the acute conditions used to increase blood supply into the kidneys, the spaces between the flat foot processes became more widely dilated and the basement membrane was seen to be three-layered. The ultrastructure of glomeruli in functioning kidneys has been demonstrated for the first time by this "in vivo cryotechnique."

Freeze-fracture immunocytochemistry for intracellular localization of serotonin in mast cells stimulated with compound 48/80.

Changes of intracellular localization of serotonin in rat mast cells were examined by freeze-fracture immunocytochemistry, to prevent the translocation of the serotonin antigen. Rat peritoneal cells including mast cells were stimulated in vitro with compound 48/80, at 17 degrees C for 0, 30 or 60 s for exocytosis to occur. The mast cells were fixed, quickly frozen and freeze-fractured to expose the antigen on the fractured surface. They were immunostained with serotonin antibody, and the immunoreactions on the fractured surface were examined on ultrathin sections by electron microscopy. Unstimulated mast cells exhibited serotonin localization mostly in each intragranular matrix. In contrast, mast cells stimulated for 30 s exhibited increased serotonin in their intergranular cytoplasm. Mast cells showed more distinct immunoreactions in the cytoplasm where degranulation would be promoted after 60 s. It is suggested that intracellular release of serotonin occurred in the stimulated mast cells.

Morphological study of rat mast cells stimulated with compound 48/80 at different temperatures.

Changes of mast cells stimulated with compound 48/80 were morphologically investigated at different temperatures. Peritoneal mast cells of male rats were stimulated in vitro at 4 or 17 degrees C. At 17 degrees C, mast cells stimulated for 10 s gave decreased fluorescent reactions for phalloidin. At 30 s stimulation, they showed typical exocytosis initiated by fusions of peripherally located secretory granules to the plasma membrane. In contrast, mast cells stimulated at 4 degrees C exhibited neither decrease of phalloidin reactions nor typical excytosis even after 30 s. It was inferred that the fusions were mediated by cytoplasmic elements, probably the actin filaments previously suggested to prevent release of secretory granules. Furthermore, the space between the perigranular membrane and granular contents was enlarged in some mast cells stimulated at 4 degrees C. The morphological changes suggested that equivocal events occurred also in the cytoplasm of these cells. The mast cells showed no typical exocytosis at 4 degrees C.

X-ray microanalysis of rat mast cells stimulated with compound 48/80 in combination with quick-freezing method.

X-ray microanalysis was performed on rat mast cells prepared by quick-freezing, cryosectioning and freeze-drying (QF-FD) method, or quick-freezing and freeze-substitution (QF-FS) method. Peritoneal cells including mast cells were stimulated with compound 48/80 for 0, 10 or 30 s at 17 degrees C, and the mast cells stimulated for 30 s started exocytosis. In X-ray spectra of the QF-FD specimen, mast cells stimulated for 10 s increased their levels of phosphorus, sodium and chlorine in the intergranular cytoplasm prior to exocytosis, and kept this increase until 30 s after stimulation. In the QF-FS specimen, where soluble elements were removed, peaks of phosphorus, sulphur and potassium could be detected as elements in X-ray spectra. Phosphorus increased and potassium decreased in intergranular cytoplasm of mast cells stimulated for 10 s, and these changes became more obvious after 30 s. However, supplemental increase of other cations such as sodium could not be detected in the QF-FS specimens.