In situ strategy for biomedical target localization via nanogold nucleation and secondary growth.

Immunocytochemistry visualizes the exact spatial location of target molecules. The most common strategy for ultrastructural immunocytochemistry is the conjugation of nanogold particles to antibodies as probes. However, conventional nanogold labelling requires time-consuming nanogold probe preparation and ultrathin sectioning of cell/tissue samples. Here, we introduce an in situ strategy involving nanogold nucleation in immunoenzymatic products on universal paraffin/cryostat sections and provide unique insight into nanogold development under hot-humid air conditions. Nanogold particles were specifically localized on kidney podocytes to target synaptopodin. Transmission electron microscopy revealed secondary growth and self-assembly that could be experimentally controlled by bovine serum albumin stabilization and phosphate-buffered saline acceleration. Valuable retrospective nanogold labelling for gastric H+/K+-ATPase was achieved on vintage immunoenzymatic deposits after a long lapse of 15 years (i.e., 15-year-old deposits). The present in situ nanogold labelling is anticipated to fill the gap between light and electron microscopy to correlate cell/tissue structure and function.

Three-dimensional culture of a pancreatic cancer cell line, SUIT-58, with air exposure can reflect the intrinsic features of the original tumor through electron microscopy.

Mini-abstract: Application of a three-dimensional culture system with air exposure facilitates the formation of large cell spheres possessing cribriform glands and producing mucin in the collagen gel. Transmission electron microscopy revealed the formation of microvilli and junctional complexes at the apical side of the cell. This study aimed to reproduce the characteristics of original adenocarcinoma tumors in vitro. The pancreatic cell line, SUIT-58, derived from a moderately differentiated adenocarcinoma of metastatic pancreatic cancer was used. The cells have a sheet structure in conventional cell culture without forming glands or exhibiting mucin production in the lumen. First, the necessity of scaffolds to create an adenocarcinoma-like microenvironment for SUIT-58 pancreatic cancer cells was assessed. Compared with conventional culture plates, the use of type I collagen as a scaffold played an important role in the formation of densely congested microvilli, as observed through scanning electron microscopy. As gland formation is one of the features of adenocarcinoma, we also assessed gland formation. Use of a recently developed three-dimensional culture system with air exposure resulted in the formation of large cell spheres possessing cribriform glands, which released mucin into the lumen. Transmission electron microscopy also revealed the formation of microvilli in the lumen of the glands and junctional complex at the intercellular part, which were similar to those observed in xenografts. These findings indicate that an in vitro three-dimensional culture system with air exposure reflects the intrinsic features of the original tumor, suggesting that this culture system could be useful for preliminary research of certain cancers.

Informative three-dimensional survey of cell/tissue architectures in thick paraffin sections by simple low-vacuum scanning electron microscopy.

Recent advances in bio-medical research, such as the production of regenerative organs from stem cells, require three-dimensional analysis of cell/tissue architectures. High-resolution imaging by electron microscopy is the best way to elucidate complex cell/tissue architectures, but the conventional method requires a skillful and time-consuming preparation. The present study developed a three-dimensional survey method for assessing cell/tissue architectures in 30-µm-thick paraffin sections by taking advantage of backscattered electron imaging in a low-vacuum scanning electron microscope. As a result, in the kidney, the podocytes and their processes were clearly observed to cover the glomerulus. The 30 µm thickness facilitated an investigation on face-side (instead of sectioned) images of the epithelium and endothelium, which are rarely seen within conventional thin sections. In the testis, differentiated spermatozoa were three-dimensionally assembled in the middle of the seminiferous tubule. Further application to vascular-injury thrombus formation revealed the distinctive networks of fibrin fibres and platelets, capturing the erythrocytes into the thrombus. The four-segmented BSE detector provided topographic bird's-eye images that allowed a three-dimensional understanding of the cell/tissue architectures at the electron-microscopic level. Here, we describe the precise procedures of this imaging method and provide representative electron micrographs of normal rat organs, experimental thrombus formation, and three-dimensionally cultured tumour cells.

Mice lacking the intracellular cation channel TRIC-B have compromised collagen production and impaired bone mineralization.

The trimeric intracellular cation (TRIC) channels TRIC-A and TRIC-B localize predominantly to the endoplasmic reticulum (ER) and likely support Ca(2+) release from intracellular stores by mediating cationic flux to maintain electrical neutrality. Deletion and point mutations in TRIC-B occur in families with autosomal recessive osteogenesis imperfecta. Tric-b knockout mice develop neonatal respiratory failure and exhibit poor bone ossification. We investigated the cellular defect causing the bone phenotype. Bone histology indicated collagen matrix deposition was reduced in Tric-b knockout mice. Osteoblasts, the bone-depositing cells, from Tric-b knockout mice exhibited reduced Ca(2+) release from ER and increased ER Ca(2+) content, which was associated with ER swelling. These cells also had impaired collagen release without a decrease in collagen-encoding transcripts, consistent with a defect in trafficking of collagen through ER. In contrast, osteoclasts, the bone-degrading cells, from Tric-b knockout mice were similar to those from wild-type mice. Thus, TRIC-B function is essential to support the production and release of large amounts of collagen by osteoblasts, which is necessary for bone mineralization.

Establishment and characterization of SUIT-58 pancreas cancer cell line and its subline S58-SF adapted to serum-free condition derived from metastatic liver tumor.

A new pancreas cancer cell line, SUIT-58, was established from metastatic liver tumor. The cultured cells exhibited polygonal shape, and proliferated in a form of sheet-structure showing prominent nucleoli and frequent mitotic features. Chromosome count ranged from 54 to 73 with modal chromosome numbers 72 and 73. It was noteworthy that this cell line grew in the serum-free media and maintained in this condition for 30 passages (designated as S58-SF). Both SUIT-58 and S58-SF cell lines were successfully transplanted into nude mice, and their tumor doubling times in xenografts were calculated as 5.4 and 2.8 days, respectively. Histopathologically, the xenografts formed glandular structure that resembled the original tumor. In culture media, the doubling time of SUIT-58 and S58-SF cell lines was calculated as 32 and 35.7 h, respectively. Although the cellular arrangements of SUIT-58 and S58-SF cell lines are different to some extent, their subcellular structures under electron microscope were similar with a large number of lysosomes and distinct desmosomes at cell-cell adhesion sites. The present SUIT-58 and its derivative cell line S58-SF will be applicable for biological studies to develop a new clinical treatment of refractory pancreatic cancer.

Establishment and biological characterization of a novel cell line derived from hepatoid adenocarcinoma originated at the ampulla of Vater.

Hepatoid adenocarcinoma is a rare gastrointestinal tumor and mostly reported in the stomach. Effective chemotherapy has yet to be developed to improve poor prognosis. The present study was undertaken to establish a useful cell line derived from a hepatoid adenocarcinoma, possibly leading to a new therapeutic strategy. The new human cell line VAT-39 was established from a metastatic lymph node of a 69-year-old Japanese male patient with hepatoid adenocarcinoma of the ampulla of Vater. The primary tumor and metastatic lymph node were composed of hepatoid adenocarcinoma cells exhibiting immunohistochemical reactivity for alpha-fetoprotein (AFP) and glypican-3 (GPC3). In the metastatic lymph node, Periodic acid-Schiff (PAS) staining clarified diffuse deposition of glycogen in the cytoplasm, indicating analogous characteristics to the primary hepatoid adenocarcinoma. Moreover, VAT-39 cells produced high levels of AFP in the cultured medium, and reverse-transcriptase polymerase chain reaction (RT-PCR) verified increased expression of GPC3 mRNA in this cell line. Further, we evaluated the sensitivity to major chemotherapeutic drugs against the bile duct cancer. Neither 5-fluorouracil nor gemcitabine showed particular sensitivity to this cell line. The tumorigenicity of the cultured cells was confirmed in athymic nude mice and the histological features of the explanted tumor were similar to the VAT-39 cell line. The present VAT-39 is the first hepatoid adenocarcinoma cell line that originates from the ampulla of Vater and it will be applicable for basic biological studies searching for new strategies of molecular targeted chemotherapy to this disease.

New insights concerning insulin synthesis and its secretion in rat hippocampus and cerebral cortex: amyloid-ß1-42-induced reduction of proinsulin level via glycogen synthase kinase-3ß.

The reduction of insulin levels in hippocampal areas is associated with Alzheimer's disease. The present study using rat brain explores the mechanisms of insulin synthesis and secretion, as well as amyloid-ß1-42 (Aß(1-42))-induced reduction of proinsulin expression. After confirming the expression of insulin mRNA and proinsulin in rat brain, we visualized and analyzed the motion of insulin secretion in rat hippocampal neurons using pH-sensitive green fluorescent protein (pHluorin) fused to the insulin. In the rat hippocampal neurons expressing insulin-pHluorin, time-lapse confocal laser scanning microscopy revealed the appearance of fluorescent spots induced by depolarization after stimulation with 50 mM KCl. In these fluorescent spots, Ca(2+)-dependent activator protein for secretion 2 (CAPS2), which is the regulator of the dense-core vesicle involving neuronal peptides, was co-localized with insulin-pHluorin. However, Aß(1-42)-induced reduction of proinsulin in rat hippocampal neurons was inhibited by treatment with lithium and transfection with glycogen synthase kinase-3ß (GSK-3ß) siRNA. These results demonstrate that synthesized insulin is secreted from rat hippocampal and cortical neuron's dense-core vesicles, and that activation of GSK-3ß in Aß(1-42)-induced Alzheimer's model hippocampal neurons decreases the insulin synthesis.

Involvement of the orexin system in adrenal sympathetic regulation.

Orexin (hypocretin) is a neuropeptide secreted from hypothalamic neurons that is known to be activated during motivated behaviors and active waking. Presently, our knowledge of orexin is mainly limited to the central nervous system, and the involvement of the orexin system in peripheral tissues has received little attention. In the present study, we analyzed the existence of the orexin system in the adrenal medulla, which is part of the sympathetic nervous system. Orexin and its receptors are expressed in the bovine adrenal medulla. Orexins stimulated intracellular calcium changes and epinephrine release from cultured bovine adrenal medullary cells. Applied orexin decreased expression of prepro-orexin, orexin receptor-1 and orexin receptor-2, suggesting negative feedback regulation in the adrenal gland. Our results indicate involvement of the orexin system in the sympathetic regulation of the adrenal medulla.

Involvement of guanylin and GC-C in rat mesenteric macrophages in resistance to a high-fat diet.

A high-fat diet (HFD) is a well-known contributing factor in the development of obesity. Most rats fed HFDs become obese. Those that avoid obesity when fed HFDs are considered diet resistant (DR). We performed a microarray screen to identify genes specific to the mesenteric fat of DR rats and revealed high expression of guanylin and guanylyl cyclase C (GC-C) in some subjects. Our histologic studies revealed that the cellular source of guanylin and GC-C is macrophages. Therefore, we developed double-transgenic (Tg) rats overexpressing guanylin and GC-C in macrophages and found that they were resistant to the effects of HFDs. In the mesenteric fat of HFD-fed Tg rats, Fas and perilipin mRNAs were downregulated, and those of genes involved in fatty acid oxidation were upregulated, compared with the levels in HFD-fed wild-type rats. In vitro studies demonstrated that lipid accumulation was markedly inhibited in adipocytes cocultured with macrophages expressing guanylin and GC-C and that this inhibition was reduced after treatment with guanylin- and GC-C-specific siRNAs. Our results suggest that the macrophagic guanylin-GC-C system contributes to the altered expression of genes involved in lipid metabolism, leading to resistance to obesity.

TRIM50 protein regulates vesicular trafficking for acid secretion in gastric parietal cells.

Of the TRIM/RBCC family proteins taking part in a variety of cellular processes, TRIM50 is a stomach-specific member with no defined biological function. Our biochemical data demonstrated that TRIM50 is specifically expressed in gastric parietal cells and is predominantly localized in the tubulovesicular and canalicular membranes. In cultured cells ectopically expressing GFP-TRIM50, confocal microscopic imaging revealed dynamic movement of TRIM50-associated vesicles in a phosphoinositide 3-kinase-dependent manner. A protein overlay assay detected preferential binding of the PRY-SPRY domain from the TRIM50 C-terminal region to phosphatidylinositol species, suggesting that TRIM50 is involved in vesicular dynamics by sensing the phosphorylated state of phosphoinositol lipids. Trim50 knock-out mice retained normal histology in the gastric mucosa but exhibited impaired secretion of gastric acid. In response to histamine, Trim50 knock-out parietal cells generated deranged canaliculi, swollen microvilli lacking actin filaments, and excess multilamellar membrane complexes. Therefore, TRIM50 seems to play an essential role in tubulovesicular dynamics, promoting the formation of sophisticated canaliculi and microvilli during acid secretion in parietal cells.

Functional transformation of gastric parietal cells and intracellular trafficking of ion channels/transporters in the apical canalicular membrane associated with acid secretion.

The parietal cell of the gastric gland is a highly differentiated cell responsible for the gastric hydrochloric acid secretion into the lumen of the stomach. In response to stimulation of acid secretion, the parietal cells undergo well-characterized morphological transformations to recruit H⁺/K⁺-ATPase from the cytoplasmic tubulovesicles to the apical canalicular membrane. Besides H⁺ extrusion via H⁺/K⁺-ATPase, Cl⁻ efflux and K⁺ recycling across the apical canalicular membrane are necessary via chloride and potassium channels/transporters, respectively. In the last decade, a number of molecular candidates for the Cl⁻ efflux and K⁺ recycling have been identified in the apical canalicular membrane of the parietal cell. This review focuses on the functional transformation of gastric parietal cells and intracellular trafficking of ion channels/transporters expressed in the apical canalicular membrane associated with gastric acid secretion.

A new device for high-pressure freezing of cultured cell monolayer using 10-microm-thin stainless discs as both culture plate and specimen carrier.

High-pressure freezing (HPF) has been generally accepted as the most reliable method for cryofixation of biological samples, yielding a deep vitreous freezing. In recent cell biology, mammalian cultured cells are widely used, but HPF of cultured cell monolayer has not reached its full potential. In this study, we developed a new reliable device for HPF of cultured cell monolayer by using a 10-microm-thin stainless disc both as culture plate and specimen carrier. We describe the practical procedure, and demonstrate fine structures of HeLa cells cultured and cryofixed on the stainless discs as results.

Exfoliation of gastric pit-parietal cells into the gastric lumen associated with a stimulation of isolated rat gastric mucosa in vitro: a morphological study by the application of cryotechniques.

It is clinicopathologically important to elucidate the cell kinetics for the maintenance of normal gastric epithelium. In a rat gastric mucosa isolated after stimulation, a number of cells were exfoliated into the gastric lumen of the pit region. The present study was undertaken to clarify the origin of exfoliated cells and their histochemical profiles by taking the advantages of cryotechniques. As results, most of the exfoliated cells were identified as pit-parietal cells labeled with both peanut-lectin and anti-H+/K+-ATPase antibody. Quantitative analysis verified a time-dependent increase in the number of exfoliated cells in the gastric mucosa isolated after stimulation. The exfoliated cells exhibited a diffuse intracellular staining for E-cadherin, suggesting a dissociation of the adhesion molecule prior to the cell exfoliation. It should be noted that most of the exfoliated cells were negative to the apoptotic markers (TUNEL staining and caspase-3). Ultrastructurally, autophagosome-like structures consisting of H+/K+-ATPase positive membranes were frequently seen in the exfoliated pit-parietal cells. In addition, the pit-parietal cell exfoliation was accompanied by sealing of their basal portion with the cytoplasmic processes of adjacent surface mucous cells. The present morphological findings provide a new insight into the cell kinetics in the gastric epithelium in vitro.

Ultrastructural transformation of gastric parietal cells reverting from the active to the resting state of acid secretion revealed in isolated rat gastric mucosa model processed by high-pressure freezing.

To elucidate a functional transformation of gastric parietal cells, we have newly developed an isolated rat gastric mucosa model whose parietal cells exhibited a reverting process from the active to the resting state of acid secretion. Briefly, the parietal cells were treated with cimetidine following prior stimulation of acid secretion in the model, and cryofixed by plunge freezing for light microscopy or high-pressure freezing for electron microscopy. As a result, immunohistochemistry of H(+)/K(+)-ATPase demonstrated a progressive translocation of H(+)/K(+)-ATPase from the apical to the cytoplasmic region. The ultrastructure of parietal cells at 5 min in the reverting phase was quite similar to that of maximally stimulated one. However, the apical microvilli of intracellular canaliculi (IC) changed bulbous by degrees, resulted in complete occlusion of IC at 60 min in the reverting phase. The apical membranes were subsequently internalized into the cytoplasm forming unique penta-laminar membranes. Interestingly, at 90 min in the reverting phase, the penta-laminar membranes formed a number of multilamellar autophagosomes that were intensely labeled for H(+)/K(+)-ATPase. Then, the parietal cells exhibited well-developed Golgi apparatus and lysosomal compartments involving the multilamellar membranes at 105 min, and mostly reverted to their resting conformation at 120 min in the reverting phase. Corresponding to the ultrastructural changes of microvilli, the immunohistochemistry of ezrin showed a dissociation of ezrin from the apical region at 30 min in the reverting phase. The present findings provide new insights into the functional transformation in gastric parietal cells reverting to their resting conformation.

The cryofixation of isolated rat gastric mucosa provides new insights into the functional transformation of gastric parietal cells: an in vitro experimental model study.

Cryofixation is currently accepted as the best initial fixation step to preserve not only the fine structure but also the antigenicity of biological samples. To elucidate the functional transformation of gastric parietal cells, we have newly developed an in vitro experimental model, named the isolated gastric mucosa. In this study, acid secretion of the parietal cell was stimulated with histamine or inhibited with cimetidine, and the samples were cryofixed by plunge freezing for light microscopy or high-pressure freezing for electron microscopy. As a result, the organization of glandular cells was well-preserved and quite similar to freshly excised rat gastric mucosa for at least 2 h after isolation. Immunohistochemistry of H+/K+-ATPase demonstrated a translocation of H+/K+-ATPase from the cytoplasm to the apical membrane associated with histamine-stimulation. In cimetidine-treated mucosa, most of the parietal cells were morphologically in the resting state, showing numerous tubulovesicles in their cytoplasm. In contrast, histamine-stimulated parietal cells exhibited well-developed intracellular canaliculi lined with long microvilli. To the best of our knowledge, the present study is first to demonstrate an electron micrograph that strongly suggests a membrane fusion between the tubulovescile and the apical membrane. Moreover, a stimulation-associated translocation of ezrin was clearly shown from the cytoplasm to the apical region, corresponding to apical microvilli development in the isolated gastric mucosa model. We here describe the preparation of the isolated rat gastric mucosa model, which provides new insights into the functional transformation of parietal cells by the application of cryotechniques.

Regeneration of gastric mucosa during ulcer healing follows pathways that correspond to the ontogenetic course of rat fundic glands.

Gastric ulcers in humans are notoriously chronic and recurring lesions. Although the average individual who undergoes no treatments requires many years for healing, most studies on the healing process of the experimentally induced ulcers have mainly focused on the early stages. Natural history of the ulcer healing has not been completely revealed. We have undertaken long-term investigation up to the 150th day after the cryo-injury to shed light on the natural history of the ulcer healing process compared with developmental changes of postnatal fundic glands. By the 30th day, restitutive gastric glands were mostly seen to cover the ulcer lesions, where well-developed gland-type mucous cells, showing Griffonia simplicifolia agglutinin (GSA)-II labeling, appeared to occupy the basal portion. Most of the bromodeoxyuridine-labeled cells were superimposed on the GSA-II-positive cell zone, forming the proliferative zone. By the 150th day, the restitutive glands were complete, with all epithelial components and topology of the normal fundic glands. The process of the ulcer healing was quite compatible with the developmental changes of the postnatal fundic glands. These results imply that the regeneration of gastric epithelium during the ulcer healing follows pathways linked to the ontogenetic course of the fundic gland.

Cardiac amyloidosis associated with a novel transthyretin aspartic acid-18 glutamic acid de novo mutation.

A 40-year-old man presented with initial symptoms of syncope caused by restrictive cardiomyopathy and autonomic nervous system impairment, but it was confirmed that he had a novel transthyretin (TTR) variant, aspartic acid-18 glutamic acid (Glu), and a de novo gene mutation. A polymerase chain reaction-induced mutation restriction analysis with a mismatched sense primer demonstrated that he was heterozygous for TTR Glu 18. Liver transplantation was not performed because of profound weakness and severe postural hypotension. Right-sided heart failure predominated in association with low output syndrome and a gradual decrease in total QRS voltage on electrocardiogram over 5 years of follow-up. Autonomic neuropathy developed and he eventually died of both-sided heart failure at the age of 45 years. Immunohistochemical and DNA studies are important to diagnose and treat TTR-related cardiac amyloidosis.