Critical roles of type III phosphatidylinositol phosphate kinase in murine embryonic visceral endoderm and adult intestine.

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.

[Pineal gland and extrapineal melatonin in visceral organs during natural human aging].

In order to define the functional morphology of the pineal gland (epiphysis) and to reveal extrapineal sources of melatonin (MT) during human aging, biopsy samples obtained from 175 male persons aged 16 to 84 were examined. The dynamics of MT-containing cells in the stomach, intestine, bronchi, prostate and the non-endocrinal cells of the adrenals were described.

Topographical significance of membrane skeletal component protein 4.1 B in mammalian organs.

The polarized architecture of epithelial cells is a fundamental determinant of cell structures and functions. Both formation and orientation of proper epithelial polarity are needed for cell-cell or cell-matrix adhesion, signal transduction and cytoskeletal interactions of multimolecular complexes at apical, lateral and basal cell membranes. These cell membrane domains are usually segregated by some junctional complexes. Recent molecular genetic studies on the anchor structure between myelin sheaths and axons have indicated the specific molecular organization for polarization of axolemma and the myelin sheaths at paranodes, termed 'septate-like junctions'. It was also speculated that other mammalian organs may use a similar junctional system. The protein 4.1 B was originally found to be localized in paranodes and juxtaparanodes of myelinated nerve fibers. Our recent immunohistochemical studies on protein 4.1B have indicated its significance for the cell-cell and/or cell-matrix adhesion in various rodent organs. The protein 4.1 family of proteins have been supposed to possess variable molecular domains relating to cell adhesion, ion balance, receptor responses and signal transduction. Therefore, more precise studies on the molecular structure and the functional domains of protein 4.1B, as well as on its changes under physiological and pathological conditions, may provide a clue for organogenesis in various mammalian organs.

Limited expression of nuclear pore membrane glycoprotein 210 in cell lines and tissues suggests cell-type specific nuclear pores in metazoans.

The nuclear pore complex (NPC) is the only known gateway for nucleocytoplasmic traffic. The nuclear pore membrane glycoprotein 210 (POM210/gp210) is considered to be important for the assembly and structure of pore complexes in metazoan cells. However, here we demonstrate cell-type specific expression of the gp210 protein during mouse organogenesis. As shown previously for its mRNA, distinct expression of the gp210 was seen in developing epithelia and some other cell types, whereas it was undetectable in nuclei of several other embryonic tissue compartments. In sharp contrast, monoclonal antibody 414 recognizing four non-membrane nucleoporins, stained the nuclear envelope of all cell types. In four cultured mouse cell lines, gp210 mRNA and protein were below detection levels, in contrast to some other nucleoporins tested. Distinct expression of gp210 mRNA and protein was seen in cultured mouse embryonic stem (ES) cells. These findings support the view of cell-type specific NPCs in metazoans and that the gp210 gene is regulated by cell-type specific control elements not shared by other nucleoporins. Although it cannot be excluded that very low expression levels of gp210 are sufficient to allow attachment of NPCs, a more likely alternative is that it has cell-type specific functions.

Contraction bands in visceral and vascular smooth muscle.

Smooth muscle contraction bands (SMCBs) have been described in the gastrointestinal tract, subsequent to acute ischemia, and in the coronary arteries of animals and individuals with a sudden death; in these circumstances SMCBs have been postulated to serve as a premortem marker, and suggested as diagnostically useful. The present investigation was undertaken to determine whether the presence of SMCBs could be correlated with a premortem clinical condition. Retrospectively, the routinely prepared histological sections from 76 autopsy and 93 surgical cases were screened semiquantitatively for the presence of SMCBs. The autopsy sections examined included the gastrointestinal tract, the prostate, and the coronary arteries, as well as all other smooth muscle-containing tissues; the surgical specimens included: coronary artery endarterectomies; saphenous vein bypass grafts; temporal artery biopsies; prostatic curettings; colectomies; varicose veins; leiomyomas of uterus, bowel, and skin; and, leiomyosarcomas. The clinical and pathology reports were reviewed for patient demographics, major clinical diagnoses, presence of shock, details of any resuscitation attempts, time interval to postmortem, and the cause of death. SMCBs were evident in 100% of the gastrointestinal and prostate, and in 96% of the coronary artery autopsy sections examined. All surgical specimens were positive for SMCBs, the exceptions being leiomyomas (positive in 13 of 22; 60%) and leiomyosarcomas (4 of 5; 80%); SMCBs in surgical specimens were less prominent when compared with those observed in autopsy tissue. No correlation was found between the presence of SMCBs and any clinical or demographic parameter assessed, because of the virtual universal occurrence of the SMCBs. The presence of less distinct SMCBs in surgical specimens may very well be artifactual, akin to myocardial and skeletal muscle contraction bands. The observation that SMCBs at autopsy are virtually ubiquitous suggests that they are best considered an agonal phenomenon, and a nonspecific pathological finding.

A combined electron microscopic investigation of the peritoneal mesothelium in the rat.

Visceral and parietal peritoneum of adult Wistar rats was studied by means of transmission and scanning electron microscopy. The structure of the peritoneum follows a general plan: mesothelium, basal lamina, and submesothelial connective tissue layer. Two basic types of mesothelial cells are described: flat and high (cubic), as well as transitional forms. A regularity of distribution of these cells in the visceral and parietal peritoneal sheets, and in the cover of individual organs and regions is described. A functional characterization of the different types of mesothelial cells is attempted, based on the differences of their cytoplasmic organization. The involvement of the mesothelium in the homeostasis in the peritoneal cavity is discussed.

Cellular localization of enzymatically active thrombin in intact human tissues by hirudin binding.

Cellular sites of coagulation activation within complex, intact tissues have been studied by immunohistochemical techniques. Hirudin, a specific and high affinity inhibitor of the active site of thrombin, together with antibody to hirudin were applied to sections of AMeX-fixed specimens of normal lung, kidney, placenta, freshly incised skin and unperturbed skin obtained at fresh autopsy; to rheumatoid synovial tissue; and to malignant tissue from a variety of tumor types. Staining for thrombin was observed selectively on pulmonary alveolar, rheumatoid synovial, and placental macrophages that express an intact extrinsic coagulation pathway. Staining was also observed restricted to the endothelium of capillaries in freshly incised skin but not in either unperturbed skin or in aged incisions. Staining of tumor cell bodies was observed in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma tissues that we found previously to show tumor cell-associated procoagulant activity. This staining occurred commonly on cells within the tumor mass that were distant from stromal fibrinogen/fibrin. By contrast, tumor-associated macrophage but not tumor cell staining was seen in adenocarcinoma and squamous cell carcinoma of the lung, and little or no staining was seen colon cancer tissue. Negative controls in which either the hirudin probe or its antibody were omitted failed to show staining. These results are in accord with previous findings and suggest that such techniques may be useful for studying the cellular sites of thrombin generation in intact tissues. We postulate that administration of potent and specific thrombin antagonists, such as hirudin, to patients with relevant tumor types might be followed by homing of hirudin to tumor cells in vivo so that effects of local thrombin generation on malignant progression can be determined.

Crystalline glomerular inclusions in multiple myeloma.

Prominent crystalline inclusions were noted in the glomeruli of a 57-year-old man with a 6-month history of swelling and pain in the hands, slight proteinuria, and elevated serum creatinine and BUN. The crystalline inclusions were most prominent in the visceral epithelium but were also noted in endothelial and mesangial cells, in the parietal epithelium, and in the epithelial cells of the proximal tubules. On electron microscopy the crystalline structures were of various geometric shapes but had no definite substructure. Immunofluorescence was negative. The patient was considered to have a hitherto undescribed metabolic disease. Several months later the patient underwent an operation for carpal tunnel syndrome and amyloid deposits, crystalline inclusions similar to those noted in the kidney were observed in the synovial tissue. Shortly after this the patient was found to have multiple myeloma of IgG, kappa type with Bence Jones proteinuria. Lymphoplasmacytic cells in the bone marrow contained the same crystalline inclusions noted in the kidney and synovium. This case therefore seems to represent a new and very rare form of glomerular involvement in multiple myeloma.

Villin expression in the visceral endoderm and in the gut anlage during early mouse embryogenesis.

Villin is an evolutionarily well conserved, Ca2+ regulated actin-binding protein, and a major structural component of the brush border of specialized absorptive cells. Using paraffin sections and an affinity purified polyclonal anti-villin antibody, we have investigated the early expression of villin during mouse embryogenesis. Villin is first detectable at the early post-implantation stage in visceral endodermal cells at the periphery of the egg cylinder. In this extra embryonic layer, the expression of villin increases and then persists until full term gestation. In the embryo, villin first appears in gut anlage during the axial rotation. Using the same methodology, villin expression is also demonstrated in differentiating embryoid bodies from a teratocarcinoma. Both in extra embryonic and embryonic extracts, villin expression is confirmed by immunoblot and Northern blot analysis which reveal, respectively, a single polypeptide of 93 kd and an mRNA of 3.4 kb in length, two well defined parameters for adult mouse villin gene expression. The results presented here show that paraffin sections allow very sensitive and highly resolutive detection of antigens in early embryogenesis. They provide a detailed developmental profile of villin expression and demonstrate the usefulness of villin as a marker for epithelial cells involved in absorptive processes.