Endogenous protein C is essential for the functional integrity of human endothelial cells

Circulating protein C (PC) plays a vital role as an anti-coagulant and anti-inflammatory mediator. We show here that human endothelial cells produce PC that acts through novel mediators to enhance their own functional integrity. When endogenous PC or its receptor, endothelial protein C receptor (EPCR), was suppressed by small interfering (si) RNA, human umbilical cord endothelial cell (HUVEC) proliferation was decreased and apoptosis elevated. Interestingly, PC or EPCR siRNA significantly increased HUVEC permeability, which is likely via reduction of the angiopoietin (Ang)1/Ang2 ratio and inhibition of the peripheral localization of the tight junction protein, zona occludins-1. In addition, PC or EPCR siRNA inhibited type IV collagen and matrix metalloproteinase-2, providing the first evidence that PC contributes to vascular basement membrane formation. These newly described actions of endogenous PC act to stabilize endothelial cells and enhance barrier function, to potentially promote the functional integrity of blood vessels.     

Primary neural induction as studied by scanning electron microscopy.

Normal primary neural induction has been studied by scanning electron microscopy and the results compared with those obtained by TEM. Mesoderm cells are usually in contact with several other cells, both mesodermal and endodermal in origin. By SEM the ectoderm layer has been shown to be in contact with the underlying mesoderm cells. Tufts of fibrous basement membrane are also present between the two cell types. TEM specimens also show an intermediate basement membrane.     

Relationship between the pentose phosphate shunt and methemoglobin reductase activity in human erythrocytes: Effect of aging on methemoglobin reductase activity

Summary The increase in methemoglobin reductase activity in human erythrocytes upon incubation with inosine, phosphate, pyruvate occurs only in the presence of methylene blue. No difference in activity of the methemoglobin reductases was observed between enzyme extracts of fresh cells and aged cells.     

Relationship between the pentose phosphate shunt and methemoglobin reductase activity in human erythrocytes: Effect of aging on methemoglobin reductase activity.

The increase in methemoglobin reductase activity in human erythrocytes upon incubation with inosine, phosphate, pyruvate occurs only in the presence of methylene blue. No difference in activity of the methemoglobin reductases was observed between enzyme extracts of fresh cells and aged cells.     

Characterization of tumor differentiation factor (TDF) and its receptor (TDF-R)

Tumor differentiation factor (TDF) is an under-investigated protein produced by the pituitary with no definitive function. TDF is secreted into the bloodstream and targets the breast and prostate, suggesting that it has an endocrine function. Initially, TDF was indirectly discovered based on the differentiation effect of alkaline pituitary extracts of the mammosomatotropic tumor MtTWlO on MTW9/PI rat mammary tumor cells. Years later, the cDNA clone responsible for this differentiation activity was isolated from a human pituitary cDNA library using expression cloning. The cDNA encoded a 108-amino-acid polypeptide that had differentiation activity on MCF7 breast cancer cells and on DU145 prostate cancer cells in vitro and in vivo. Recently, our group focused on identification of the TDF receptor (TDF-R). As potential TDF-R candidates, we identified the members of the Heat Shock 70-kDa family of proteins (HSP70) in both MCF7 and BT-549 human breast cancer cells (HBCC) and PC3, DU145, and LNCaP human prostate cancer cells (HPCC), but not in HeLa cells, NG108 neuroblastoma, or HDF-a and BLK CL.4 cells fibroblasts or fibroblast-like cells. Here we review the current advances on TDF, with particular focus on the structural investigation of its receptor and on its functional effects on breast and prostate cells.     

Role of laminin-nidogen complexes in basement membrane formation during embryonic development

Laminin and nidogen (entactin) are major glycoprotein components of basement membranes. At least seven different isoforms of laminin have been identified. Laminin and nidogen form high affinity complexes in basement membranes by specific binding between the laminin γ1 chain and the G3 globule of nidogen. Additional interactions between nidogen and collagen IV, perlecan and other basement membrane components result in the formation of ternary complexes between these matrix components. Nidogen is highly susceptible to proteolytic cleavage, and binding to laminin protects nidogen from degradation. Nidogen is considered to have a crucial role as a link protein in the assembly of basement membranes. Basement membrane components are synthesized at high levels during tissue growth and development, and sites of morphogenesis correlate with localized remodelling of basement membranes. The formation of distinct basement membrane matrices in the developing embryo is influenced by the laminin isoforms produced and by whether laminin and nidogen are co-expressed and secreted as a complex or are produced by cooperation between two cell layers. The potential roles of laminin-nidogen complexes, cell-matrix interactions, and other intermolecular interactions within the matrix in basement membrane assembly and stability are discussed in this review.     

3H-thymidine incorporation (DNA synthesis) and radiotoxicity in the ovary of the Japanese quail before and during follicle formation

No evidence was found for ribosomal DNA amplification in the oocytes of the Japanese quail, before or during folliculogenesis. DNA synthesis in the somatic cells, involved in follicle formation, starts at the medullar side of the basement membrane. The localized sterilization of the quail ovary after administration of 3H-thymidine (3H-TdR) seems to be due to radiation-induced lesions in the follicle forming somatic cells, rather than to direct radiation damage of the oocyte.     

Extracellular matrix components in intestinal development

Intestinal morphogenesis and differentiation are dependent on heterotypic cell interactions between embryonic epithelial cells (endoderm) and stromal cells (mesenchyme). Extracellular matrix molecules represent attractive candidates for regulators of these interactions. The structural and functional diversity of the extracellular matrix as intestinal development proceeds is demonstrated by 1) spatio-temporal specific expression of the classically described constituents, 2) the finding of laminin and collagen IV variants, 3) changes in the ratio of individual constituent chains, and 4) a stage-specific regulation of basement membrane molecule production, in particular by glucocorticoids. The orientation/assembly of these extracellular matrix molecules could direct precise cellular functions through interactions via integrin molecules. The involvement of extracellular matrix, and in particular basement membrane molecules in heterotypic cell interactions leading to epithelial cell differentiation, has been highlighted by the use of experimental models such as cocultures, hybrid intestines and antisense approaches. These models allowed us to conclude that a correct elaboration and assembly of the basement membrane, following close contacts between epithelial and fibroblastic cells, is necessary for the expression of differentiation markers such as digestive enzymes.     

An electron microscopic study on the autonomic innervation of the rabbit parotid gland

Summary As visualized in the electron microscope, the parotid gland of the rabbit has a dual innervation. Both adrenergic and cholinergic nerves are equally distributed in the parenchyma and often run together within the same nerve bundle. Nerve terminals are observed not only subjacent to the basement membrane but interposed between the latter and the acinar cells, where they establish a close membrane to membrane contact with the latter.This investigation was supported by grants from Svenska Medicinska Sällskapet and the University of Umeå.     

Haemolytic complement consumption by Parietaria pollen extracts in relation to peptide-bound flavonoids

Inhaled allergens from house dust, mites or animal danders activate human complement in vitro by engaging the C1-component through a non-antibody-dependent mechanism. These earlier findings are extended by showing that the allergenic components in extracts of Parietaria pollen are almost equally potent complement activators as those from house dust or mites. Spectroscopic evidence indicates that haemolytic complement consumption by the Parietaria allergens and their enzymic fragments is most likely related to post-translational side-chains comprising flavonoid derivatives. These adsorbed and/or peptide-bound tannin-like structures may also explain the exceptional stability of the high- and low-molecular mass allergenic components in Parietaria pollen extracts. Received 12 November 1996; received after revision 12 December 1996; accepted 17 December 1996     

Immunological demonstration of large quantities of glycogen or of a glycogen-like substance in human embryonic colon cells and in colon carcinomas by means of rabbit antisera raised against a strain of Escherichia coli 013]

Rabbit antisera raised against a strain of E. coli 013, with a strong antiglycogen activity, were tested on human fetal and normal adult colons, on colon carcinomas, and on colon tumor cells in culture (HT29). Only very rare granules were present in adult normal colons when tested with the immunofluorescence method. In faetal colons, in 12 out of 14 carcinomas, and on HT29 cells, the immunofluorescent reactions were similar to those observed in normal liver. The reactions were negative after previous treatment with alpha-amylase. They were inhibited with glycogen, with phenol-alcohol, perchloric, and trichloroacetic extracts from faetal colons, and with a tumor trichloroacetic extract. The extracts precipitated with anti-E. coli 013 antisera. They had a strong inhibiting activity in a radioimmunoassay test with labeled glycogen. The extracts from normal adult colons did not precipitate with the antisera and they had no inhibiting activity in either immunofluorescence and radioimmunoassay tests.     

Double stranded ribonuclease activity in human lymphocyte nuclei.

Ribonuclease activity directed against the synthetic duplex polyrC:polyI was detected in nuclear extracts from both unstimulated and PHA-stimulated human lymphocytes. In the latter cells, the activity was about twice that of small lymphocytes.     

UV guided dendritic growth patterns and the networking of melanocytes

Whole skin organ cultures of vitiliginous, skin show that the marginal melanocytes are highly sensitive to a pulse of UV exposure (210–380 nm) during the G₂ phase of the cell cycle, as seen by prominent dendricity. Melanocytes are highly dendritic in the epidermis overlying rapidly growing tumors, as well as within proliferative lesions such as basal cell carcinomas and aggressive seborrheic keratosis. In the organ cultures the dendrites extend towards the source of UV, i.e. the surface, while the main body lies along the basement membrane. The epidermal melanocytes overlying tumors lie, almost vertically, dendrites aligned towards the underlying tumor on one side and the surface on the other. Within tumors dendritic elongation is guided by mitotic and PCNA positive (S-phase) tumor cells, which are a source of ultraweak UV emissions in the range of 210–330 nm. These observations indicate that ultraweak biophoton emissions from neighbouring cells can simulate environmental cues and contribute to the plasticity of networks such as the melanocytes or the visual pathways.     

Double stranded ribonuclease activity in human lymphocyte nuclei

Summary Ribonuclease activity directed against the synthetic duplex polyrC: polyrI was detected in nuclear extracts from both unstimulated and PHA-stimulated human lymphocytes. In the latter cells, the activity was about twice that of small lymphocytes.     

Proteoglycans of basement membranes

Proteoglycans carrying either heparan sulfate and/or chondroitin sulfate side chains are typical constituents of basement membranes. The most prominent proteoglycan (perlecan) consists of a 400–500 kDa core protein and three heparan sulfate chains. Electron microscopy and cDNA sequencing show a complex and elongated domain structure for the core protein which in part is homologous to that of the laminin A chain. This structure may be varied by alternative splicing and proteolysis. Integration into basement membranes probably occurs by heparan sulfate binding to laminin and collagen IV, core protein binding to nidogen and by limited self assembly. The proteoglycan is in addition a cell-adhesive protein which is recognized by 1 integrins. Several more proteoglycans with smaller core proteins (10–160 kDa) apparently exist in basement membranes but are less well characterized. Biological functions include control of filtration through basement membranes and binding of growth factors and protease inhibitors.     

Primary neural induction as studied by scanning electron microscopy

Summary Normal primary neural induction has been studied by scanning electron microscopy and the results compared with those obtained by TEM. Mesoderm cells are usually in contact with several other cells, both mesodermal and endodermal in origin. By SEM the ectoderm layer has been shown to be in contact with the underlying mesoderm cells. Tufts of fibrous basement membrane are also present between the two cell types. TEM specimens also show an intermediate basement membrane.Acknowledgments. M. A. E. would like to thank ProfessorF. Beck in whose department this work was conducted. An especial acknowledgment to Mrs.Wendy Nugent for her skilled technical assistance in the preparation of the SEM and TEM specimens. Mr.Duncan Boreham, Electron Microscope Unit, University of Leicester kindly offered assistance and advice.S. V. C. would like to thank ProfessorR. P. Dale in whose department the SEM photographs were prepared.     

Demonstration of antigen associated with human breast cancers]

The insoluble pellet of human mammary carcinomas was solubilized by an acid buffer. Antiserum prepared with this acidosoluble fraction, after suitable absorption gave one precipitin line with the immunizing extracts: this line is different from those given by the tumor associated antigens actually known. The same antiserum reacted only with sections of human mammary carcinomas by immunofluorescence . It did not stain sections of normal mammary glands or benign mammary diseases. Reactivity with cancers of other organs was absent or doubtful. Hence it is likely that an antigen associated to human mammary carcinomas was characterized.     

The diet and gut microflora influence the distribution of enteroendocrine cells in the rat intestine

Several functions of the gut are locally influenced by peptides and biogenic amines released from enteroendocrine cells. The aim of the present study was to assess whether the luminal stimulus of diet or microbial flora or diet-microbial interactions have an influence on the distribution of enteroendocrine cells along the crypt-surface axes of the small and large intestine. The effects of diet and indigenous flora were investigated by comparing the numbers of argyrophil and serotonin immunoreactive cells in the jejunum and colon of germ free and conventional rats fed either a purified diet containing fine ingredients or a commercial diet containing crude fibre of cereal origin. The effects of human flora were analysed in germ-free rats inoculated with human faecal organisms. 1. Feeding the commercial diet reduced the number of argyrophil endocrine cells in the jejunum and serotonin immunoreactive cells in the colon of gern-free animals but increased the serotonin immunoreactive cells in the colon of conventional animals. 2. The rat flora increased the serotonin immunoreactive cells in the colon of animals fed a commercial diet and decreased in those fed a purified diet. 3. Inculation of human flora increased the numbers of serotonin immunoreactive cells both in the jejunum and colon. The results provide evidence that the dietary changes and diet-microbial interactions can affect the regional number of enteroendocrine cells.     

Effects of mistletoe (Viscum album L.) extracts on cultured tumor cells

Bacterially fermented mistletoe preparations (BFMP) were tested on rat hepatoma tissue culture (HTC) cells and human leukemia Molt 4 cells. A dose-dependent inhibition of the growth rate of the cells was observed. For both cell lines, cytostatic concentrations, expressed in weight of fresh plant, were 0.5 mg/ml culture medium for oak BFMP and 1 mg/ml for apple tree BFMP. However, the action of the two preparations was markedly different on each cell line. Non-viable HTC cells were not stained by trypan blue while non-viable Molt 4 cells were fully colored by this reagent. A lysis of cellular membranes of HTC cells was observed by electron microscopy. Furthermore, oak BFMP inhibited the growth of virus transformed 3T3-SV40 cells more than that of non-transformed 3T3 cells. In contrast to BFMP, non-fermented extracts and a purified mistletoe lectin showed a greater inhibition of the growth of Molt 4 cells than of HTC cells. Samples withdrawn at different times during fermentation gradually lost their inhibitory effect on the growth of Molt 4 cells while their action on HTC cells increased up to the 4th day of fermentation. These results are discussed in relation to the cytotoxic substances of mistletoe already characterized.