Tubular invaginations in cerebral endothelium and their relation to smooth-surfaced cisternae in hagfish (Myxine glutinosa)

Summary The organization of vesicular profiles in the endothelium of cerebral capillaries of the hagfish, Myxine glutinosa, has been reinvestigated. Judged from random thin sections the endothelial cells contain numerous vesicles and tubules, in contrast to brain endothelia of most other vertebrates. However, three-dimensional reconstructions based on ultrathin serial sections (thickness 18 nm) showed that the profiles represent a system of irregular tubular invaginations of the cell membrane, comparable to the vesicular invaginations demonstrated in extracerebral capillary endothelia of frogs and rats. In addition, smooth-surfaced cisternae were present in close relation to the invaginations. The function of endothelial invaginations is unknown. They do not transport macromolecules, because the blood-brain barrier is practically impermeable to proteins. However, since the system of the invaginations and smooth-surfaced cisternae is structurally similar to the system of caveolae and sarcoplasmic reticulum in smooth muscle cells, a common function seems likely. It is proposed that endothelial invaginations and smooth-surfaced cisternae are involved in regulation of cytosolic Ca⁺⁺-concentration.     

Human myocardial Na,K-ATPase concentration in heart failure

The Na,K-ATPase is of major importance for active ion transport across the sarcolemma and thus for electrical as well as contractile function of the myocardium. Furthermore, it is receptor for digitalis glycosides. In human studies of the regulatory aspects of myocardial Na,K-ATPase concentration a major problem has been to obtain tissue samples. Methodological accomplishments in quantification of myocardial Na,K-ATPase using vanadate facilitated ³H-ouabain binding to intact samples have, however, made it possible to obtain reliable measurements on human myocardial necropsies obtained at autopsy as well as on biopsies of a wet weight of only 1–2 mg obtained during heart catheterisation. However, access to the ultimately, normal, vital myocardial tissue has come from the heart transplantation programs, through which myocardial samples from cardiovascular healthy organ donors have become available. In the present paper we evaluate the various values reported for normal human myocardial Na,K-ATPase concentration, its regulation in heart disease and the association with digitalization. Normal myocardial Na,K-ATPase concentration level is found to be 700 pmol/g wet weight. No major variations were found between or within the walls of the heart ventricles. During the first few years of life a marked decrease in myocardial Na,K-ATPase concentration is followed by a stable level obtained in early adulthood and normally maintained throughout life. In patients with enlarged cardiac x-ray silhouette a significant positive, linear correlation between left ventricular ejection fraction (EF) and Na,K-ATPase concentration was established. A maximum reduction in Na,K-ATPase concentration of 89% was obtained when EF was reduced to 20%. Generally, heart failure associated with heart dilatation, myocardial hypertrophy as well as ischaemic heart disease is associated with reductions in myocardial Na,K-ATPase concentration of around 25%. During digoxin treatment of heart failure patients a further reduction in functional myocardial Na,K-ATPase concentration of 15% has been found. Thus, the total reduction in functional myocardial Na,K-ATPase concentration in digitalised heart failure patients may well be of the magnitude 40%. In conclusion, it has become possible to quantify human myocardial Na,K-ATPase in health and disease. Revealed reductions are in heart failure of importance for contractile function, generation of arrhythmia and for digoxin treatment.     

Structure ofNecturus gallbladder epithelium during transport at low external osmolarities

Summary Gallbladders transport isotonically over a wide range of osmolarities. This ability has been assumed to depend on the geometry of the lateral intercellular spaces. We report that this geometry in theNecturus gallbladder varies extensively with the external osmolarity and dependsin vitro on the integrity of the subepithelial tissues. The structure of the living epithelium was studied by Nomarski light microscopy while ultrastructural effects were revealed by electron microscopy. The short-term effects (<60 min) of low external osmolarities were: 1) the cells became bell-shaped with an increased cell height measured centrally, 2) lateral intercellular spaces lost their convoluted character; and 3) numerous membrane-bound cavities appeared in the cells. Furthermore, long-term exposure to the low external osmolarities caused an uneven density of epithelial cells. With subepithelial tissues intact, blistering of the epithelium cell layer was evident. Qualitative electron-microscopic data indicate that the membrane of the cavities was recruited from the basolateral cell membrane. This agrees well with light-microscopic observation that the cavities were initiated as invaginations of this cell membrane.     

Impermeability of hagfish cerebral capillaries to horseradish peroxidase

Summary Brain capillaries and their permeability to intravenously injected horseradish peroxidase, HRP, (MW: 40,000) were examined electron-microscopically in an attempt to find a structural explanation for the poorly developed blood-brain barrier in the hagfish, Myxine glutinosa. In particular, it was the aim of this study to examine the role of the numerous endothelial vesicles and tubules in the transport of this tracer between blood and brain. Many of the vesicles and tubules were found to be in continuity with the luminal or abluminal surfaces, but tubules generating channels through the endothelial cells were never observed. The cleft between adjacent endothelial cells was obliterated by punctate junctions. HRP, which was allowed to circulate for up to 35 min, was not found in the basal lamina or in the surrounding brain parenchyma. Few of the luminal vesicles and tubules were marked by the tracer. In the intercellular cleft HRP was stopped by the junctions. It is concluded that the hagfish like other vertebrates has a blood-brain barrier to HRP, and the numerous vesicles and tubules occurring in hagfish brain endothelium are not involved in the transendothelial transport of this macromolecule.     

A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.     

Near Infrared Optical Projection Tomography for Assessments of β-cell Mass Distribution in Diabetes Research

By adapting OPT to include the capability of imaging in the near infrared (NIR) spectrum, we here illustrate the possibility to image larger bodies of pancreatic tissue, such as the rat pancreas, and to increase the number of channels (cell types) that may be studied in a single specimen. We further describe the implementation of a number of computational tools that provide: 1/ accurate positioning of a specimen''s (in our case the pancreas) centre of mass (COM) at the axis of rotation (AR)²; 2/ improved algorithms for post-alignment tuning which prevents geometric distortions during the tomographic reconstruction² and 3/ a protocol for intensity equalization to increase signal to noise ratios in OPT-based BCM determinations³. In addition, we describe a sample holder that minimizes the risk for unintentional movements of the specimen during image acquisition. Together, these protocols enable assessments of BCM distribution and other features, to be performed throughout the volume of intact pancreata or other organs (e.g. in studies of islet transplantation), with a resolution down to the level of individual islets of Langerhans.     

MicroRNA Profiling in Human Neutrophils during Bone Marrow Granulopoiesis and In Vivo Exudation

The purpose of this study was to describe the microRNA (miRNA) expression profiles of neutrophils and their precursors from the initiation of granulopoiesis in the bone marrow to extravasation and accumulation in skin windows. We analyzed three different cell populations from human bone marrow, polymorphonuclear neutrophil (PMNs) from peripheral blood, and extravasated PMNs from skin windows using the Affymetrix 2.0 platform. Our data reveal 135 miRNAs differentially regulated during bone marrow granulopoiesis. The majority is differentially regulated between the myeloblast/promyelocyte (MB/PM) and myelocyte/metamyelocyte (MC/MM) stages of development. These 135 miRNAs were divided into six clusters according to the pattern of their expression. Several miRNAs demonstrate a pronounced increase or reduction at the transition between MB/PM and MC/MM, which is associated with cell cycle arrest and the initiation of terminal differentiation. Seven miRNAs are differentially up-regulated between peripheral blood PMNs and extravasated PMNs and only one of these (miR-132) is also differentially regulated during granulopoiesis. The study indicates that several different miRNAs participate in the regulation of normal granulopoiesis and that miRNAs might also regulate activities of extravasated neutrophils. The data present the miRNA profiles during the development and activation of the neutrophil granulocyte in healthy humans and thus serves as a reference for further research of normal and malignant granulocytic development.     

TGF‐β induces SOX2 expression in a time‐dependent manner in human melanoma cells

The sry‐related high‐mobility box (SOX)‐2 protein has recently been proven to play a significant role in progression, metastasis, and clinical prognosis spanning several cancer types. Research on the role of SOX2 in melanoma is limited and currently little is known about the mechanistic function of this gene in this context. Here, we observed high expression of SOX2 in both human melanoma cell lines and primary melanomas in contrast to melanocytic nevi. This overexpression in melanoma can, in part, be explained by extra gene copy numbers of SOX2 in primary samples. Interestingly, we were able to induce SOX2 expression, mediated by SOX4, via TGF‐β1 stimulation in a time‐dependent manner. Moreover, the knockdown of SOX2 impaired TGF‐β‐induced invasiveness. This phenotype switch can be explained by SOX2‐mediated cross talk between TGF‐β and non‐canonical Wnt signaling. Thus, we propose that SOX2 is involved in the critical TGF‐β signaling pathway, which has been shown to correlate with melanoma aggressiveness and metastasis. In conclusion, we have identified a novel downstream factor of TGF‐β signaling in melanoma, which may have further implications in the clinic.