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.     

P-glycoprotein as multidrug transporter: a critical review of current multidrug resistant cell lines

MDR has been studied extensively in mammalian cell lines. According to usual practice, the MDR phenotype is characterized by the following features: cross resistance to multiple chemotherapeutic agents (lipophilic cations), defective intracellular drug accumulation and retention, overexpression of P-gp (often accompanied by gene amplification), and reversal of the phenotype by addition of calcium channel blockers. An hypothesis for the function of P-gp has been proposed in which P-gp acts as a carrier protein that actively extrudes MDR compounds out of the cells. However, basic questions, such as what defines the specificity of the pump and how is energy for active efflux transduced, remain to be answered. Furthermore, assuming that P-gp acts as a drug transporter, one will expect a relationship between P-gp expression and accumulation defects in MDR cell lines. A review of papers reporting 97 cell lines selected for resistance to the classical MDR compounds has revealed that a connection exists in most of the reported cell lines. However, several exceptions can be pointed out. Furthermore, only a limited number of well characterized series of sublines with different degrees of resistance to a single agent have been reported. In many of these, a correlation between P-gp expresson and transport properties can not be established. Co-amplification of genes adjacent to the mdr1 gene, mutations [122], splicing of mdr1 RNA [123], modulation of P-gp by phosphorylation [124] or glycosylation [127], or experimental conditions [26,78] could account for some of the complexity of the phenotype and the absence of correlation in some of the cell lines. However, both cell lines with overexpression of P-gp without increased efflux [i.e., 67,75] and cell lines without P-gp expression and accumulation defects/increased efflux [i.e., 25,107] have been reported. Thus, current results from MDR cell lines contradict - but do not exclude - that P-gp acts as multidrug transporter. Other models for the mechanism of resistance have been proposed: (1) An energy-dependent permeability barrier working with greater efficacy in resistant cells. This hypothesis is supported by studies of influx which, although few, all except one demonstrate decreased influx in resistant cells; (2) Resistant cells have a greater endosomal volume, and a greater exocytotic activity accounts for the efflux. Furthermore, large amounts of P-gp in the plasma membrane altering the ultrastructure and generalized changes, such as increases or decreases in membrane fluidity, alterations in lipid composition, changes in transmembrane pH gradient and membrane potential have been described in MDR cell lines and could account for some of the findings.     

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.     

Cognitive Performance of G?ttingen Minipigs Is Affected by Diet in a Spatial Hole-Board Discrimination Test

Consumption of a high energy diet, containing high amounts of saturated fat and refined sugar has been associated with impairment of cognitive function in rodents and humans. We sought to contrast the effect of a high fat/cholesterol, low carbohydrate diet and a low fat, high carbohydrate/sucrose diet, relative to a standard low fat, high carbohydrate minipig diet on spatial cognition with regards to working memory and reference memory in 24 male Göttingen minipigs performing in a spatial hole-board discrimination test. We found that both working memory and reference memory were impaired by both diets relative to a standard minipig diet high in carbohydrate, low in fat and sugar. The different diets did not impact levels of brain-derived neurotrophic factor in brain tissue and neither did they affect circulatory inflammation measured by concentrations of C-reactive protein and haptoglobin in serum. However, higher levels of triglycerides were observed for minipigs fed the diets with high fat/cholesterol, low carbohydrate and low fat, high carbohydrate/sucrose compared to minipigs fed a standard minipig diet. This might explain the observed impairments in spatial cognition. These findings suggest that high dietary intake of both fat and sugar may impair spatial cognition which could be relevant for mental functioning in humans.     

Overlapping expression patterns among the genes encoding Arabidopsis chromosomal high mobility group (HMG) proteins

High mobility group (HMG) proteins are usually considered ubiquitous components of the eukaryotic chromatin. Using HMG gene promoter-GUS reporter gene fusions we have examined the expression of the reporter gene in transgenic Arabidopsis plants. These experiments have revealed that the different HMGA and HMGB promoters display overlapping patterns of activity, but they also show tissue- and developmental stage-specific differences. Moreover, leader introns that are present in some of the HMGB genes can modulate reporter gene expression. The differential HMG gene expression supports the view that the various HMG proteins serve partially different architectural functions in plant chromatin.     

Vaccination with p53-peptide–pulsed dendritic cells,of patients with advanced breast cancer: report from a phase I study

Peptides derived from over-expressed p53 protein are presented by class I MHC molecules and may act as tumour-associated epitopes. Due to the diversity of p53 mutations, immunogenic peptides representing wild-type sequences are preferable as a basis for a broad-spectrum p53-targeting cancer vaccine. Our preclinical studies have shown that wild-type p53-derived HLA-A2–binding peptides are able to activate human T cells and that the generated effector T cells are cytotoxic to human HLA-A2⁺, p53⁺ tumour cells. In this phase I pilot study, the toxicity and efficacy of autologous dendritic cells (DCs) loaded with a cocktail of three wild-type and three modified p53 peptides are being analysed in six HLA-A2⁺ patients with progressive advanced breast cancer. Vaccinations were well tolerated and no toxicity was observed. Disease stabilisation was seen in two of six patients, one patient had a transient regression of a single lymph node and one had a mixed response. ELISpot analyses showed that the p53-peptide–loaded DCs were able to induce specific T-cell responses against modified and unmodified p53 peptides in three patients, including two of the patients with a possible clinical benefit from the treatment. In conclusion, the strategy for p53-DC vaccination seems safe and without toxicity. Furthermore, indications of both immunologic and clinical effect were found in heavily pretreated patients with advanced breast cancer. An independent clinical effect of repeated administration of DCs and IL-2 can not of course be excluded; further studies are necessary to answer these questions.