Introduction: Novel Frontiers in Cancer Metastasis

Clinical & Experimental Metastasis -     

Lysophosphatidylcholine‐induced cytotoxicity and protection by heparin in mouse brain bEND.3 endothelial cells

A pathological feature in atherosclerosis is the dysfunction and death of vascular endothelial cells (EC). Oxidized low‐density lipoprotein (LDL), known to accumulate in the atherosclerotic arterial walls, impairs endothelium‐dependent relaxation and causes EC apoptosis. A major bioactive ingredient of the oxidized LDL is lysophosphatidylcholine (LPC), which at higher concentrations causes apoptosis and necrosis in various EC. There is hitherto no report on LPC‐induced cytotoxicity in brain EC. In this work, we found that LPC caused cytosolic Ca²⁺ overload, mitochondrial membrane potential decrease, p38 activation, caspase 3 activation and eventually apoptotic death in mouse cerebral bEND.3 EC. In contrast to reported reactive oxygen species (ROS) generation by LPC in other EC, LPC did not trigger ROS formation in bEND.3 cells. Pharmacological inhibition of p38 alleviated LPC‐inflicted cell death. We examined whether heparin could be cytoprotective: although it could not suppress LPC‐triggered Ca²⁺ signal, p38 activation and mitochondrial membrane potential drop, it did suppress LPC‐induced caspase 3 activation and alleviate LPC‐inflicted cytotoxicity. Our data suggest LPC apoptotic death mechanisms in bEND.3 might involve mitochondrial membrane potential decrease and p38 activation. Heparin is protective against LPC cytotoxicity and might intervene steps between mitochondrial membrane potential drop/p38 activation and caspase 3 activation.     

Junctional epidermolysis bullosa keratinocytes in culture display adhesive, structural, and functional abnormalities.

An unusual, elongated, refractile cell morphology was observed in keratinocytes cultured from three patients with non-lethalis forms of junctional epidermolysis bullosa (JEB). To determine whether these changes might be related to altered cell adhesion, keratinocyte strains established from one patient were examined for adhesive, structural, and functional characteristics. JEB keratinocytes expressed keratin tonofilaments, as determined by staining with AE1 monoclonal antibodies and direct observation of tonofilaments by electron microscopy. JEB keratinocytes showed diminished cell-substratum adhesions, judged by interference reflection microscopy. Areas of diminished cell-substratum adhesion corresponded to F-actin-rich cell adhesions (focal adhesions) and not to cellular areas that abundantly express hemidesmosomal antigens. Analysis of cell-substratum adhesion by electron microscopy revealed extensive areas of cell-substratum separation in JEB keratinocytes that were not present in normal keratinocytes maintained in serum-free medium. Normal keratinocytes displayed numerous regions of focal contact between the ventral plasma membrane and the culture substratum, but these structures were not seen in JEB keratinocytes. Bundled actin filaments (stress fibers) were greatly diminished in expected regions of cell-substratum adhesion in JEB keratinocytes and, instead, displayed disorganized individual filaments. The growth rate of JEB keratinocytes was quite slow in culture, with a population doubling time of 2.7 d versus 1.5 d for normal keratinocytes under identical conditions. JEB keratinocytes also displayed a reduced ability to aggregate into colonies upon exposure to medium with increased extracellular calcium. JEB keratinocytes thus display adhesive, structural, and functional abnormalities that suggest this cell type may be central to the pathogenesis of junctional epidermolysis bullosa. Study of affected keratinocytes could be important to characterize associated molecular pathologies.     

Hemagglutination and proteoglycan binding by the Lyme disease spirochete, Borrelia burgdorferi.

The ability of the Lyme disease spirochete to attach to host components may contribute to its ability to infect diverse tissues. We present evidence that the Lyme disease spirochete expresses a lectin activity that promotes agglutination of erythrocytes and bacterial attachment to glycosaminoglycans. Among a diverse collection of 21 strains of Lyme disease spirochete, hemagglutinating activity was easily detected in all but 3 strains, and these three strains were noninfectious. The ability to agglutinate erythrocytes was associated with the ability of the spirochete to bind to the sulfated polysaccharide dextran sulfate and to mammalian cells. Soluble dextran sulfate was a potent inhibitor of both hemagglutination and attachment to mammalian cells, while dextran had no effect on either activity, suggesting that dextran sulfate may inhibit attachment by mimicking host cell glycosaminoglycans. Consistent with this, the spirochete bound to immobilized heparin, and soluble heparin inhibited bacterial adhesion to mammalian cells. The bacterium did not bind efficiently to Vero cells treated with heparinase or heparitinase or to mutant CHO cell lines that are deficient in proteoglycan synthesis. Sulfation of glycosaminoglycans was critical for efficient bacterial recognition, as Vero cells treated with an inhibitor of sulfation, or a mutant CHO cell line that produces undersulfated heparan sulfate, did not mediate maximal spirochetal binding. Binding of the spirochete to extracellular matrix also appeared to be dependent upon this attachment pathway. These findings suggest that a glycosaminoglycan-binding activity which can be detected by hemagglutination contributes to the attachment of the Lyme disease spirochete to host cells and matrix.     

Melatonin Levels in the Gastrointestinal Tissues of Fish, Amphibians, and a Reptile

Melatonin was detected by radioimmunoassay in the gastrointestinal tract (GIT) of several species of fish (sturgeon, rainbow trout, carp), amphibians (axolotl, leopard frog, bullfrog), and one reptile (red-sided garter snake), which were sacrificed during the daytime. The highest levels of melatonin were detected in the snake [means = 1018 pg/g stomach, 328 pg/g proximal gut (PG), 511 distal gut (DG)] and carp (means = 102 pg/g stomach, 146 pg/g PG and 141 pg/g DG). Lowest levels were found in the axolotl (means = 44 pg/g stomach and PG, 92 pg/g DG) and the bullfrog (means = 73 pg/g esophagus, 78 pg/g stomach, 20 pg/g PG, and 152 pg/g DG). In most cases there were no statistically significant differences in the melatonin levels among various GIT tissues of the same species but there were differences in tissue levels between different species.