Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor

mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma-aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels.     

Active muscle migration during insect metamorphosis

The R1 abdominal retractor muscles of the insect Tenebrio molitor change position during the course of metamorphosis. These muscles detach from the epidermal tendon cells at their anterior ends, and migrate in a posterior direction, parallel to the body axis, to form completely new attachments shortly before adult emergence. Movement is preceded by the loss of sarcomere structure, and the muscles migrate in a partially dedifferentiated condition, closely accompanied by satellite cells and haemocytes. Movement appears to result from the extension of muscle processes towards the epidermis posterior to the larval attachment sites, which contact reciprocal processes extended from the epidermis. Contacts at the new posterior sites are then reinforced, and relinquished at the anterior. This cycle is subsequently repeated. It is envisaged that migration ceases when the muscles encounter a contour in the epidermal gradient known to specify the position of the adult muscle attachment sites. This positional information may be encoded in the epidermal basal lamina. The muscles then redifferentiate, with concurrent differentiation of new epidermal tendon cells. Development of adult muscle attachments appears to require reciprocal morphogenetic interactions between muscle and epidermis.     

Formation of junctional complexes at sites of contact of hemocytes with tissue elements in degenerating nerves of the crayfish, Orconectes virilis

Hemocytes, which contain large cytoplasmic granules, invade the multilamellate glial sheath of ventral ganglion nerve roots of the crayfish following surgical interruption of these nerves. Electron microscopic examination of sections of plasticembedded tissues and replicas of freeze-cleaved ganglion roots reveals numerous slender cytoplasmic extensions of the hemocytes present in damaged nerve sheaths. Many of these microvillous extensions contact glial cells and filamentous extracellular masses. At sites of contact, the microvilli are flattened and occasionally electron-dense material is present in the hemocyte cytoplasm subjacent to the plasma membrane that is closely apposed to a glial cell or connective tissue. Intramembranous surfaces of hemocyte plasmalemmae exposed by freeze-fracture, exhibit particle aggregates 700–2500 Å in diameter. Individual particles are 95–105 Å in diameter. Since the particle aggregates correspond in overall dimension and position in the cell to the sites of contact of hemocyte processes with other sheath components, it is assumed that the two structures are equivalent and represent a junctional complex very similar in structure to some hemidesmosomes. Results from this study strongly suggest that granulated crustacean hemocytes, in response to surgical injury of nerves, invade the damaged nerve sheath and identify damaged glial cells and connective tissue by forming slender cytoplasmic processes which contact elements of the sheath. Tissue components contacted by the hemocytes may subsequently be phagocytosed by them. This is the first report of an invertebrate hemocyte-mediated response to tissue damage in which evidence is presented that the hemocyte may identify necrotic cells and extracellular matrix by forming junctional complexes with them. Crustacean hemocytes, therefore, are likely much more complex functionally than has been previously estimated.     

Primary culture of capillary endothelium from rat brain

To provide an in vitro system for studies of brain capillary function we developed a method for culture of brain capillary endothelial cells. Capillaries were isolated from rat brain and enzymatically treated to remove the basement membrane and contaminating pericytes. Subsequent Percoll gradient centrifugation resulted in a homogeneous population of capillary endothelial cells that attached to a collagen substrate and incorporated [3H]thymidine. Evidence for the endothelial nature of these cells was provided by the presence of Factor VIII antigen and angiotensin converting enzyme activity and by the failure of platelets to adhere to the cell surface. In addition, the cells were joined together by tight junctions. Thus, primary cultures of these cells retained both endothelial and blood-brain barrier features.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.     

Ligation of CD28 by its natural ligand CD86 in the absence of TCR stimulation induces lipid raft polarization in human CD4 T cells

Stimulation of resting CD4 T cells with anti-CD3/CD28-coated beads leads to rapid polarization of lipid rafts (LRs). It has been postulated that a major role of costimulation is to facilitate LR aggregation. CD86 is up-regulated or expressed aberrantly on immune cells in a wide array of autoimmune and infectious diseases. Using an Ig fusion with the extracellular domain of CD86 (CD86Ig) bound to a magnetic bead or K562 cells expressing CD86, we demonstrated that ligation of CD28 by its natural ligand, but not by Ab, induced polarization of LRs at the cell-bead interface of fresh human CD4 T cells in the absence of TCR ligation. This correlated with activation of Vav-1, increase of the intracellular calcium concentration, and nuclear translocation of NF-kappaB p65, but did not result in T cell proliferation or cytokine production. These studies show, for the first time, that LR polarization can occur in the absence of TCR triggering, driven solely by the CD28/CD86 interaction. This result has implications for mechanisms of T cell activation. Abnormalities in this process may alter T and B cell tolerance and susceptibility to infection.