Robert H. Warren

Adrenergic and Cholinergic Receptors of Cerebral Microvessels

The presence of α- and β-adrenergic and muscarinic cholinergic receptors in cerebral microvessels of the rat and pig was assessed by ligand binding techniques. The results demonstrate the presence of specific binding to α2- and β-adrenergic receptors but no appreciable specific binding to α1-adrenergic or muscarinic cholinergic receptors. β-Adrenergic receptors of pig cerebral microvessels are similar to those of the brain and other organs in their binding characteristics to the tritiated ligand and in their stereospecificity of binding to the biologically active isomers of β-adrenergic agonists. Further evidence derived from the differential potency of binding displacement by the various β-adrenergic agonists and selective β1- and β2-adrenergic antagonists indicates that β-adrenergic receptors of pig cerebral microvessels are mostly of the β2-subtype.

Specificity of cell-cell interactions in sea urchin embryos: Appearance of new cell-surface determinants at gastrulation

Abstract Studies on normal and hybrid sea urchin embryos show that, beginning at gastrulation, hybrid cells express cell-surface antigens specific to both species. The appearance of these antigens is shown to be correlated with a change in the adhesive specificity of hybrid cells: Beginning at gastrulation, hybrid cells recognize and adhere to embryonic cells of both normal genotypes. Prior to gastrulation, hybrid cells adhere to cells of the maternal genotype only. Two adhesion assays demonstrate these adhesive preferences. (i) When cell aggregates are placed together in a dish, Lytechnius aggregates fuse together, and Tripneustes aggregates fuse together, but aggregates of the two species do not fuse with each other. Hybrid cell aggregates, if they are past the beginning of gastrulation, fuse to both Tripneustes and Lytechinus aggregates. (ii) In a collection assay, midgastrula cells of the hybrid embryos are collected at a high rate to aggregates of either species. Pregastrula hybrid cells collect at a high rate to aggregates of the maternal species only. This change in adhesive preference is temporally correlated with the appearance of new cell surface antigens. Antiserum was prepared in rabbits against membranes from Lytechinus gastrulae. Indirect immunofluorescence tests show that hybrid cells of the cross (T♀ × L♂) express Lytechinus -specific antigens at the cell surface beginning at gastrulation. Furthermore, an apparent relationship between the new cell-surface antigens and adhesion exists in that Lytechinus cell adhesion is inhibited specifically after binding Fab fragments of the Lytechinus antiserum. The antiserum has no effect on Tripneustes adhesion. The Lytechinus adhesion-inhibiting activity can be removed by absorption of the antiserum with Lytechinus cells.

Cortical reorganization following fertilization of sea urchin eggs: Sensitivity to cytochalasin B

A transient sensitivity to cytochalasin B was detected in freshly fertilized sea urchin eggs. Large membrane-limited protrusions formed when cytochalasin B was added between 1 and 6 min postinsemination. When cytochalasin B was removed from the cultures, eggs continued to develop with protrusions remaining, and cleavage furrows formed through many of the protrusions. Electron microscopy showed that cytochalasin B disrupted the microfilamentous structures that formed in the egg cortex after fertilization. Protrusions also appeared when eggs were activated by the Ca 2+ ionophore A23187 followed by cytochalasin B addition. Protrusions did not appear when cytochalasin B was added at any time prior to fertilization, during insemination, or after the 6-min period postinsemination. Mechanical compression or incubation in a cortical isolation medium were effective as treatments enhance the cytochalasin B-sensitive response. These results suggest a time frame for postfertilization changes in the cortex of the zygote.

The BAG-1 isoform BAG-1M regulates keratin-associated Hsp70 chaperoning of aPKC in intestinal cells during activation of inflammatory signaling

ABSTRACT Atypical PKC (&igr;/&lgr; and &zgr;; hereafter referred to as aPKC) is a key player in the acquisition of epithelial polarity and participates in other signaling cascades including the control of NF-&kgr;B signaling. This kinase is post-translationally regulated through Hsp70-mediated refolding. Previous work has shown that such a chaperoning activity is specifically localized to keratin intermediate filaments. Our work was performed with the goal of identifying the molecule(s) that block Hsp70 activity on keratin filaments during inflammation. A transcriptional screen allowed us to focus on BAG-1, a multi-functional protein that assists Hsp70 in nucleotide exchange but also blocks its activity at higher concentrations. We found the BAG-1 isoform BAG-1M upregulated threefold in human Caco-2 cells following stimulation with tumor necrosis factor receptor &agr; (TNF&agr;) to induce a pro-inflammatory response, and up to sixfold in mouse enterocytes following treatment with dextran sodium sulfate (DSS) to induce colitis. BAG-1M, but no other isoform, was found to co-purify with intermediate filaments and block Hsp70 activity in the keratin fraction but not in the soluble fraction within the range of concentrations found in epithelial cells cultured under control and inflammation conditions. Constitutive expression of BAG-1M decreased levels of phosphorylated aPKC. By contrast, knockdown of BAG-1, blocked the TNF&agr;-induced decrease of phosphorylated aPKC. We conclude that BAG-1M mediates Hsp70 inhibition downstream of NF-&kgr;B.

Microtubules and actin in giant nerve fibers of the spiny lobster, Panulirus argus.

Giant axons of the spiny lobster, Panulirus argus, are filled with microtubules that are decorated with fine, irregular filaments. Mitochondria and membrane-limited clear vesicles are the only other distinguishable elements in the axoplasm and are located around the periphery of the axon near the axolemma. Neither 100 A neurofilaments nor 70 A microfilaments are evident in fixed, intact axons or in negatively stained axoplasm. Actin-like microfilaments are a prominent constituent of the glial cells that closely ensheathe the axons, and gel electrophoresis studies suggest that most of the actin in the nerve fibers is located in the glia rather than in the axons. Studies of isolated axoplasm indicate that microtubules are the primary elements stabilizing the axoplasm. The microtubules in the isolated axoplasm are disrupted by Ca2+ in the medium in the presence of protease inhibitors.

Myoblasts are aligned with collagen fibrils in regenerating frog tadpole tails

Myoblasts in the regenerating frog tadpole tail differentiate from mesenchymal cells that lie next to the basement membrane of the epidermis of the tail. As these cells elongate and form myotubes, they orientate uniformly in the longitudinal axis of the tail. The collagen fibrils of the basement membrane adjacent to the myogenic cells are also orientated in the tail axis just prior to and during the time when the myogenic cells are elongating. This has been demonstrated by transmission electron microscopy of thin sections, by differential interference contrast microscopy of isolated basement membranes, and by scanning electron microscopy of the inner surface of the basement membrane. Since elongating myoblasts are in contact with the longitudinally orientated fibrils, the latter could provide directional cues to the elongating myoblasts. This proposition is supported by the finding that isolated basement membranes readily orientate cells that are cultured upon their inner surfaces.