Binding of the Neuronal Protein B-50, but Not the Metabolite B-60, to Calmodulin

Protein kinase C phosphorylates the neurone-specific protein B-50 at a single Ser41 residue, which is also the point for a major proteolytic cleavage in vitro, and probably in vivo, that produces a B-50 phosphorylation-inhibiting N-terminal fragment and a large C-terminal metabolite B-60 (B-50(41-226]. The intact purified protein will bind to calmodulin in the absence of calcium, but the interaction has an absolute requirement for dephospho-B-50. In an attempt to unify two aspects of B-50 biochemistry, we have examined the interaction of B-50 binding to calmodulin and B-50 proteolysis. HPLC- and affinity-purified B-50 bound to calmodulin, but purified B-60 did not. To ensure that this effect was not due to the phosphorylation state of pure, isolated B-60, the metabolite was generated in vitro using a Triton extract of synaptosomal plasma membranes, which contains the as yet uncharacterized B-50 protease. B-60 derived from dephospho-B-50 also failed to bind calmodulin. The results demonstrate a direct connection between B-50 binding to calmodulin and B-50 proteolysis. The position of the proposed calmodulin-binding domain within intact B-50 is discussed in light of the failure of calmodulin to bind B-60.     

Yeasts in molecular biology. Spheroplast preparation withCanadida utilis,Schizosaccharomyces pombe andSaccharomyces cerevisiae

Efficient preparation of spheroplasts fromCandida utilis, Saccharomyces cerevisiae, andSchizosaccharomyces pombe, using a purified mixture of enzymes fromTrichoderma harzianum, is described. Limitations of other methods, and differences between yeasts are demonstrated.     

Connexin43 Is Another Gap Junction Protein in the Peripheral Nervous System

Abstract: That many cells express more than one connexin (Cx) led us to examine whether Cxs other than Cx32 are expressed in the PNS. In addition to Cx32 mRNA, Cx43 and Cx26 mRNAs were detected in rat sciatic nerve by northern blot analysis. Cx43 mRNA, but not Cx26 mRNA, was expressed in both the primary Schwann cell culture and immortalized Schwann cell line (T93). The steady-state levels of the Cx43 mRNA in the primary Schwann cell culture increased 2.0-fold with 100 µ M forskolin, whereas that of P₀ increased 7.0-fold. Immunoreactivity to Cx43 was detected on western blots of cultured Schwann cells, T93 cells, and sciatic nerves but not on blots of PNS myelin. Immunohistochemical study using human peripheral nerves revealed that anti-Cx43 antibody stained cytoplasm around nucleus of Schwann cells but not myelin, confirming western blot results. Although P₀ expression was markedly decreased by crush injury of the sciatic nerves, Cx43 expression showed no apparent change. Developmental profiles showed that Cx43 expression in the sciatic nerve increased rapidly after birth, peaked at about postnatal day 6, and then decreased gradually to a low level. In adult rats, the Cx43 mRNA value was much lower than that of Cx32. These findings suggest that Cx43 is localized in Schwann cell bodies and that, compared with P₀, its expression is less influenced by axonal contact and cyclic AMP levels. The high expression on postnatal day 6 indicates that Cx43 may be related to PNS myelination. Cx43 is another gap junction, but its function appears to differ from that of Cx32, as judged by the differences in their localization and developmental profiles.     

A 43 kDa protein inserted at the plasma membrane-cytoskeleton interface in rumen entodiniomorphid ciliates

Summary The P-43 ofEudiplodinium and homologous proteins in three other entodiniomorphid species, free-living ciliates, flagellates, and HeLa cells, were identified at the plasma membrane-cytoskeleton interface. Proteins cross-reacting with MAb B6 were also located at the ciliary inner surface of the plasma membrane. Due to the strong adhesion of the plasma membrane to the underlying cytoskeleton, classical extraction with detergents, urea, NaOH, and PTA, failed to separate the two components completely. However, the extraction properties of P-43, associated with its membrane-cytoskeleton interactive functions, suggest that this unglycosylated protein may present some analogies with proteins of the intermediate filaments. Their ubiquity and localization suggest that P-43 and MAb B6 crossreacting proteins may not be strictly epiplasmic but could be amphitropic proteins, strongly anchored to both the plasma membrane and the underlying microfilament framework, via protein-protein binding or by direct insertion in the lipid bilayer.Abbreviations BSA bovine serum albumin - Con A concanavalin A - EDTA ethylene diamine tetraacetic acid - EM electron microscopy - IF intermediate filaments - MAb monoclonal antibody - MET 2-mercaptoethanol - MW molecular weight - PAb polyclonal antibody - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PTA phosphotungstic acid - SDS sodiumdodecyl sulfate - TAME Na-p-tosyl-arginine methyl ester - TLCK Na-p-tosyl-lysine chloromethyl ketone     

Studies on the Role of B-50 (GAP-43) in the Mechanism of Ca2+-Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca²⁺-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca²⁺ concentration from 10^?8 to 10^?5M Ca²⁺ The Ca²⁺ sensitivity in the micromolar range is identical for [³H]NA and endogenous NA release, indicating that Ca²⁺-induced [³H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca²⁺-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca²⁺-induced NA release. PKC pseudosubstrate PKC_19-36, which inhibited B-50 phosphorylation (IC₅₀ value, 10^?5M), failed to inhibit Ca²⁺-induced NA release, even when added before the Ca²⁺ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca²⁺-induced NA release. Concerning the Ca²⁺ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca²⁺ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca²⁺- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca²⁺ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca²⁺ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca²⁺ influx resulting in exocytosis of NA.     

Epidermal Growth Factor Inhibits Gap Junctional Communication and Stimulates Serine-Phosphorylation of Connexin43 in WB Cells by a Protein Kinase C-Independent Mechanism

Epidermal growth factor (EGF) stimulated the phosphorylation of connexin43 (Cx43) in WB cells as evidenced by the formation of multiple irnmunoreactive Cx43 proteins of higher molecular mass which were abolished by treatment with alkaline phosphatase. Phosphorylation of Cx43 occurred within 10 min of EGF stimulation, was sustained for 1 h, and was associated with almost complete inhibition of gap junctional communication in these cells. EGF-induced phosphorylation and communication inhibition were retained in cells pretreated with phorbol 12-myristate 13-acetate (PMA) to deplete protein kinase C. These results show that the EGF inhibition of communication is tightly linked to protein kinase C-independent phosphorylation of Cx43. Further, Cx43 phosphorylated in the presence of EGF did not react with phosphotyrosine antibodies and in ³²P_i incorporation experiments was shown to contain only phosphoserine indicating that the tyrosine kinase activity of the EGF receptor was not directly involved.     

Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43

The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mm 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mm 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1–3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mm 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mm octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3–5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 m monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mm 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.We acknowledge the technical assistance of Richard Lewis and Meghan Abella. We thank Dr. Hugh Dookwah for contributions to the myometrial cell isolation protocol and Drs. Stephen H. Safe, Timothy D. Phillips, and Evelyn Tiffany-Castiglioni for helpful discussions. This work was funded by NIH (HD-26182, P42-ES04917, ES05871-01A1), the March of Dimes Birth Defects Foundation Basic Research grant #1-0796, and USDA 92-37203-7952.     

Glycoforms of serum α1-acid glycoprotein as markers of inflammation and cancer

1-acid glycoprotein (AGP) is a serum acute phase glycoprotein which possesses five N-linked complex type heteroglycan side chains which may be present as bi-, tri- and tetraantennary structures. Depending upon the content of biantennary structure on AGP, up to four glycoforms of AGP are present in serum. These glycoforms can be easily estimated in body fluids by means of crossed affinity-immunoelectrophoresis (CAIE) with the lectin, Concanavalin A (Con A). Con A selectively binds biantennary structures; the more biantennary structures on AGP, the stronger the binding. In acute inflammation, a relative increase of AGP glycoforms with biantennary units is observed - a type I glycosylation change. In some chronic inflammatory states there is an relative decrease of AGP glycoforms with biantennary heteroglycans — a type II glycosylation change. Moreover, in certain other states such as pregnancy, estrogen administration or liver damage, type II glycosylation changes are also seen. A detailed analysis of the clinical applications of the assessment of AGP glycoforms in sera of patients with rheumatic diseases, AIDS and various types of cancers is presented. Accumulated data shows that AGP glycoforms may be very useful in the detection of intercurrent infections in the course of rheumatoid arthritis, systemic lupus erythematosus, or myeloblastic leukaemia, and in the detection of secondary infections in human immunodeficiency virus infected individuals. AGP glycoforms are also very useful in differentiation between various forms of trophoblastic disease and are helpful in monitoring the treatment of these patients. Finally, AGP glycoforms provide valuable information for differentiation between primary and secondary liver cancer.     

Synaptic Vesicle Recycling in Cultured Cerebellar Granule Cells: Role of Vesicular Acidification and Refilling

Abstract: The role of the transvesicular protonmotive force in synaptic vesicle recycling was investigated in cultured cerebellar granule cells. The vesicular V-ATPase was inhibited by 1 µ M bafilomycin A1; as an alternative, the pH component of the gradient was selectively collapsed by equilibration of the cells with 10 m M methylamine and monitored with the fluorescent probe Lysosensor Green. Electrical field-evoked exocytosis of d -[³H]aspartate was inhibited by bafilomycin A1 but not by methylamine, indicating that a transvesicular membrane potential rather than pH gradient is required for transmitter retention within vesicles. In contrast, neither compound affected the field-evoked uptake, recycling, or destaining of the vesicle-specific dye FM2-10; thus, vesicles whose lumens were neutral and/or depleted of transmitter could still recycle in the nerve terminal. No exhaustion of d -[³H]aspartate exocytosis was observed when cells were subjected to six consecutive trains of field stimuli (40 Hz/10 s separated by 10 s). In contrast, the release of preloaded FM2-10 was reduced by ∼50%, with each stimulus indicating that unlabeled vesicles with accumulated d -[³H]aspartate were competing with labeled vesicles for exocytosis. As d -[³H]aspartate was accumulated rapidly across the vesicle membrane from the large cytoplasmic pool, the transmitter-loaded but unlabelled vesicles may represent refilled recycling vesicles. FM2-10 destaining and d -[³H]aspartate exocytosis were reduced in parallel at low frequencies, challenging a role for transient vesicle fusion.     

Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth

Summary Incorporation of the gene for connexin 43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with normal cells the growth of the connexin overexpressors was completely arrested, as these cells established strong communication with the normal ones. Thus, in culture by themselves or in coculture, the connexin overexpressor cells grew like normal cells. These results demonstrate that the cell-cell channel is instrumental in growth control; they are the expected behavior if the channel transmits cytoplasmic growth-regulatory signals.