Detection of multiple forms of human ceruloplasmin. A novel Mr 200,000 form

Three polypeptides with apparent Mr = 200,000, 135,000, and 115,000, reacting with antibody to human ceruloplasmin (Cp), were consistently found in sera of normal adult and newborn subjects, patients with Wilson's disease, as well as in the oxidase-active fraction of purified human Cp, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentrations of the three Cp polypeptides were proportional to the total Cp oxidase activity measured in whole serum. Peptide mapping revealed that the three Cp polypeptides were closely related. Cross-linking of Cp135 resulted in dimers with electrophoretic mobility similar to that of Cp200. A common shift in electrophoretic mobility following N-glycanase treatment indicated that all three polypeptides were N-glycosylated, and that the apparent differences in molecular mass could not be related to the carbohydrate moiety. Immunoprecipitates of cell lysates of [35S]cysteine labeled HepG2 cells revealed the presence of two species of newly synthesized Cp polypeptides, Mr 200,000 and 135,000, which were secreted into the media. Secretion of Cp200 by the human liver appears to be physiologic and may be the result of posttranslational modification of Cp135.     

Evidence for the presence of diacylglycerol kinase in rat brain myelin

The previous demonstration that incubation of brain slices with [³²P]phosphate brings about rapid tabeling of phosphatidic acid in myelin suggests that the enzyme involved should be present in this specialized membrane. DAG kinase (ATP:1,2-diacyglycerol 3-phosphotransferase, E.C. 2.7.1.107) is present in rat brain homogenate at a specific activity of 2.5 nmol phosphatidic acid formed/min/mg protein, while highly purified myelin had a much lower specific activity (0.29 nmol/min/mg protein). Nevertheless, the enzyme appears to be intrinsic to this membrane since it can not be removed by washing with a variety of detergents or chelating agents, and it could not be accounted for as contamination by another subcellular fraction. Production of endogenous, membrane-associated, diacylglycerol (DAG) by PLC (phospholipase C) treatment brought about translocation from soluble to particulate fractions, including myelin. Another level of control of activity involves inactivation by phosphorylation; a 10 min incubation of brain homogenate with ATP resulted in a large decrease in DAG kinase activity in soluble, particulate and myelin fractions.     

Acrylamide-Induced Increases in Deposition of Axonally Transported Glycoproteins in Rat Sciatic Nerve

The axonal transport of proteins, glycoproteins, and gangliosides in sensory neurons of the sciatic nerve was examined in adult rats exposed to acrylamide via intraperitoneal injection (40 mg/kg of body weight/day for nine consecutive days). The L5 dorsal root ganglion was injected with either [35S]methionine to label proteins or [3H]glucosamine to label, more specifically, glycoproteins and gangliosides. At times ranging from 2 to 6 h later, the sciatic nerve and injected ganglion were excised and radioactivity in consecutive 5-mm segments determined. In both control and acrylamide-treated animals, outflow profiles of [35S]methionine-labeled proteins showed a well defined crest which moved down the nerve at a rate of approximately 340 mm/day. Similar outflow profiles and transport rates were seen for [3H]glucosamine-labeled glycoproteins in control animals. However, in animals treated with acrylamide, the crest of transported labeled glycoprotein was severely attenuated as it moved down the nerve. This finding suggests that in acrylamide-treated animals, axonally transported glycoproteins were preferentially transferred (unloaded or exchanged against unlabeled molecules) from the transport vector to stationary axonal structures. We also examined the clearance of axonally transported glycoproteins distal to a ligature on the nerve. The observed impairment of clearance in acrylamide-treated animals relative to controls is supportive of the above hypothesis. Acrylamide may directly affect the mechanism by which axonally transported material is unloaded from the transport vector. Alternatively, the increased rate of unloading might reflect an acrylamide-induced increase in the demand for axonally transported material.     

Bradykinin Stimulates Phosphoinositide Hydrolysis and Mobilization of Arachidonic Acid in Dorsal Root Ganglion Neurons

Cultures of fetal rat dorsal root ganglion neurons (7 days in culture) were prelabeled with myo-[3H]inositol or [3H]arachidonic acid for 24 h and stimulated with 10 microM bradykinin for time intervals of 5-300 s. The incubation was terminated by addition of 5% perchloric acid to extract inositol phosphates or organic solvent to extract lipids. Inositol phosphates were resolved by anion-exchange HPLC; lipids were resolved by TLC. Bradykinin stimulation resulted in a 10-fold increased accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol bisphosphate (IP2) (fivefold) by 5 s. The increase in IP3 was transient (half maximal by 1 min), whereas stimulated IP2 levels were sustained for several minutes. Even longer term increases were observed in inositol monophosphate. Stimulation also resulted in a threefold increase in arachidonic acid which was preceded by transient increases in diacylglycerol (twofold) and arachidonoyl-monoacylglycerol (threefold). The temporal lag in the accumulation of arachidonic acid with respect to diglyceride and monoglyceride suggested the involvement of di- and monoglyceride lipases in arachidonic acid mobilization. A role for phospholipase A2 is also possible, because pretreatment of cultures with quinacrine partially blocked arachidonic acid release. Bradykinin-stimulated arachidonic acid release was decreased in the presence of calcium channel blockers nifedipine or verapamil (50 microM), or EDTA (2.5 mM). The role of calcium was verified further in that accumulation of phosphatidic acid, diacylglycerol, and arachidonic acid was maximally stimulated by treatment with the calcium ionophore A23187 (20 microM).     

Inhibitory effects of ATP and other nucleotides on atrial natriuretic peptide (ANP) binding to R1-type ANP receptors in human neuroblastoma NB-OK-1 cell membranes.

ATP dose-dependently inhibited rat 125I-ANP-(99-126) binding to membranes from the human neuroblastoma cell line NB-OK-1 by increasing the KD value for the hormone without altering the Bmax value. After a 20 min preincubation with 37.5 pM 125I-ANP-(99-126) and 0.5 mM ATP, followed by the addition of 0.3 microM unlabelled ANP-(99-126), the proportion of rapidly dissociating receptors was 4-times higher than in the absence of ATP. The other nucleotides ADP, AMP, AMP-PNP, ATP gamma S, GTP, GDP, GMP, GMP-PNP and GTP gamma S were also inhibitory but with a lower potency and/or efficacy. Binding equilibrium data were satisfactorily simulated by a computer program based on partially competitive binding of ANP-(99-126) and the nucleotides, and this, together with the data on dissociation kinetics, strongly suggests that several nucleotides, when added at concentrations up to 1 mM, form a ternary ANP-receptor-nucleotide complex.     

Rat PHI, PHI-GLY and PHV (1-42) stimulate adenylate cyclase in six rat tissue and cell membranes

PHI and the two C-terminally extended forms PHI-GLY and PHV(1-42) coexist in rat tissues. We compared the relative potency and efficacy of these three PHI forms and of VIP to stimulate adenylate cyclase activity and, when feasible, to occupy VIP receptors in six rat tissue and cell membranes. With the exception of lung membranes, all three PHI forms were markedly less potent than VIP but all were systematically as efficacious. PHI-GLY and PHV(1-42) were never more potent than PHI itself and their relative potencies revealed four spectra, depending on the membrane preparation tested: 1) PHI = PHI-GLY = PHV(1-42) in hepatic, pulmonary and pancreatic membranes; 2) PHI greater than PHV(1-42) = PHI-GLY in membranes from circulating lymphocytes; 3) PHI = PHV(1-42) greater than PHI-GLY in membranes from the thymocyte cell line 51E; and 4) PHI greater than PHI-GLY = PHV(1-42) in anterior pituitary membranes. These results indicate that the two naturally observed C-terminal extensions of rat PHI variously affected peptide potency on 6 rat membrane preparations.     

Modulation of Expression and Assembly of Vinculin duringin VitroFibrillar Collagen-Induced Angiogenesis and Its Reversal

A model of collagen-inducedin vitroangiogenesis was used to investigate the modulation of expression and assembly of focal adhesion plaque-associated proteins during the process of differentiation. Human umbilical vein endothelial cells (HUVEC), first attached on an adhesive substratum (gelatin-, fibronectin-, or laminin-coated dish) or adherent collagen gel and then covered by an overlaying collagen gel, organized within 3–4 days in tube-like structures (TLS). Removing the overlaying collagen gel from fully differentiated HUVEC induced a reversion of the process and HUVEC returned to a monolayer pattern. Modulations of focal adhesion-associated proteins occurring in HUVEC during thein vitrodifferentiation process and its reversal were investigated by Western blot analysis. A significant decrease of expression of vinculin, the integrin α₂subunit, talin, α-actinin, and actin was observed in TLS whereas the amount of FVIII-related antigen did not vary as compared to control monolayer cultures. During reversal, all the reduced proteins were markedly reexpressed. Human skin fibroblasts (HSF), submitted to the same experimental conditions, did not form TLS. Most of the focal adhesion proteins in HSF were similarly modulated by an overlaying collagen gel with the exception of vinculin, which was not modified. This particular protein was therefore more thoroughly investigated. In a nondifferentiated monolayer of HUVEC, a significant proportion of vinculin was organized into a detergent-resistant juxtamembranous structure (focal adhesion plaque) which disassembled early in TLS formation and reassembled during the reversal of the process. The reduction of vinculin during TLS formation was preceded by a downregulation of its mRNA while this mRNA was upregulated during reversal of the morphotype. These results suggest that the modulations of the cytoskeletal and focal adhesion proteins and more specifically of vinculin coupled to its subcellular redistribution are critical and early events in the cascade of mechanochemical signaling duringin vitroangiogenesis induced by fibrillar collagen.     

Origin of cholesterol in myelin

We review some of the older literature concerning metabolic turnover of cholesterol in the nervous system. The overall picture is that incorporation of radioactive precursors into brain cholesterol is roughly proportional to the rate of myelination and that, once incorporated, radioactive cholesterol is relatively stable metabolically. We outline a strategy for demonstrating the source (local synthesis or uptake from the circulation) of cholesterol in brain. The experimental design involves determining the rate of accumulation of cholesterol this is calculated as the increasing amounts of sterol in brain at successive time intervals during development. The rate of appearance of newly synthesized cholesterol is determined from incorporation of radioactivity from³H₂O (injected i.p. several hours prior to sacrifice) into cholesterol. The radioactivity associated with the sterol fractions and the specific activity of body water determined from the serum can be used to calculate the absolute amount of sterol newly synthesized during the time when³H₂O was present. The results obtained demonstrated that all of the bulk cholesterol accumulating in brain can be accounted for by newly synthesized cholesterol. None of the radioactive cholesterol came from the circulation, since cholesterol feeding suppressed cholesterol biosynthesis in the liver and specific radioactivity of circulating cholesterol was negligible. Thus, almost all cholesterol accumulating in brain during development is locally synthesized. Special issue dedicated to Dr. Marion R. Smith.