Search

Immunocytochemical distribution of the fetal protein fetuin in the neocortex of developing rat brain and the presence of its mRNA, as detected by using reverse transcriptase‐polymerase chain reaction analysis, was studied in fetuses at embryonic day 15 (E15) through E22, in neonates at postnatal day 0 (P0) through P20, and in adults. Quantitative estimates of fetuin in cerebrospinal fluid (CSF) and plasma were obtained over the same period. Exogenous (bovine) fetuin injected intraperitoneally into fetal and postnatal rats was used to study the uptake of fetuin into CSF and brain and its distribution compared with endogenous fetuin; bovine albumin was used as a control. Fetuin was identified immunocytochemically in the cortical plate and subplate cells of the developing neocortex. In the rat fetus, fetuin first was apparent at E17, mainly in cell processes, but a few subplate cells also were positive. By E18, there was strong staining in subplate neurons and in inner cells of the cortical plate. At E21, these inner cells of the cortical plate were beginning to differentiate into layer VI neurons, many of which were positive for fetuin. By P0–P1, more layer VI neurons and some layer V neurons had become positive for fetuin. Fetuin immunoreactivity generally was weaker at P1, and, by P2–P3, it had disappeared from all of the layers of the developing neocortex. Bovine fetuin (but not albumin), probably taken up through CSF over the neocortical dorsal surface, had a cytoplasmic distribution; endogenous rat fetuin was both cytoplasmic and membrane bound. Thus, much of this fetuin can be accounted for by uptake, although the presence of fetuin mRNA indicates that in situ synthesis may also contribute. J. Comp. Neurol. 423:373–388, 2000. © 2000 Wiley‐Liss, Inc.

The last of a series on the popular operating system for personal computers concentrates on the inner core of CP-M, the BIOS. An example of BIOS is given for an older microcomputer, the IMSAI 8080. Instructions for modification of the printer driver subroutine in BIOS are given. A simple printer driver is created as a demonstration. Merging of new and modified parts of CP-M is covered. Program listings are given for a sample BIOS, a new driver, and other CP-M segments. Block diagrams of the CP-M memory map and systems image are given.

Print String is an I-O system call in the CP-M operating system for microcomputers. Print String allows sending complete words and sentences to the monitor screen. Read Console Buffer (another system command) stores an edited line of console input at the address held in register pair DE. The STA instruction stores the value from register A at the address given in the STA instruction. The List Output to Printer system call sends a character to the list device which is normally a printer. The Get I-O Byte and Set I-O Byte are useful instructions when interfacing the computer system to a non-standard device like a modem.

Disk system calls are a powerful feature of the CP-M operating system. Files are made up of 128 byte records. The BDOS (Basic Disc Operating System) uses a DMA buffer of 128 bytes and a FCB (File Control Block) of 36 bytes. Make File, Open File, Close File, Random Read, and Write Random are system calls executed by BDOS. Program listings and sample output show the working of BDOS. The August, 1983 issue will conclude this introduction to CP-M with a discussion of BIOS (Basic Input Output System).

Bis(monoacylglycero)phosphate (BMP) is synthesized from exogenous phosphatidylglycerol (PG) by macrophages (Cochran, F. R., Roddick, V. L., Connor, J. R., Thornburg, J. T., and Waite, M. (1987) J. Immunol. 138, 1877-1883). Previous work from our laboratory showed that arachidonic acid in BMP was released by the macrophages upon challenge of the cells with PMA (Cochran, F. R., Connor, J. R., Roddick, V. L., and Waite, M. (1985) Biochem. Biophys. Res. Commun. 130, 800-806). Here we extend those studies using a model cultured cell line of macrophages, RAW 264.7. When PG labeled with 32P- and [3H]glycerol in both moieties was added to the culture medium, 32P/[3H]BMP was synthesized in a time-dependent manner. Fractionation of cell homogenates on a discontinuous sucrose gradient in which the light membranes were floated from dense sucrose showed an enrichment of [3H]BMP in light membrane fractions. The precursor [3H]PG was also found in the light fractions but, relative to the [3H]BMP, was more abundant in the denser membrane fractions. The appearance of [3H]PG and [3H]BMP in the light membrane fraction was time-dependent which suggested that the initial uptake and metabolism of [3H]PG was into the denser membranes. Incubation of the light membranes under conditions that are optimal for the lysosomal phospholipase A1 led to significant metabolism of [3H]PG. Both degradation of [3H]PG to water-soluble compounds and its conversion to acylphosphatidylglycerol occurred while no lyso-PG was detected. On the other hand, little BMP was found to be degraded. From these studies we postulate that in lysosomes acylphosphatidylglycerol is a precursor of BMP and that the previously reported turnover of arachidonic acid by BMP may occur via transacylation rather than hydrolysis.

Following legislative measures to ban the use of tributyltin-based anti-fouling paints on small boats in the U.K., a programme of investigations has been commissioned to examine inputs from shipping. Shipping activity has been divided into three categories for investigation: (a) commercial harbours; (b) anchorages; and (c) dry docks. Significant concentrations of TBT have been measured in harbours and at anchorages, in some cases exceeding 50 ng 1-1 TBT in the water column in the immediate vicinity of ships. However, the contribution to environmental concentrations of TBT from anti-fouled yachts caused difficulties in assessment of the inputs from ships alone at many of the sites under investigation. Dry-docking practices have been shown to result in discharge of hazardous concentrations of TBT and such activities are now subject to a voluntary code of practice.

The wettability of alumina by ternary alloys of copper, titanium and aluminium, gallium gold, indium, nickel or silver has been investigated using sessile drop tests conducted in vacuum at 1050–1250° C. Substantial additions of titanium are known to induce copper to wet alumina due to the formation of a titanium rich reaction product at the alloy/ ceramic interface, but the present work has shown that the concentration of titanium can be reduced by the addition of ternary alloying elements. Additions of indium are very beneficial, of aluminium, gold or silver are moderately beneficial, and of gallium or nickel are of negligible benefit or detrimental. These observations, and previous work with copper-tin-titanium alloys [1] can be interpreted in terms of effects on the activity of titanium which it is argued will be enhanced if the ternary alloying element has a low surface energy and is readily saturated by titanium. The correlation of the experimental wetting observations with the surface energy and titanium solubility data for the ternary alloying elements provides a basis for the rational development of reactive metal brazes for joining unmetallized ceramics.

1-O-Alkyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (i.e., platelet-activating factor) was prepared and confirmed to possess potent platelet aggregating activity. It was also potent in aggregating and degranulating rabbit and human neutrophils. When injected into rabbits, the lipid induced profound neutropenia, thrombocytopenia, and anaphylactic symptoms. The lyso derivative of this lipid, 1-O-alkyl-sn-glyceryl-3-phosphorylcholine, was inactive or several orders of magnitude weaker in inducing these responses. The acetylated lipid appears to be a potent stimulator of both platelets and neutrophils. Its anaphylactic-like toxicity may be related, at least in part, to its ability to aggregate or otherwise stimulate these cells.

Reticuloendotheliosis virus (REV) contains an endogenously instructed, RNA-directed DNA polymerase activity. Both the endogenous and exogenous DNA polymerase activities exhibited up to 10-fold greater activity at the optimum concentration of manganous ion (0.025 mM for exogenous; 0.25 mM for endogenous) than at any concentration of magnesium ion. Antiserum to the DNA polymerase of an REV group virus (spleen necrosis virus) inhibited both endogenous and exogenous DNA polymerase activity of REV, whereas antiserum to the Rous sarcoma virus (Rous-associated virus-0) [RSV(RAV-0)]DNA polymerase did not. The DNA product of the endogenous reaction is associated with the high-molecular-weight RNA of REV and anneals with REV RNA but not with RNA from Rous sarcoma virus.

The uptake and metabolism of chylomicron-remnant lipids by individual liver cell types was examined by incubating remnants with monolayer cultures of hepatocytes, Kupffer cells, and endothelial cells from rat liver. Remnants were prepared in vitro from radiolabelled mesenteric-lymph chylomicra, utilizing either purified lipoprotein lipase from bovine milk, or plasma isolated from heparinized rats. The resulting particles contained [3H]phosphatidylcholine and cholesterol, and [14C]oleate in the acylglycerol, phospholipid, fatty-acid and cholesterol-ester fractions. The capacities of the three cell types for uptake of both [3H]lipids and [14C]lipids were determined to be, on a per-cell basis, in the order: Kupffer greater than hepatocytes greater than endothelial. The relative proportions of [3H]phospholipid and total [3H]cholesterol taken up by hepatocytes and non-parenchymal cells remained constant with time. The uptake of [14C]oleoyl lipids by all three cell types was slightly greater than that of the total [3H]cholesterol and [3H]phospholipid components. There was evidence of cholesterol-ester hydrolysis and turnover of [14C]oleate in the phospholipid fraction in hepatocytes and Kupffer cells, but not endothelial cells, over the first 2 h. With both remnant preparations, these observations indicate that significant differences exist between the three major liver cell types with respect to the uptake and metabolism of remnant lipid components.

The purpose of this study was to characterize the lipolytic activities released by heparin from rat livers. Heparin perfusates of rat livers degraded monooleoylglycerol, trioleoylglycerol and phosphatidylcholine in emulsions as well as in chylomicrons, chylomicron remnants, low-density lipoprotein/high-density lipoprotein-1 (LDL/HDL-1) and high-density lipoprotein-2 (HDL-2). The preferred substrate was mono-oleoylglycerol. Heparin perfusates were separated by chromatography on either heparin-Sepharose or N-desulphated, N-acetylated heparin-Sepharose into at least two related lipases which differed in their ability to hydrolyse HDL-2 phosphatidylcholine, but not in their ability to degrade mono-oleoylglycerol, trioleoylglycerol and phosphatidylcholine in emulsions. The sodium dodecyl sulphate (SDS)/polyacrylamide-gel-electrophoretic patterns of heparin perfusates purified on either normal or N-desulphated N-acetylated heparin-Sepharose were the same, despite differences in their ability to degrade HDL-2 phosphatidylcholine. There was a single band of Mr 56000 without 2-mercaptoethanol in the SDS disruption buffer and three major bands, of Mr 62000, 59000 and 56000, with 2-mercaptoethanol present. When mono-oleoylglycerol lipase was purified 161-fold, there was a concomitant enrichment of the Mr-56000 protein.

The exchange of phosphatidylcholine and unesterified cholesterol between rat mesenteric lymph chylomicrons and human high-density lipoproteins was studied in vitro by incubation of radiolabelled chylomicrons (with [N-methyl-14C]phosphatidylcholine and [7(n)-3H]cholesterol) with unlabelled high-density lipoproteins. The kinetic analysis was based on the extent of radioisotope exchange, which was determined by the proportion of label appearing in the high-density lipoprotein elution peak after rapid fractionation on analytical agarose columns. Under our experimental conditions, no net transfer of either phosphatidylcholine or cholesterol is observed. The kinetics of exchange of both phosphatidylcholine and cholesterol are biphasic. Over the first 30 min a maximum of 25% of the phosphatidylcholine and 33% of the cholesterol in chylomicrons exchanges rapidly into the high-density-lipoprotein fraction. Thereafter both lipids continue to exchange for up to 3 h at a much lower rate. For the rapid exchange process the calculated exchange rates for phosphatidylcholine and cholesterol are proportional to the concentrations of both chylomicrons and high-density lipoproteins. The second-order rate constants are (10.5 +/- 0.5) X 10(-5) microM-1 X min-1 for phosphatidylcholine and (32.1 +/- 4.5) X 10(-5) microM-1 X min-1 for cholesterol. The kinetics of the exchange process thus suggest that a significant proportion of both phosphatidylcholine and unesterified cholesterol is rapidly exchangeable between these lipoproteins, and that this exchange is mediated by a ‘bimolecular’, or collisional, mechanism.

When cultures producing reticuloendotheliosis virus were incubated for 24 h in medium of lowered NaCl concentration, virus production was inhibited. The extent of inhibition increased as the salt concentration of the medium was decreased. The inhibition was rapidly reversed by replacement of low-salt medium with normal medium. During the first hour after the inhibited cultures were returned to normal medium, virus was released at an accelerated rate, making the total amount of virus released by inhibited and control cultures the same. After 1 h in normal medium, the rate of virus production in the previously inhibited cultures was the same as in the control cultures. Incubation of infected cells in low-salt medium resulted in a 60% decrease in the overall rate of protein synthesis. Although returning the cells to normal medium rapidly reversed the inhibition of virus production, it did not rapidly increase the rate of protein synthesis. These results suggest that host cell-directed protein synthesis is preferentially inhibited by the low-ionic-strength medium, whereas that required for virus production continues.

Many stimulators of prostaglandin production are thought to activate the Ca2+- and phospholipid-dependent protein kinase first described by Nishizuka and his colleagues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. In this paper we report evidence that the activation of protein kinase C caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) is involved in the increased prostaglandin production induced by 12-O-tetradecanoylphorbol-13-acetate in Madin-Darby canine kidney (MDCK) cells. We have shown that TPA activates protein kinase C in MDCK cells with similar dose response curve as observed for TPA induction of arachidonic acid release in MDCK cells. Activation of protein kinase C was associated with increased phosphorylation of proteins of 40,000 and 48,000 daltons. We used two compounds (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and 1-(5-isoquinolinesulfonyl)piperazine) known to inhibit protein kinase C by different mechanisms to further examine if activation of protein kinase C was involved in the increased synthesis of prostaglandins in TPA-treated MDCK cells. We found that both compounds inhibited protein kinase C partially purified from MDCK cells and that ET-18-OMe inhibited the phosphorylation of proteins by protein kinase C in the intact cells. Addition of either compound during or after TPA treatment decreased both release of arachidonic acid from phospholipids and prostaglandin synthesis. Release of [3H]arachidonic acid from phosphatidylethanolamine in TPA-treated cells was blocked by ET-18-OMe or 1-(5-isoquinolinesulfonyl)piperazine addition. However, arachidonic acid release stimulated by A23187 is not blocked by Et-18-OMe. When assayed in vitro, treatment of cells with Et-18-OMe did not prevent the enhanced conversion of arachidonic acid into prostaglandins induced by pretreatment of cells with TPA. Our results suggest that the stimulation of phospholipase A2 activity by TPA occurs via activation of protein kinase C by TPA.

Hormonal induction of three hepatic enzymes, phosphoenolpyruvate car-boxykinase, tryptophan oxygenase, and ty-rosine aminotransferase, was significantly inhibited in mice infected with Sindbis virus. Induction of phosphoenolpyruvate carboxy-kinase was suppressed whether it was elicited by overnight fasting or by administration of hydrocortisone. This enzyme responded normally to steroid induction 4 hr after virus administration, but after 6 hr, it was significantly less responsive, The timing of this effect corresponds with the appearance of interferon in the serum of virus-infected mice and mice injected with uvirradiated virus, but results with commercially available interferon make it questionable whether interferon is the factor responsible.

12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment of Madin-Darby canine kidney cells resulted in an increased incorporation of 32Pi and [methyl-3H]choline into choline-containing phosphoglycerides (PC). In pulse-chase experiments, TPA treatment caused an increased release of [methyl-3H]choline from the PC fraction of prelabeled cells. When cells were prelabeled with [3H]arachidonic acid and [14C]palmitic acid, TPA treatment resulted in an increased synthesis of 14C, 3H-diglycerides. Further studies were done to determine the relationship between PC breakdown and diglyceride synthesis. Cells were preincubated with ether-linked 1-O-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine which was acylated to form 1-O-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine. Subsequent treatment of these cells with TPA resulted in an increased synthesis of 1-O-[3H]hexadecyl-2-acyl-sn-glycerol compared to cells not stimulated with TPA. These findings demonstrate that TPA stimulates PC turnover in Madin-Darby canine kidney cells and provide evidence for a novel mechanism of diglyceride formation.

The catalytic properties of a 1440-fold purified preparation of lysosomal phospholipase A1 were examined. The preparation was at least 95% specific for the sn-1 position of neat phosphatidylethanolamine (PE). The apparent specificity of the enzyme toward substrates was affected by three factors: the physical arrangement of molecules in the substrate aggregate, the charge on the lipid-water interface and the chemical structure of the substrate as it relates to the active site of the enzyme. Of various phospholipids tested in the absence of detergent PE was the preferred substrate, phosphatidylcholine (PC) was hydrolyzed at one-fifth the rate of PE, while phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylglycerol (PG) were degraded very slowly. Triton WR1339 stimulated the hydrolysis of PC, PI, PS, and PG but inhibited the hydrolysis of PE, with PG the preferred substrate at a 6:1 Triton/phospholipid ratio. The preference for PC over PE in detergent mixtures was attributed to the active site fit of the chemical structures of the substrate molecules. The enzyme preferentially hydrolyzed neat PE containing palmitic and oleic acids at position 1. A negative surface charge was required for the hydrolysis of PC and PE. Ca2+ stimulated the hydrolysis of PI, PS, and PG but inhibited the hydrolysis of PE. The inhibition of PE hydrolysis by Ca2+ was the result of an alteration in the surface charge of the PE vesicle. Chromatography of phospholipase A1 on concanavalin A-Sepharose resulted in a loss of activity toward acidic phospholipids which could be restored with Ca2+. Plasmalogen PE was found to inhibit the hydrolysis of diacyl-PE at the level of interfacial binding but not by competition for the active site of the enzyme. These results suggest that the hexagonal structure of PE represents a preferred physical form for catalysis by phospholipase A1, while the bilayer form is less readily attacked. Dispersion of the substrate in the inert detergent enhanced the activity of those substrates normally forming bilayer structures. We demonstrate the importance of the “quality of interface” in regulating the activity of the enzyme.

The substrate specificity of purified phospholipase A1 was studied using mixed micelles of phospholipid and Triton X-100. The kinetic analysis employed determined Vmax, Ks (a dissociation constant for the phospholipase A1-mixed micelle complex), and Km (the Michaelis constant for the catalytic step which reflects the binding of the enzyme to the substrate in the interface). The order of Vmax values was phosphatidic acid greater than phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine. The order of Ks values was phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidic acid greater than phosphatidylserine; the order of Km values was phosphatidic acid greater than phosphatidylethanolamine = phosphatidylserine greater than phosphatidylcholine. When present together, phosphatidylcholine inhibited the hydrolysis of phosphatidylethanolamine but phosphatidylethanolamine did not affect the hydrolysis of phosphatidylcholine. Sphingomyelin, phosphatidylcholine plasmalogen, and phosphatidylethanolamine plasmalogen had no effect on the hydrolysis of phosphatidylethanolamine. The effects of the reaction products, lysolipids and/or fatty acids, were also considered for their influence on phosphatidylethanolamine hydrolysis catalyzed by phospholipase A1. Free fatty acid was found to inhibit, whereas lysophospholipids stimulated hydrolysis of phosphatidylethanolamine. In a mixture of 1,2- and 1,3-diacylglycerides in mixed micelles, only the acyl chain at the sn-1 position of the 1,2 compound was hydrolyzed. Surface charge did not modulate the hydrolysis of phosphatidylcholine vesicles or mixed micelles. In conclusion, it is hypothesized that steric hindrance at position 3 of the glycerol regulates substrate binding in the active site and that an acyl group in position 1 is favored over a vinyl ether linkage for binding.

Madin-Darby canine kidney cells deacylate arachidonic acid from cellular phospholipid in response to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and convert the free arachidonic acid to prostaglandins. We have used this system to characterize the acyl specificity of deacylation. Cells were labeled with either [14C]linoleic, [14C]eicosatrienoic (delta 8,11,14 or delta 5,8,11), or [14C]arachidonic acid and stimulated with 10 nM TPA. We found that TPA stimulated the deacylation of all four acids, primarily from phosphatidylethanolamine and phosphatidylcholine.l Only products from linoleic (presumably through chain elongation and desaturation), eicosatrienoic (delta 8,11,14), and arachidonic acids produced prostaglandins. Those produced from linoleic and eicosatrienoic acid (delta 8,11,14)-labeled cells were determined to be primarily of the 1-series, while arachidonic acid-labeled cells produced prostaglandins of the 2-series. Together these results indicate that the stimulated deacylation of phospholipids is not specific for arachidonic acid and that the membrane acyl composition controls the particular series of prostaglandin which is produced.

Range data have become increasingly popular in recent years. Range sensors can acquire shape directly, thus avoiding the difficulties of methods using multiple intensity images, and have been adopted in many applications bin picking, robotic assembly, inspection, recognition, robot navigation, automated cartography and medical diagnosis to name but a few.

The effect of the calcium ionophore A23187 on the release and metabolism of [3H]arachidonic acid was examined in normal polymorphonuclear leukocytes and those obtained from patients with chronic granulomatous disease. The ionophore A23187 which stimulates oxidative metabolism in normal polymorphonuclear leukocytes was ineffective in increasing oxidative metabolism (chemiluminescence) in polymorphonuclear leukocytes from patients with chronic granulomatous disease. However, the ionophore A23187 stimulated the release of [3H]arachidonic acid from chronic granulomatous disease neutrophil phospholipids and stimulated its metabolism into hydroxyeicosatetraenoic acids and leukotrienes.

Lowering the NaCl concentration of the medium inhibits the release of Sindbis virus from infected chicks cells at a stage after the nucleocapsids have bound to the membranes of the infected cells. The failure of trypsin treatment to release the inhibited virus and the ratio of the proteins in the inhibited cells make it seem likely that the inhibited virus is all intracellular. Experiments using antisera specific for E1 and E2, the envelope glycoproteins of Sindbis, suggest that the inhibitory effect of low-salt medium is mediated through an effect on E2. Lactoperoxidase radioiodination experiments indicate that, even when cleaved from PE2, E2 is not exposed on the surface of low-NaCl-treated chick cells.

Indirect fluorescent-antibody studies of living and fixed chick cells infected with temperature-sensitive mutants of Sindbis virus suggest that functional envelope glycoprotein E1 must be inserted through the plasma membrane before E2. PE2 and E2 do not affect the insertion of E1. The experiments also suggest that normal PE2, a glycosylated precursor to E2, reacts with anti-E2 serum; the abnormal PE2 made by a temperature-sensitive PE2 cleavage-defective mutant did not. Abnormal E1 proteins made by E1-defective mutants also failed to react with anti-E1 serum.

The hydrolysis of thioester containing phospholipids by rat liver plasmalemma phospholipase A1 was measured in a continuous spectrophotometric assay. In this assay thioester substrates were employed which, upon hydrolysis, liberated a free thiol which was reacted with 4,4′-dithiopyridine to yield the product 4-thiopyridone that absorbs at 324 nm. Thioester substrates, prepared by chemical synthesis, were used in phospholipid and Triton X-100 micelles for kinetic analysis carried out according to the method of Hendrickson and Dennis (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739). Vmax, Ks, and Km values obtained for various isomers and racemic mixtures of the synthetic thioester analogs are compared with corresponding oxyester substrates. Unnatural sn-1 isomers competitively inhibited the hydrolysis of natural sn-3 isomers of phosphatidylethanolamine and phosphatidic acid. Furthermore, the sn-1 isomer of phosphatidic acid was hydrolyzed by phospholipase A1, but with lower catalytic efficiency than the sn-3 isomer. The presence of a thioester at the sn-1 position did not change the Vmax significantly, as compared to the oxyester phospholipids. When two thioesters were present on the phospholipid molecule, the Vmax was decreased significantly. A convenient synthesis of 1-monothioester analogs of phospholipids is reported. The results presented show the usefulness of the spectrophotometric assay for measuring phospholipase A1 activity as well as the influence of racemic mixtures and thioesters on the hydrolytic rate.

Bis(monoacylglycero)phosphate (BMP) has the unique stereoconfiguration of 3-acyl-sn-glycero-1-phosphoryl-1'-sn-[3'-acylglycerol] (Brotherus, J., Renkonen, O., Herrmann, J., and Fischer, W. (1974) Chem. Phys. Lipids 13, 178-182) which differs from other known mammalian phospholipids that have the sn-glycero-3-phosphoryl configuration. This stereochemistry may contribute to its physiologic function. Here we describe studies using the macrophage-like cell line RAW 264.7 designed to determined how this unique stereoconfiguration occurs. These studies show that the stereoconfiguration of BMP produced from exogenous phosphatidylglycerol (PG) by RAW 264.7 cells has the expected stereoconfiguration of 3-acyl-sn-glycero-1-phosphoryl-1'-sn-[3'-acylglycerol]. Experiments using diacyl-sn-[2-3H]glycero-3-phosphoryl-sn-1'-[2-3H]glycerol demonstrate that this unique stereoconfiguration is not produced due to an oxidation/reduction mechanism involving the sn-2-glycerol carbon. When dioleoyl-sn-[1-14C]glycero-3-phosphoryl-rac-glycerol was converted to 14C-labeled BMP, the 14C label was found esterified to the phosphate moiety. These results suggest that a stereospecific enzyme is capable of reorienting the radiolabeled glycerol backbone of this PG substrate, effectively changing the stereochemistry of the lipid. We also show that this enzyme is stereoselective with regard to the base glycerol moiety of the substrate PG used. Finally, we propose a new pathway for the synthesis of BMP from PG.

The magnetic and structural properties of CoxPt100−xfilms with x=40,45 and 50 at%, have been investigated. The magnetic studies were focused on the dependence of magnetic hysteresis on the film thickness. The sample thicknesses varied between 10 and 200 nm. The coercivities of the as-grown, disordered fcc structure films did not change very much with thickness for all compositions studied. Upon annealing at 650 and 700°C, a structural transformation takes place to the ordered face centered tetragonal (fct) phase which has a high anisotropy resulting in large coercivities that show a pronounced thickness dependence for all the compositions studied.

A monolayer technique was used to study the substrate specificity of hepatic lipase (HL) and the effect of surface pressure and apolipoproteins on hydrolysis of lipid monolayers by this enzyme. HL hydrolyzed readily phosphatidylethanolamine monolayers. Pure trioctanoylglycerol was found to be a poor substrate but when progressively diluted with nonhydrolyzable 1,2-didodecanoylphosphatidylcholine hydrolysis of triacylglycerol by HL reached maximum at a molar ratio of 1:1 triacylglycerol to phosphatidylcholine. The activation of triacylglycerol hydrolysis was not due to altered penetration of HL. The surface pressure optimum of HL for the hydrolysis of phosphatidylethanolamine monolayers was broad between 12.5 and 25 mN/m. When apolipoprotein E was injected beneath the monolayer of phosphatidylethanolamine prior to enzyme addition, a 3-fold activation of HL was observed at surface pressures equal to or below 15 mN/m. Below surface pressures of 20 mN/m apolipoprotein E did not affect the penetration of HL into the lipid-water interface. Apolipoprotein E slightly activated the hydrolysis of triacylglycerol by HL at 10 mN/m. At a high surface pressure of 25 mN/m all apolipoproteins tested (apolipoproteins A-I, A-II, C-I, C-II, C-III, and E) inhibited the penetration into and HL activity on phosphatidylethanolamine At 18.5 mN/m all apolipoproteins except apolipoprotein E inhibited the hydrolysis of triacylglycerol in the triacylglycerol:phosphatidylcholine mixed film. Based on these results we present a hypothesis that phospholipid present in apolipoprotein E-rich high density lipoprotein-1 and triacylglycerol in intermediate density lipoprotein would be preferred substrates for HL.

1. A radioimmunoassay for angiotensin I is described. 2. An extraction procedure for angiotensin I from blood was developed, using alcoholic precipitation to stop the renin/renin substrate reaction. 3. The normal range found for angiotensin I in venous blood was 11–88 pg/ml and in arterial blood 12–72 pg/ml. 4. Angiotensin I levels fell following bilateral nephrectomy but angiotensin I was still detected several weeks following operation.

The development of polyuria and polydipsia in rabbits immunized with vasopressin has been reported by Miller & Moses (1969) and Edwards, Chard, Kitau, Forsling & Landon (1972). The degree of polyuria was reported by Miller & Moses (1969) to be related to the titre of the antiserum. The antigenic nature of argininevasopressin (AVP) and the observed unresponsiveness of certain patients with diabetes insipidus treated with vasopressin have been discussed by Roth, Glick, Klein & Petersen (1966). The present communication reports the development of a syndrome resembling diabetes insipidus of varying severity, in three out of four rabbits actively immunized against AVP, and also indicates that antibody affinity, as well as titre, may be a related factor.Arginine-vasopressin (A. B. Ferring, Sweden) was conjugated to rabbit serum albumin (RSA) by the method of Goodfriend, Fasman, Kemp & Levine (1966). The final dialysate was diluted with saline to give 0·5 mg AVP—RSA

We describe here a modification of a method used to estimate plasma renin-substrate (Tree, 1973) in which radioimmunoassay of angiotensin I (Waite, 1972) is substituted for the bioassay.Measurement of plasma renin-substrate (angiotensinogen) concentration depends upon complete hydrolysis of substrate to angiotensin I by the action of a high concentration of the enzyme renin. In this system conversion of angiotensin I to angiotensin II and destruction of angiotensin I by angiotensinases is prevented by phenanthroline and EDTA. The angiotensin I thus formed is measured by bioassay of samples taken from the incubating mixture at 8 and 16 h.Similar samples for radioimmunoassay of angiotensin I were treated in one of two ways. (1) Radioimmunoassay without preliminary extraction: 50 μl aliquots of each of 91 incubation products were serially diluted before radioimmunoassay. (2) Radioimmunoassay with preliminary extraction: 50 μl aliquots of each of 68 incubation products were extracted onto Dowex (AG 50