Effect of lipid bilayer properties on the photocycle of green proteorhodopsin

The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocycle was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Escherichia coli was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-β-d-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurified phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coli cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli.

2D and 3D crystallization of the wild-type IIC domain of the glucose PTS transporter from Escherichia coli.

The bacterial phosphoenolpyruvate: sugar phosphotransferase system serves the combined uptake and phosphorylation of carbohydrates. This structurally and functionally complex system is composed of several conserved functional units that, through a cascade of phosphorylated intermediates, catalyze the transfer of the phosphate moiety from phosphoenolpyruvate to the substrate, which is bound to the integral membrane domain IIC. The wild-type glucose-specific IIC domain (wt-IIC(glc)) of Escherichia coli was cloned, overexpressed and purified for biochemical and functional characterization. Size-exclusion chromatography and scintillation-proximity binding assays showed that purified wt-IIC(glc) was homogenous and able to bind glucose. Crystallization was pursued following two different approaches: (i) reconstitution of wt-IIC(glc) into a lipid bilayer by detergent removal through dialysis, which yielded tubular 2D crystals, and (ii) vapor-diffusion crystallization of detergent-solubilized wt-IIC(glc), which yielded rhombohedral 3D crystals. Analysis of the 2D crystals by cryo-electron microscopy and the 3D crystals by X-ray diffraction indicated resolutions of better than 6Å and 4Å, respectively. Furthermore, a complete X-ray diffraction data set could be collected and processed to 3.93Å resolution. These 2D and 3D crystals of wt-IIC(glc) lay the foundation for the determination of the first structure of a bacterial glucose-specific IIC domain.

MOUSE MAMMARY TUMOR VIRUS-RELATED PROTEINS: ISOLATION AND CHARACTERIZATION OF A UNIQUE GLYCOPROTEIN

Mouse mammary tumor virus (MMTV) contained six major proteins, identified as gp55, gp33, p25, pp20, p12, and p10. Immunoprecipitation of cytoplasmic extracts from MMTV-infected, pulse-labeled cells identified three MMTV core-specific precursor proteins, termed Pr78('gag), Pr110('gag), Pr110('gag), and Pr180('gag+). The major intracellular core-specific precursor polyprotein, Pr78('gag), contained antigenic determinants and tryptic peptides characteristic of p25, p12, and p10. Pr110('gag) contained all but one of the leucine-containing tryptic peptides of Pr78('gag), plus several additional peptides. In addition to Pr78('gag) and Pr110('gag), monospecific antisera to virion p12 and p25 also precipitated from pulse-labeled cells a small amount of Pr180('gag+). This large polyprotein contained nearly all of the leucine-containing tryptic peptides of Pr78('gag) and Pr110('gag) plus several additional peptides. By analogy to type-C viral systems, Pr180('gag+) is presumed to represent a gag-pol-specific common precursor which is the major translation product in the synthesis of MMTV RNA-dependent-DNA polymerase. Immunoprecipitation of cytoplasmic extracts from pulse-labeled cells with antisera to gp55 identified two envelope-specific proteins, designated gPr76('env) and gP79('env). The major envelope-specific precursor, gPr76('env), could be labeled with radioactive glucosamine and contained antigenic determinants and tryptic peptides characteristic of gp55 and gp33. A quantitatively minor glycoprotein, gP79('env), contained both fucose and glucosamine and was precipitable from cytoplasmic extracts with monospecific serum to gp55. It is suggested that gP79('env) represents fucosylated gPr76('env) which is transiently synthesized and cleaved rapidly into gp55 and gp33.^ A glycoprotein of 130,00 molecular weight (gP130) was precipitable from the cytoplasm of GR-strain mouse mammary tumor cells by a rabbit antiserum (anti-MMTV) to Gr-strain mouse mammary tumors virus (GR-MMTV). Two dimensional thin layer analysis of ('35)S-methionine-containing peptides revealed that five of nine gp33 peptides and one of seven gp55 peptides were shared by gP130 and gPr76('env). Six of ten p25 peptides and four more core-related peptides were shared by Pr78('gag) and gP130. Protein gP130 also contained several tryptic peptides not found in gPr76('env), or in the core protein precursors Pr78('gag), Pr110('gag), or Pr180('gag+). both gP130 and a second protein, p30, were found in immunoprecipitates of detergent disrupted, isotopically labeled GR-MMTV treated with anti-MMTV serum. Results suggest that antibodies to gP130 in the anti-MMTV serum are capable of recognizing those protein sequences which are not related to viral structural proteins. These gP130-unique peptides are evidently host specific. Polyproteins consisting of juxtaposed host- and virus-related protein tracts have been implicated in the process of cell transformation in other mammalian systems. Therefore, gP130 may be instrinsic to the oncogenic potential of MMTV. ^

NADPH-CYTOCHROME P-450 REDUCTASE: EVIDENCE FOR TWO SEPARATE STRUCTURAL DOMAINS AND ISOLATION AND CHARACTERIZATION OF THE MEMBRANE ASSOCIATED SEGMENT

Homogenous detergent-solubilized NADPH-Cytochrome P-450 reductase was incorporated into microsomes and liposomes. This binding occurred spontaneously at temperatures between 4(DEGREES) and 37(DEGREES) and appeared to involve hydrophobic forces as the binding was not disrupted by 0.5 M sodium chloride. This exogenously-added reductase was active catalytically towards native cytochrome P-450, suggesting an association with the microsomal membrane similar to endogenous reductase. Homogeneous detergent-solubilized reductase was disaggregated by Renex-690 micelles, confirming the presence of a hydrophobic combining region on the enzyme. In contrast to these results, steapsin protease-solubilized reductase was incapable of microsomal attachment and did not interact with Renex-690 micelles. Detergent-solubilized reductase (76,500 daltons) was converted into a form with the electrophoretic mobility of steapsin protease-solubilized reductase (68,000 daltons) and a 12,500 dalton peptide (as determined by polyacrylamide-SDS gel electrophoresis) when the liposomal-incorporated enzyme was incubated with steapsin protease. The 68,000 dalton fragment thus obtained had properties identical with steapsin protease-solubilized reductase, i.e. it was catalytically active towards cytochrome c but inactive towards cytochrome P-450 and did not bind liposomes. The 12,500 dalton fragment remained associated with the liposomes when the digest was fractionated by gel filtration, suggesting that this is the segment of the enzyme which is embedded in the phospholipid bilayer. Thus, detergent-solubilized reductase appears to contain a soluble catalytic domain and a separate and separable membrane-binding domain. This latter domain is required for attaching the enzyme to the membrane and also to facilitate the catalytic interaction between the reductase and its native electron acceptor, cytochrome P-450. The membrane-binding segment of the reductase was isolated by preparative gel electrophoresis in SDS following its generation by proteolytic treatment of liposome-incorporated reductase. The peptide has a molecular weight of 6,400 as determined by gel filtration in 8 M guanidine hydrochloride and has an amino acid composition which is not especially hydrophobic. Following removal of SDS and dialysis out of 6 M urea, the membrane-binding peptide was unable to inhibit the activity of a reconstituted system containing purified reductase and cytochrome P-450. Moreover, when reductase and cytochrome P-450 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptide were used. Thus, the membrane-binding peptide was ineffective as an inhibitor of mixed function oxidase activity, suggesting perhaps that it facilitates catalysis by anchoring the catalytic domain of the reductase proximal to cytochrome P-450 (i.e. in the same mixed micelle) rather than through a specific interaction with cytochrome P-450. ^

SUBCELLULAR LOCALIZATION, PURIFICATION AND PROPERTIES OF A CDP-DIACYLGLYCEROL - DEPENDENT PHOSPHATIDYLSERINE SYNTHASE IN BACILLUS LICHENIFORMIS

A CDP-diacylglycerol dependent phosphatidylserine synthase was detected in three species of gram-positive bacilli, viz. Bacillus licheniformis, Bacillus subtilis and Bacillus megaterium; the enzyme in B. licheniformis was studied in detail. The subcellular distribution experiments in cell-free extracts of B. licheniformis using differential centrifugation, sucrose gradient centrifugation and detergent solubilization showed the phosphatidylserine synthase to be tightly associated with the membrane. The enzyme was shown to have an absolute requirement for divalent metal ion for activity with a strong preference for manganese. The enzyme activity was completely dependent upon the addition of CDP-diacylglycerol to the assay system; the role of the liponucleotide was rigorously shown to be that of phosphatidyl donor and not just a detergent-like stimulator. This enzyme was then solubilized from B. licheniformis membranes and purified to near homogeneity. The purification procedure consisted of CDP-diacylglycerol-Sepharose affinity chromatography followed by substrate elution from blue-dextran Sepharose. The purified preparation showed a single band with an apparent minimum molecular weight of 53,000 when subjected to SDS polyacrylamide gel electrophoresis. The preparation was free of any phosphatidylglycerophosphate synthase, CDP-diacylglycerol hydrolase and phosphatidylserine hydrolase activities. The utilization of substrates and formation of products occurred with the expected stoichiometry. Radioisotopic exchange patterns between related substrate and product pairs suggest a sequential BiBi reaction as opposed to the ping-pong mechanism exhibited by the well studied phosphatidylserine synthase of Escherichia coli. Proteolytic digestion of the enzyme yielded a smaller active form of the enzyme (41,000 daltons) which appears to be less prone to aggregation.^ This has been the first detailed study in a well-defined bacillus species of the enzyme catalyzing the CDP-diacylglycerol-dependent formation of phosphatidylserine; this reaction is the first committed step in the biosynthetic pathway to the major membrane component, phosphatidylethanolamine. Further study of this enzyme may lead to understanding of new mechanisms of phosphatidyl transfer and novel modes of control of phospholipid biosynthetic enzymes. ^

STUDIES ON THE MECHANISM OF ASSEMBLY OF MURINE LEUKEMIA VIRUS

The purpose of this research was to elucidate the mechanism of assembly of retroviruses, specifically of murine leukemia virus, as studied through the treatment of virus-infected cells with interferon and through the use of temperature sensitive (ts) mutants. Our studies have shown a rapid and specific association of Rauscher murine leukemia virus (R-MuLV) precursor polyprotein Pr65('gag) with cytoskeletal elements in infected mouse fibroblasts. The Pr65('gag) associated with Nonidet P-40 (NP40)-insoluble cytoskeletal structures appeared to be subphosphorylated in comparison to NP40-soluble Pr65('gag). The association of Pr65('gag) with skeletal elements could be disrupted by extraction of the cytoskeleton with sodium deoxycholate, an ionic detergent. Both the skeleton-associated Pr65('gag) and its NP40-soluble counterpart were labeled with {('3)H}-palmitate, indicating their probable association with lipids presumably in the plasma membrane. Pr65('gag) molecules bound to skeletal elements in the infected cell appeared to be more stable to proteolytic processing than NP40-soluble Pr65('gag). Our studies with certain ts mutants of murine leukemia virus, defective in virus assembly, including Mo-MuLV ts3 and R-MuLV ts17, ts24, ts25 and ts26, have shown that virions released at 39(DEGREES)C (nonpermissive temperature) had high levels of uncleaved Pr65('gag) relative to that seen in virions released at 33(DEGREES)C (permissive temperature). Examination of cell extracts revealed that Pr54('gag) was more stable to processing at 39(DEGREES)C than at 33(DEGREES)C, whereas the 'env' and glycosylated 'gag' proteins were processed to the same extent at both temperatures. Detergent extraction of pulse-labeled cells to generate an NP40-insoluble cytoskeleton-enriched fraction showed that in ts3-, ts17- and ts24-infected cells, Pr65('gag) accumulated in the cytoskeleton-enriched fraction. In contrast, cells infected with ts25 or ts26 showed no preferential localization of Pr65('gag) in the cytoskeleton in a short pulse, but instead, Pr65('gag) accumulated in both the NP40-soluble and -insoluble fractions during a chase-incubation. The association of Pr65('gag) with cytoskeletal elements in the cell was neither increased nor decreased by blocking virus assembly and release with interferon. Based on these and other results, we have proposed a model for the active role of cytoskeleton-associated Pr65('gag) in retrovirus assembly.^

INVESTIGATIONS ON THE INTRACELLULAR LOCALIZATION OF PHOSPHATIDYLSERINE SYNTHASE FROM ESCHERICHIA COLI

Phosphatidylserine synthase catalyzes the committed step in the synthesis of the major lipid of Escherichia coli, phosphatidylethanolamine, and may be involved in regulating the balance of the zwitterionic and anionic phospholipids in the membrane. Unlike the other enzymes involved in the biosynthesis of phospholipids in E. coli, phosphatidylserine synthase is not membrane associated but seems to have a high affinity for the ribosomal fraction of cells broken by various methods. Investigations on the enzyme in cell free extracts using glycerol gradient centrifugation revealed that the binding of the synthase to ribosomes may be prevented by the presence of highly basic compounds such as spermidine and by the presence of detergent-lipid substrate micelles under assay conditions. Thus phosphatidylserine synthase may not be ribosome associated under physiological conditions but associated with its membrane bound substrate (Louie and Dowhan (1980) J. Biol. Chem. 255, 1124).^ In addition homogeneous enzyme shows many of the properties of a membrane associated protein. It binds nonionic detergent such as Triton X-100, which is also required during purification of the enzyme. Optimal catalytic activity is also dependent on micelle or surface bound substrate. Phosphatidylserine synthase has been synthesized in vitro using a coupled transcription-translation system dependent on the presence of the cloned structural gene. The translation product was found to preferentially associate with the ribosomal fraction even in the presence of added E. coli membranes. Preferential membrane binding could be induced if the membranes were supplemented with the lipid substrate CDP-diacylglycerol. Similar effects were obtained with the acidic lipids phosphatidylglycerol and cardiolipin. On the other hand the zwitterionic lipid phosphatidylethanolamine and the lipid product phosphatidylserine did not cause any detectable membrane association. These results are consistent with the enzyme recognizing membrane bound substrate (Carman and Dowhan (1979) J. Biol. Chem. 254, 8391) and with the lipid charge influencing membrane interaction.^ Phosphatidylserine synthase is at a branch point in lipid metabolism, which may determine the distribution of the zwitterionic and anionic phospholipids in the membrane. The results obtained here indicate phosphatidylserine synthase may play a significant role in membrane lipid biosynthesis by maintaining charge balance of the E. coli membrane. In determining the localization of phosphatidylserine synthase in vitro one may have a better understanding of its function and control in vivo and may also have a better understanding of its role in membrane assembly.^

CHARACTERIZATION OF CYTOCHROME P-450 MIXED FUNCTION OXIDASE OF RAT COLON MUCOSA

Non-pregnant, female adult rats pretreated with either phenobarbital (PB) or (beta)-naphthoflavone ((beta)NF) through short-course intraperitoneal injections were shown by sodium dithionite-reduced carbon monoxide difference spectroscopy and NADPH-cytochrome c in vitro assay to contain cytochrome P-450 and NADPH-dependent reductase associated with the microsomal fraction of colon mucosa. These two protein components of the mixed function oxidase system were released from the microsomal membrane, resolved from each other, and partially purified by using a combination of techniques including solubilization in nonionic detergent followed by ultracentrifugation, anion exchange and adsorption column chromatographies, native gel electrophoresis, polyethylene glycol fractionation and ultrafiltration.^ In vitro reconstitution assays demonstrated the cytochrome P-450 fraction as the site of substrate and molecular oxygen binding. By the use of immunochemical techniques including radial immunodiffusion, Ouchterlony double diffusion and protein electroblotting, the cytochrome P-450 fraction was shown to contain at least 5 forms of the protein, having molecular weights as determined by SDS gel electrophoresis identical to the corresponding hepatic cytochrome P-450. Estimation of total cytochrome P-450 content confirmed the preferential induction of particular forms in response to the appropriate drug pretreatment.^ The colonic NADPH-dependent reductase was isolated from native gel electrophoresis and second dimensional SDS gel electrophoresis was performed in parallel to that for purified reductase from liver. Comparative electrophoretic mobilities together with immunochemical analysis, as with the cytochrome P-450s, reconstitution assays, and kinetic characterization using artificial electron acceptors, gave conclusive proof of the structural and functional homology between the colon and liver sources of the enzyme.^ Drug metabolism was performed in the reconstituted mixed function oxidase system containing a particular purified liver cytochrome P-450 form or partially pure colon cytochrome P-450 fraction plus colon or liver reductase and synthetic lipid vesicles. The two drugs, benzo{(alpha)}pyrene and benzphetamine, which are most representative of the action of system in liver, lung and kidney, were tested to determine the specificity of the reconstituted system. The kinetics of benzo{(alpha)}pyrene hydroxylation were followed fluorimetrically for 3-hydroxybenzo{(alpha)}pyrene production. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Contribution of transmembrane and cytoplasmic domains to membrane protein association

Membrane proteins are critical to every aspect of cell physiology, with their association mediating important biological functions. The transmembrane and cytoplasmic domains are known to be important for their association. In order to characterize their role in detail, we have applied different biophysical techniques in detergent micelles to two model systems. The first study involves FcRγ, a single transmembrane domain protein existing as a disulfide linked homodimer. We investigated the role of a conserved transmembrane polar residue and the cytoplasmic tail in FcRγ homo-interactions. Our results by various biophysical techniques including SDS-PAGE, circular dichroism and sedimentation equilibrium in detergent micelles indicate importance of both the transmembrane polar residue and cytoplasmic tail in maintaining proper conformation for FcRγ homo-interactions. A contrasting second study was on L-selectin, another single transmembrane domain protein with a large extracellular domain and a short cytoplasmic tail. Previous cross-linking experiments indicate its possible dimerization. However, the purified fragment of L-selectin and corresponding mutants did not dimerize when analyzed by TOXCAT assay, sedimentation equilibrium and fluorescence resonance energy transfer. It was likely that the presence of juxtamembrane positively charged residues led to decreased migrational rates in SDS PAGE. In conclusion, complementary biophysical techniques should be used with care when studying membrane protein association in detergent micelles. As an extension to our study on L-selectin, we also investigated its interaction with Calmodulin (CaM) in detergent micelles. CaM was found to interact with different detergents. We applied fluorescence and NMR spectroscopy to characterize the interaction of both the apo and Ca 2+ bound form of CaM, with commonly used detergents, below and above their respective critical micelle concentrations. The interaction of apo-CaM with detergents was found to vary with the nature of the detergent head group, whereas Ca2+-CaM interacted with individual detergent molecules irrespective of the nature of their head group. NMR titration experiments of CaM with detergents indicated involvement of specific residues from the N-lobe, linker and C-lobe of CaM. ^

Association of pellicle growth morphological characteristics and clinical presentation of Mycobacterium tuberculosis isolates

Trehalose dimycolate (TDM) is a mycobacterial glycolipid that is released from the surface of virulent M. tuberculosis. We evaluated the rate of growth, colony characteristics and production of TDM by Mycobacterium tuberculosis strains isolated from different clinical sites. Since detergent removes TDM from organisms, we analyzed growth rate and colony morphology of 79 primary clinical isolates grown as pellicles on the surface of detergent free Middlebrook 7H9 media. The genotype of each had been previously characterized. TDM production was measured by thin layer chromatography on 32 of these isolates. We found that strains isolated from pulmonary sites produced large amounts of TDM, grew rapidly as thin spreading pellicles, showed early cording (<1 week) and climbed the sides of the dish. In contrast, the extrapulmonary isolates (lymph node and bone marrow) produced less TDM (p<0.01), grew as discrete patches with little tendency to spread or climb the walls (p<0.02). The Beijing pulmonary (BP) isolates produced more TDM than non Beijing pulmonary isolates. The largest differences were observed in Beijing strains. The Beijing pulmonary isolates produced more TDM and grew faster than the Beijing extrapulmonary isolates (p<0.01). This was true even when the pulmonary and extrapulmonary isolates were derived from the same clade. These growth characteristics were consistently observed only on the first passage after primary isolation. This suggests that the differences in growth rate and TDM production observed reflect differences in gene expression patterns of pulmonary and extrapulmonary infections, that Mycobacterium tuberculosis in the lung grows more rapidly and produces more TDM than it does in extrapulmonary sites. This provides new opportunities to investigate gene expression of Mycobacterium tuberculosis in human.^

Aberrant regulation of the Src -FAK signaling pathway in the human colon adenocarcinoma cell line HT-29

c-Src, a protein tyrosine kinase (PTK) the specific activity of which is increased $>$20-fold in $\sim$80% of colon tumors and colon tumor cell lines, plays a role in both growth regulation and tumorigenicity of colon tumor cells. To examine the effect of increased c-Src specific activity on colon tumor cells, coumarin-derived tyrosine analog PTK inhibitors were assessed in a standard colon tumor cell line, HT-29. Of the nine compounds tested for inhibiting c-Src activity in a standard immune complex kinase assay from c-Src precipitated from HT-29 cells, the 7,8-dihydroxy-containing compounds daphnetin and fraxetin were most effective, with IC$\sb{50}$s of 0.6 $\pm$ 0.2 mM and 0.6 $\pm$ 0.3 mM, respectively. Treatment of HT-29 cells with daphnetin resulted in inhibition of cell growth in a dose-dependent manner. In contrast, scopoletin, a relatively poor Src inhibitor in vitro, did not inhibit HT-29 cell growth in the concentration range tested. In daphnetin treated cells, a dose-dependent decrease of c-Src activity paralleling cell growth inhibition was also observed; the IC$\sb{50}$ was 0.3 $\pm$ 0.1 mM for c-Src autophosphorylation. In contrast, the IC$\sb{50}$ for c-Src protein level was $>$ 0.6 mM, indicating that the effects of daphnetin were primarily an enzymatic activity of c-Src, rather than protein level in HT-29 cells. These results are the first to demonstrate that c-Src specific activity regulates colon tumor cell growth.^ To elucidate the signaling pathways activated by c-Src in colon tumor cells, the Src family substrate FAK, which has been shown to play a role in both extracellular matrix-dependent cell growth and survival, was examined. Coprecipitation assays showed Src-FAK association in detergent insoluble fractions of both attached and detached HT-29 cells, indicating that Src-FAK association in HT-29 cells is stable and, unlike untransformed cells, not dependent on cell-substratum contact. FAK also coprecipitated with Grb2, an adaptor protein also playing a role in cell proliferation and survival, in both attached and detached HT-29 cells, suggesting that a Src-FAK-Grb2-mediated signaling pathway(s) in HT-29 cells is/are constitutively activated.^ FAK was also analyzed in c-src antisense HT-29 clones AS15 and AS33 in which c-Src is specifically reduced by transfection of an antisense expression vector. FAK protein level is unexpectedly decreased in both AS15 and AS33 cells by 5-fold and 1.5-fold compared to HT-29, respectively, corresponding with the decreased expression of c-Src observed in these cells. FAK protein level was not decreased compared to parental in the c-src "sense" clone S8. Northern blot analyses showed decreased FAK mRNA levels compared to parental in AS15 and AS33, correlating with decreased FAK protein level, indicating that FAK activity in the antisense cells is regulated, at least in part, by altering FAK expression, and that this regulation is Src dependent. Because FAK has been implicated in anoikis, the ability of c-src antisense cells to survive in the absence of cell-substratum contact was examined. Decreased cell survival is seen in both AS15 and AS33, correlating with the decreases in c-Src and FAK levels and tumorigenicity in these cells. These results suggest that at least one mechanism by which activation of c-Src contributes to tumorigenic phenotype of colon tumor cells is by aberrantly promoting a survival signal through unregulated Src-FAK-Grb2 complexes. (Abstract shortened by UMI.) ^

Stable oxygen and carbon isotope ratios of foraminifera and test results after different sample cleaning procedures from ODP Leg 207 sediments

We report oxygen and carbon stable isotope analyses of foraminifers, primarily planktonic, sampled at low resolution in the Cretaceous and Paleogene sections from Sites 1257, 1258, and 1260. Data from two samples from Site 1259 are also reported. The very low resolution of the data only allows us to detect climate-driven isotopic events on the timescale of more than 500 k.y. A several million-year-long interval of overall increase in planktonic 18O is seen in the Cenomanian at Site 1260. Before and after this interval, foraminifers from Cenomanian and Turonian black shales have d18O values in the range -4.2 per mil to -5.0 per mil, suggestive of upper ocean temperatures higher than modern tropical values. The d18O values of upper ocean dwelling Paleogene planktonics exhibit a long-term increase from the early Eocene to the middle Eocene. During shipboard and postcruise processing, it proved difficult to extract well-preserved foraminifer tests from black shales by conventional techniques. Here, we report results of a test of procedures for cleaning foraminifers in Cretaceous organic-rich mudstone sediments using various combinations of soaking in bleach, Calgon/hydrogen peroxide, or Cascade, accompanied by drying, repeat soaking, or sonication. A procedure that used 100% bleach, no detergent, and no sonication yielded the largest number of clean, whole individual foraminifers with the shortest preparation time. We found no significant difference in d18O or d13C values among sets of multiple samples of the planktonic foraminifer Whiteinella baltica extracted following each cleaning procedure.

Files of figures 2, 3 and table

Certain allelochemicals of the marine dinoflagellate Alexandrium tamarense cause lysis of a broad spectrum of target protist cells but the lytic mechanism is poorly defined. We first hypothesized that membrane sterols serve as molecular targets of these lytic compounds, and that differences in sterol composition among donor and target cells may cause insensitivity of Alexandrium and sensitivity of targets to lytic compounds. We investigated Ca2+ influx after application of lytic fractions to a model cell line PC12 derived from a pheochromocytoma of the rat adrenal medulla to establish how the lytic compounds affect ion flux associated with lysis of target membranes. The lytic compounds increased permeability of the cell membrane for Ca2+ ions even during blockade of Ca2+ channels with cadmium. Results of a liposome assay suggested that the lytic compounds did not lyse such target membranes non-specifically by means of detergent-like activity. Analysis of sterol composition of isolates of A. tamarense and of five target protistan species showed that both lytic and non-lytic A. tamarense strains contain cholesterol and dinosterol as major sterols, whereas none of the other tested species contain dinosterol. Adding sterols and phosphatidylcholine to a lysis bioassay with the cryptophyte Rhodomonas salina for evaluation of competitive binding indicated that the lytic compounds possessed apparent high affinity for free sterols and phosphatidylcholine. Lysis of protistan target cells was dose-dependently reduced by adding various sterols or phosphatidylcholine. For three tested sterols, the lytic compounds showed highest affinity towards cholesterol followed by ergosterol and brassicasterol. Cholesterol comprised a higher percentage of total sterols in plasma membrane fractions of A. tamarense than in corresponding whole cell fractions. We conclude therefore that although the molecular targets of the lytic compounds are likely to involve sterol components of membranes, A. tamarense must have a complex self-protective mechanism that still needs to be addressed.

Nutritive value of palm kernel meal in diets for growing rabbits

The aim of this work was to determine the nutritive value of palm kernel meal (PKM) in diets for growing rabbits. In Experiment 1, 20 New Zealand × Californian growing rabbits 50 d-old were used to determine energy, crude protein, fibre and fat digestibility of PKM. The nutritive value was estimated by the difference method using a basal diet and another diet made by substituting 200 g/kg of basal diet with PKM. Energy, crude protein, ether extract and neutral detergent fibre of PKM digestibilities were, respectively, 0.549 (±0.056, SE), 0.541 (±0.069), 0.850 (±0.048) and 0.430 (±0.101), and the digestible energy concentration was 10.9 MJ/kg (±1.03) DM. In Experiment 2, 412 rabbits were allocated at random to the two experimental diets to measure growing performance. Inclusion of 200 g PKM/kg in the diet did not affect feed or digestible energy intake but decreased slightly (by around 5%) average daily gain (P = 0.003) and feed efficiency (P < 0.001). Neither mortality nor Clostridium perfringens counts in soft faeces were affected by type of diet. Palm kernel meal can be considered a palatable source of fibre, protein and fat for rabbits and can substitute significant amounts of other fibrous ingredients in the diet without adverse effects on growth performance.

Correlation between ileal digestibility of amino acids and chemical composition of soybean meals in broilers at 21 days of age

The correlations between chemical composition and coefficient of standardized ileal digestibility (CSID) of crude protein (CP) and amino acids (AA) were determined in 22 soybean meal (SBM) samples originated from USA (n = 8), Brazil (BRA; n = 7) and Argentina (ARG; n = 7) in 21-day old broilers. Birds were fed a commercial maize-SBM diet from 1 to 17 days of age followed by the experimental diets in which the SBM tested was the only source of protein (205 g CP/kg) for three days. Also, in vitro nitrogen (N) digestion study was conducted with these samples using the two-step enzymatic method. The coefficient of apparent ileal digestibility (CAID) of the SBM, independent of the origin, varied from 0.820 to 0.880 for CP, 0.850 to 0.905 for lysine (Lys), 0.859 to 0.907 for methionine (Met) and 0.664 to 0.750 for cysteine (Cys). The corresponding CSID values varied from 0.850 to 0.966 for CP, 0.891 to 0.940 for Lys, 0.931 to 0.970 for Met and 0.786 to 0.855 for Cys. The CSID of CP and Lys of the SBM were positively correlated with CP (r = 0.514; P menor que 0.05 and r = 0.370; P = 0.09, respectively), KOH solubility (KOH sol.) (r = 0.696; P menor que 0.001 and r = 0.619; P menor que 0.01, respectively), trypsin inhibitor activity (TIA) (r = 0.541; P menor que 0.01 and r = 0.416; P = 0.05, respectively) and reactive Lys (r = 0.563; P menor que 0.01 and r = 0.486; P menor que 0.05) values, but no relation was observed with neutral detergent fiber and oligosaccharide content. No relation between the CSID of CP determined in vivo and N digestibility determined in vitro was found. The CSID of most key AA were higher for the USA and the BRA meals than for the ARG meals. For Lys, the CSID was 0.921, 0.919 and 0.908 (P menor que 0.05) and for Cys 0.828, 0.833 and 0.800 (P menor que 0.01) for USA, BRA and ARG meals, respectively. It is concluded that under the conditions of this experiment, the CSID of CP and Lys increased with CP content, KOH sol., TIA and reactive Lys values of the SBM. The CSID of most limiting AA, including Lys and Cys, were higher for USA and BRA meals than for ARG meals.