Cytochrome P450 3A9 catalyzes the metabolism of progesterone and other steroid hormones

The catalytic requirements and the role of P450 3A9, a female-specific isoform of CYP3A from rat brain, in the metabolism of several steroid hormones were studied using recombinant P450 3A9 protein. The optimal steroid hormone hydroxylase activities of P450 3A9 required cholate but not cytochrome b₅. P450 3A9 was active in the hydroxylation reactions of testosterone, androstenedione, progesterone and dehydroepiandrosterone (DHEA). No activity of P450 3A9 toward cortisol was detectable under our reconstitution conditions. Among all the steroid hormones examined, female-specific P450 3A9 seemed to catalyze most efficiently the metabolism of progesterone, one of the major female hormones, to form three mono-hydroxylated products, 6-, 16-, and 21-hydroxyprogesterone. Our data also showed that P450 3A9 can catalyze the formation of a dihydroxy product, 4-pregnen-6, 21-diol-3, 20-dione, from progesterone with a turnover number, 1.3 nmol/min/nmol P450. Based on the V_max/K_m values for P450 3A9 using either 21-hydroxprogesterone or 6-hydroxyprogesterone as a substrate, 4-pregnen-6, 21-diol-3, 20-dione may be formed either by 6-hydroxylation of 21-hydroxprogesterone or 21-hydroxylation of 6-hydroxyprogesterone. As a major isoform of CYP3A expressed in rat brain, the activities of P450 3A9 toward two major neurosteroids, progesterone and DHEA suggested a possible role for P450 3A9 in the metabolism of neurosteroids.     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Mechanisms of regulation of liver fatty acid-binding protein

Liver fatty acid-binding protein (L-FABP) expression is modulated by developmental, hormonal, dietary, and pharmacological factors. The most pronounced induction is seen after treatment with peroxisome proliferators, which induce L-FABP coordinately with microsomal cytochrome P-450 4A1 and the enzymes of peroxisomal fatty acid -oxidation. These effects of peroxisome proliferators may be mediated by a receptor which has been shown to be activated by peroxisome proliferators in mammalian cell transfection studies. However, the peroxisome proliferators tested thus far do not bind to this receptor, known as the peroxisome proliferator-activated receptor (PPAR), and its endogenous ligand(s) also remain unknown. Peroxisome proliferators inhibit mitochondrial -oxidation, and one hypothesis is that the dicarboxylic fatty acid metabolites of accumulated LCFA, formed via the P-450 4A1 -oxidation pathway, serve as primary inducers of L-FABP and peroxisomal -oxidation. We have tested this hypothesis in primary hepatocyte cultures exposed to clofibrate (CF). Inhibition of P-450 4A1 markedly diminished, via a pre-translational mechanism, the CF induction of L-FABP and peroxisomal -oxidation. In further experiments, long-chain dicarboxylic acids, the final products of the P-450 4A1 -oxidation pathway, but not LCFA, induced L-FABP and peroxisomal -oxidation pre-translationally. These results suggest a role, in part, for long-chain dicarboxylic acids in mediating the peroxisome proliferator induction of L-FABP and peroxisomal -oxidation. We also found that LCFA, which undergo rapid hepatocellular metabolism, could become inducers of L-FABP and peroxisomal -oxidation under conditions where their metabolism was inhibited. The role of the PPAR in mediating these effects is unknown, but clearly warrants further study. The induction of L-FABP and peroxisomal -oxidation by LCFA and/or their -oxidized metabolites may provide a means for limiting the deleterious effects of increased intracellular concentrations of free LCFA, and thus act as an important hepatocellular adaptation to impairment or overload of mitochondrial LCFA oxidation.     

Isolation and characterisation of the Photosystem two reaction centre complex from a double mutant of Chlamydomonas reinhardtii

A rapid procedure has been developed for the isolation of the photosystem two reaction centre complex (PS II RC) from a double mutant of Chlamydomonas reinhardtii, F54-14, which lacks the Photosystem one complex and the chloroplast ATPase. Thylakoid membranes are solubilised with 1.5% (w/v) Triton X-100 and the PS II RC purified by anion-exchange chromatography using TSK DEAE-650(S) (Merck). The complex has a pigment stoichiometry of approximately six chlorophyll a: two pheophytin a: one cytochrome b-559: one to two -carotene. It photoaccumulates reduced pheophytin and oxidised P680 in the presence of sodium dithionite and silicomolybdate, respectively. Immunoblotting experiments have confirmed the presence of the D1 and D2 polypeptides in this complex. The -subunit of cytochrome b-559 was identified by N-terminal sequencing. Comparison of the complex with the PS II RC from pea using SDS-polyacrylamide gel electrophoresis showed that their polypeptide compositions were similar. However, the -subunit of cytochrome b-559 from C. reinhardtii has a lower apparent molecular weight than the pea counterpart whereas the -subunit is larger.Abbreviations DM n-dodecyl -d-maltoside - RC reaction centre - SiMo silicomolybdate, SiMo₁₂O₄₀ ^4– - TAP Tris-acetate-phosphate     

Orientation of the porphyrin ring in artificial chlorophyll membranes

Summary A sensitive photometric method is described by which the dichroism of lipid bilayer membranes in aqueous phase can be measured. The method is applied to black films with incorporated chlorophylla andb. With chlorophylla a relatively large dichroism is found in the Soret band and a much weaker dichroism in the red band. From the experimental data, the angles _B and _R between the blue and red transition moments and the membrane can be obtained. _B and _R are then used to calculate the angle of the porphyrin ring with respect to the membrane surface. For chlorophylla and three different lipids, values of between 44 and 49° are found.     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Developmental changes in neutral glycosphingolipids of mouse placenta

The mammalian placenta is a unique organ for the study of developmental changes. Placentas of laboratory animals such as the mouse allow for the determination of the exact stage of pregnancy, which cannot be achieved with human placenta. In this study, neutral glycosphingolipids were isolated from mouse (inbred strain C57BL/6) placentas, from day 10 to day 18 of gestation, and were separated by high performance thin layer chromatography. Densitometric measurements after orcinol staining showed, at day 10 of gestation, the presence of mono-, tetra-, tri- and dihexosylceramide in decreasing quantities, as well as four unidentified spots. On day 12, the glycosphingolipid composition changed with the disappearance of the unidentified spots and the appearance of an orcinol positive spot migrating similarly to the Forssman antigen; no further changes occurred between days 12 and 18 of gestation. The identity of the Forssman-like glycosphingolipid with the Forssman antigen was established by binding of¹²⁵I labelledHelix pomatia agglutinin (-GalNAc specific) to glycosphingolipids separated on high performance thin layer chromatography plates, and by the reaction of the isolated glycosphingolipid with a monoclonal anti-Forssman antibody. The appearance of the Forssman antigen at day 12 of gestation coincided with the day of final maturation of the mouse placenta and subsequent cessation of growth, suggesting a possible role of the glycosphingolipid during embryonic development.Abbreviations asialo-GM₁ Gal 3GalNAc4Gal4Glc1Cer - BCIP 5-bromo-4-chloro-3-indolylphosphate - DHC lactosylceramide, Gal4Glc1Cer - Forssman antigen GalNAc3GalNAc3Gal4Gal4Glc1Cer - globoside GalNAc3Gal4Gal4Glc1Cer - GSL glycosphingolipids - HPA Helix pomatia agglutinin - HPTLC high performance thin layer chromatography - MHC galactosylceramide, Gal1Cer - MHC glucosylceramide, Glc1Cer - PBS phosphate-buffered saline - PNA peanut agglutinin - PVP poly(vinylpyrrolidone), mol. wt 40 000 - SBA soybean agglutinin - THC trihexosylceramide, Gal4Gal4Glc1Cer. To whom correspondence should be addressed.     

Immobilization of digitoxin 12β-hydroxylase,a cytochrome P-450-dependent enzyme from cell cultures of Digitalis lanata EHRH

Attempts were made to immobilize digitoxin 12-hydroxylase, a membrane-bound, cytochrome P-450-dependent monooxygenase from cell cultures of Digitalis lanata. The optimum procedure was the entrapment of microsomes in 2% alginate by crosslinking the polysaccharide chains with CaCl₂. After the immobilization of the enzyme about 70% of its activity was retained. The kinetic data such as the pH optimum and the optimum substrate concentrations were identical for the immobilized enzyme and freely suspended microsomes. Using -methyldigitoxin as a substrate enzyme activity could be observed for more than 20 h. A continuous flow system for immobilized digitoxin 12-hydroxylase is described.Abbreviations -mdg -methyldigoxin - -mdt -methyldigitoxin     

Purification and characterization of liver cytochrome P-446 isolated from protein energy malnourished rats

A liver cytochrome P-450 isozyme has been purified to homogeneity from protein-energy malnourished rats induced with -naphthoflavone (-NF). The purification steps included chromatography on DEAE-Sephadex-A-25, DEAE-cellulose (DE-53), hydroxylapatite (HA) and carboxymethyl-sephadex (CM) columns. The reduced carbon monoxide difference and absolute spectra showed a Soret peak at 446.5 nm. The wavelength maxima for the oxidized and reduced spectra were at 416 and 408 nm, respectively. Cytochrome P-446 appears to have a predominantly low spin ferric iron, migrates as a single band of molecular weight 56000 in sodium dodecyl sulfate polyacrylamide gels and has a specific content of 14 nmol/mg of protein. P-446 oxidized various substrates at different rates in a reconstituted system with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine. In this system turnover rates for benzo[]pyrene, testosterone and benzphetamine oxidation were: 81.10; 1.85 and 1.42 nmoles product/min/nmol P-446 respectively. While NH₂ terminal amino acid sequence analysis of 18 of the first 20 residues suggests that the cytochrome P-446 isolated from malnourished rats is identical with form c, the catalytic activities suggest that this isozyme may be a more effective or efficient catalyst for some substrates.Abbreviations -NF -napthoflavone - SDS-PAGE Sodium Dodecyl Sulfate polyacrylamide gel electrophoresis - 3-MC 3-Methyl Cholanthrene - PEG Poly Ethylene Glycol - DTT Dithiothreitol - PMSF Phenyl Methyl Sulfonylfluoride - EDTA disodium ethylenediaminetetraacetate - NADPH reduced nicotinamide adenine dinucleotide phosphate - P-450 cytochrome P450, PB-1, PB-4, PB-5 and P-450 isozymes purified from phenobarbital induced rat liver - HPLC High Pressure Liquid Chromatography - B[]P benzo[]pyrene - CM Carboxymethyl Sephadex - PTH-amino acid phenylthiohydantoin amino acid, Cytochrome P-450 EC 1.14.14.1, NADPH Cytochrome P-450 (c) reductase ED 1.6.2.4     

Sparse distribution of γ/δ T lymphocytes around human epithelial tumors predominantly infiltrated by primed/memory T cells

Summary Evidence from the mouse system has suggested that T lymphocytes accumulating in non-lymphoid tissue, in particular epithelia, may preferentially express the T cell receptor (TCR) . In this study, we characterize the T cell receptor or phenotype of lymphocytes infiltrating human tumors of epithelial origin using monoclonal antibodies (mAb) for immunohistology and flow cytometry on cells extracted by enzyme digestion. This report shows that the majority of CD3⁺ tumor-infiltrating lymphocytes are TCR ⁺ but a small percentage of TCR can be clearly defined scattered throughout the tumor tissue with apparently no microanatomical selection. So far there has been little evidence for an accumulation of activated T cells in human tumor tissues as defined by mAb against molecules appearing transiently during the acute phase of activation. Now mAb are available that can identify primed or memory T cells such as mAb UCHL-1 recognizing the CD45RO antigen. Here we show that CD3⁺ tumor-infiltrating lymphocytes have a statistically significant accumulation of primed T cells, as compared to the autologous peripheral blood lymphocytes, suggesting their immune stimulation by tumor cells.     

Metal Cations Defibrillize the Amyloid Beta-Protein Fibrils

Amyloid beta-protein (A) is the major constituent of amyloid fibrils composing -amyloid plaques and cerebrovascular amyloid in Alzheimer's disease (AD). We studied the effect of metal cations on preformed fibrils of synthetic A by Thioflavin T (ThT) fluorescence spectroscopy and electronmicroscopy (EM) in negative staining. The amount of cross beta-pleated sheet structure of A 1–40 fibrils was found to decrease by metal cations in a concentration-dependent manner as measured by ThT fluorescence spectroscopy. The order of defibrillization of A 1–40 fibrils by metal cations was: Ca²⁺ and Zn²⁺ (IC₅₀ = 100 M) > Mg²⁺ (IC₅₀ = 300 M) > Al³⁺ (IC₅₀ =1.1 mM). EM analysis in negative staining showed that A 1–40 fibrils in the absence of cations were organized in a fine network with a little or no amorphous material. The addition of Ca²⁺, Mg²⁺, and Zn²⁺ to preformed A 1–40 fibrils defibrillized the fibrils or converted them into short rods or to amorphous material. Al³⁺ was less effective, and reduced the fibril network by about 80 % of that in the absence of any metal cation. Studies with A 1–42 showed that this peptide forms more dense network of fibrils as compared to A 1–40. Both ThT fluorescence spectroscopy and EM showed that similar to A 1–40, A 1–42 fibrils are also defibrillized in the presence of millimolar concentrations of Ca²⁺. These studies suggest that metal cations can defibrillize the fibrils of synthetic A.     

Effects of IL-1β on Receptor-Mediated Poly-Phosphoinositide Signaling Pathway in Immortalized Astrocytes (DITNC)

Astrocytes are known to play multi-functional roles in support of many homeostatic mechanisms in the central nervous system including host defense mechanisms. Despite the ability of cytokines to alter gene expression and cellular activity, their effect on receptor-mediated poly-phosphoinositide (poly-PI) signaling pathway has not been examined in detail. In this study, an immortalized astrocyte cell line (DITNC) was used to test the effect of IL-1 exposure on the poly-PI signaling pathway. Similar to primary astrocytes, DITNC cells exhibit P₂-purinergic receptor response to ATP and UTP leading to transient increases in inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] and intracellular calcium concentration, [Ca²⁺]i. Upon exposure of DITNC cells to IL-1 (100U/ml) for 24 hrs, an increased response to the poly-PI agonists was observed. The increase in ATP-mediated Ins(1,4,5)P₃ release could not be attributed to a shift in the ATP dose or an alteration of the time profile for the release of Ins(1,4,5)P₃. Since the increase in response required a lag time of 4 hr after IL-1 exposure, it is unlikely that this effect was due to a direct interaction of IL-1 with the purinergic receptor. On the other hand, an increase in ATP response could be observed in DITNC cells exposed to conditioned medium obtained after IL-1 treatment. It can be concluded that exposure of astrocytes to cytokines may lead to an increase in receptor-mediated poly-PI signaling activity and this may involve compounds secreted into the culture medium, e.g., the secretory phospholipase A₂.     

Phytochrome-regulated transfer of fructosidase from cytoplasm to cell wall in Raphanus sativus L. hypocotyls

The far-red absorbing form of phytochrome, P_fr, rapidly increases the rate of transfer of -fructosidase (E.C.3.2.1.26) from the cytoplasm to the cell wall in radish hypocotyls. Far-red light increases the level of enzyme in a particulate fraction: after two hours of light treatment, the particulate enzyme is associated almost exclusively with the endoplasmic reticulum. Transfer from the endoplasmic reticulum to the cell wall involves an incorporation into Golgi bodies and the plasmalemma: these membrane fractions were separated by centrifugation on a discontinuous sucrose density gradient and their degree of purity was determined by the use of known biochemical markers. With respect to -fructosidase, light controls, via P_fr: (1) the total amount, (2) the incorporation into the endoplasmic reticulum and (3) the transfer to the cell-wall. These three processes have different sensitivities to cycloheximide.Abbreviations -FFase -fructosidase - IDPase inosine diphosphatase - SGTase UDPG-sterol glucosyltransferase - NCRase NADPH-cytochrome c-oxydoreductase - NPA N-naphtylphtalamic acid - BSA bovine serum albumine     

Evidence of β-carotene 7,8(7′,8′) oxygenase (β-cyclocitral,crocetindial generating) in Microcystis

A -carotene oxygenase is described which occurs in the Cyanobacterium Microcystis. It cleaves -carotene and zeaxanthin specifically at the positions 7,8 and 7,8, while echinenone and myxoxanthophyll are not affected. The oxidative cleavage of -carotene leads to the formation of -cyclocitral and crocetindial and that of zeaxanthin to hydroxy--cyclocitral and crocetindial in nearly stoichiometric amounts. Oxidant is dioxygen as has been demonstrated by high incroporation (86%) of ¹⁸O₂ into -cyclocitral. -Carotene oxygenase is membrane bound, sensitive to sulfhydryl reagents, antioxidants and chelating agents. Iron seems to be an essential part of the enzyme activity. Cofactors necessary for the reaction could not be detected.Abbreviations TLC thin layer-chromatography - PIPES piperazine-N,N-bis-(2-ethanesulfonate) Na - TES 2{[tris-(hydroxymethyl)-methyl]-amino} ethanesulfonic acid Dedicated to Professor G. Drews on occasion of his 60th birthday     

Anti-diabetic activity of <Emphasis Type="Italic">β</Emphasis>-glucans and their enzymatically hydrolyzed oligosaccharides from <Emphasis Type="Italic">Agaricus blazei</Emphasis>

-Glucans were prepared from Agaricus blazei Murill by repeated extraction with hot water. The average molecular weights of -glucans were 30–50 kDa by gel filtration chromatography. Oligosaccharides (AO), derived from hydrolyzing -glucans with an endo--(16)-glucanase from Bacillus megaterium, were mainly di- and tri-saccharides. Though -glucans and AO both showed anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats, AO had about twice the activity of -glucans with respect to anti-diabetic activity.     

Lectin like properties and differential sugar binding characteristics of C-reactive proteins purified from sera of normal and pollutant induced Labeo rohita

Different forms of C-reactive proteins (CRPs) have been purified to electrophoretic homogeneity from the sera of Labeo rohita confined in freshwater (CRP_N) and water polluted with nonlethal doses of cadmium (CRP_Cd) or mercury (CRP_Hg). CRP_N[emsp4 ], CRP_Cd[emsp4 ], and CRP_Hg show remarkable differences in their electrophoretic mobility but exhibit strong immunological cross reactivity. All these CRPs exhibit variable agglutination properties with erythrocytes from diverse sources in presence of Ca⁺², which could be inhibited by a variety of sugars showing specificity for galactose. Inhibition results show that the potency of galactose as an inhibitor increases about 4 fold in the process of transformation of CRP_N to CRP_Cd and CRP_Hg[emsp4 ]. In case of CRP_N[emsp4 ], Gal (11) Gal and oNO₂ phenyl -Gal show highest inhibitory potency while oNO₂-phenyl -Gal is the most potent inhibitor for CRP_Cd and CRP_Hg but the potency of Gal (11) Gal reduced drastically. 6-phosphate D-Gal and stachyose are 20 times weaker inhibitors than D-Gal for induced CRP mediated agglutination, in contrast, these sugars are only 6 times weaker for CRP_N[emsp4 ]. Dissociation constants of the binding of CRP_N with phosphoryl choline (PC) and galactose are about 9[emsp4 ]mM and PC binding causes a change in the and conformations of these CRPs.     

Endogeneous interferon α/β produced by murine Kupffer cells augments liver-associated natural killing activity

Summary Nonparenchymal liver cells from untreated C3HeB/FeJ mice, when incubated in medium containing-10% fetal bovine serum or portal serum, produced significant amounts of interferon alpha/beta (IFN/). In contrast, other cell populations (spleen, mononuclear blood cells and peritoneal cells) from C3HeB/FeJ mice or nonparenchymal liver cells from other strains of mice (C3H/HeJ, germ-free C3H/HeN and C57Bl/6J) produced little or no detectable IFN in fetal bovine serum under the same culture conditions. The cells in the nonparenchymal liver cell population responsible for IFN/ production were adherent, phagocytic, silica-sensitive, carbonyl-iron-sensitive, and Thy1.2^–, presumably Kupffer cells or resident liver macrophages. IFN/ production by cultured Kupffer cells was not observed if medium containing fetal bovine serum or portal serum was treated with polymyxin B or if Kupffer cells were cultured in serum-free medium. This suggested that small amounts of endotoxin in fetal bovine or portal serum stimulated Kupffer cells to produce IFN/. Possibly, Kupffer cells are in a different state of activation/maturation than peritoneal and splenic macrophages since the sensitivity of resident Kupffer cells from C3HeB/FeJ mice to the stimulatory effects of endotoxin. The endogenous production of IFN/ by Kupffer cells from C3HeB/FeJ mice can augment liver-associated natural killer (NK) activity against YAC-1 cells (4 h) and induce liver-associated cytotoxic activity, not restricted by the major histocompatibility complex, against NK resistant P815 mastocytoma cells (18 h).This work was supported by National Institutes of Health grant CA28835, VA Merit Grant and by the Margaret Duffy and Robert Cameron Troup Fund Abbreviations used: NPC, nonparenchymal liver cells; FBS, fetal bovine serum; IFN, interferon; -AsGm-1, anti-asialo-GM1; -Thy1.2, anti-Thy1.2; Hepes, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; HBSS, Hanks' balanced salt solution; GBSS, Gey's balanced salt solution; SRBC, sheep red blood cells; Ab, mouse anti-SRBC; NK, natural killer; MHC, major histocompatibility complex; polyI·polyC, polyinosinec·polycytidylic acid     

Enzyme-catalyzed synthesis of alkyl β-D-glycosides with crude homogenate ofSulfolobus solfataricus

Summary Crude homogenate of thermophilic archaebacteriumSulfolobus solfataricus, possessing a -glycosidase, has been used to synthesize different alkyl -D-glycosides starting from phenyl -D-glucoside, phenyl -D-galactoside and lactose as carbohydrate donors. High product yield (95% with respect to the carbohydrate donor) of octyl -D-glucoside has been obtained in a two-phase system containing 5% of water. The enantioselection for the galactosyl transfer to the secondary hydroxyl group of propane-1,2-diol is higher than that found using -galactosidase fromE. coli.