Comparative Study of Effects of Various Sterols and Triterpenoids on Permeability of Model Lipid Membranes

Using permeability to labeled glucose as a criterion of stability for liposomal membranes, a comparative study on stabilizing properties of different sterols and triterpenes in phospholipid bilayer has been carried out as well as on structural peculiarities of sterols responsible for membranolytic properties of cucumarioside G₁ from the cucumaria Eupentacta fraudatris. Stabilizing action of the studied sterols and triterpenoides incorporated in the bilayer decreases in the following order: cholesterol sulfate > cholesterol > ⁵-sterols > -sitosterol > ergosterols > ⁷-sterols > epicholesterol > pregnane > androstane > coprosterol > 14-methylcholest-9(11)-en-3-ol > 4, 14-dimethylcholest-9(11)-en-3-ol > holothurinogenin A₁ > glucoside of cholesterol > -xylosidase of ⁷-sterols > betulin > protopanaxatriol > phosphatidylcholine liposomes without sterol > protopanaxadiol > oleanolic acid. Sterol-dependent membranolytic cucumarioside G₁ practically loses its ability to increase permeability of phospholipid membranes containing sterols obtained from this holothuria as well as coprosterol, epicholestrol, sulfated and glycosylated forms of sterols. The obtained results confirm the sterol hypothesis of the mechanism of membranotropic action of holothuria glycosides and of resistance to them of holothuria cell membranes.     

Antiinsectan properties of a novel Fusarium-derived sterol sulfate

The antiinsectan properties of the Fusarium graminearum-derived novel sterol sulfate, 4,4,24-trimethylcholesta-8,14,24(28)-trien-2,3,11,12-tetrol 12 acetate, 3-sulfate were tested on the corn earworm (Heliothis zea (Boddie), the fall armyworm (Spodoptera frugiperda (J. E. Smith), and the driedfruit beetle (Carpophilus hemipterus L.). The sterol sulfate could not be used as a sterol source by any of the insects. The sterol sulfate inhibited growth of S. frugiperda and C. hemipterus larvae at 2500 and 4000 ppm, respectively. Defensive implications of this compound are discussed.     

Two novel extracellular cholesterol oxidases of Bacillus sp. isolated from fermented flatfish

Two novel extracellular cholesterol oxidases designated CO1 and CO2, from Bacillus sp. SFF34, were purified 5.6 and 5.9-fold giving Mr values of 36 and 37 kDa. The optimum temperature for the activity was 60 °C (CO1) and 40 °C (CO2), and the optimum pH was 6.25 (CO1) and 6 (CO2) over 30 min reaction time. The apparent K _m values for cholesterol were 6.76 mM (CO1) and 4.50 mM (CO2). Both the enzymes could oxidize 5-cholestane, 5-cholestane-3-ol-7-one, coprostane, dihydrocholesterol, hecogenin, -sitosterol and stigmasterol.     

Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin

In Drosophila as in many organisms tubulins are encoded by a gene family. We have determined the complete nucleotide sequences coding for the 1 and 2 tubulins of Drosophila melanogaster and the 2 tubulin of D. hydei, and found these insect tubulins to be highly conserved and like tubulins of other organisms. This is discussed with reference to the possible functional domains of these proteins. — The 1 tubulin gene of Drosophila is constitutively expressed, whereas the 2 tubulin is expressed specifically in the testes. In D. melanogaster the amino acid sequences of these proteins are 95% homologous, differing at only 25 positions. In the testes the 2 tubulin participates in different microtubules as shown by genetic analysis (Kemphues et al. 1982). Interestingly, all of the amino acids characteristic of the testis-specific 2 tubulin are also present in the corresponding gene of D. hydei. Of special interest is the high degree of conservation of the carboxy-terminal domain in these functionally equivalent tubulins.     

Structure and evolution of the HLA Class II SXβ gene

The class II major histocompatibility complex antigens are cell-surface heterodimers consisting of an a and a chain. Cosmid cloning has shown that the three families of clas II antigens, DR, DQ, and DP, are encoded within the HLA-D region of chromosome 6 as a series of discrete gene clusters. The DP cluster contains two pairs of a and genes, one of which encodes the biochemically-defined DP antigen. In order to assess whether the other two genes, SXa and SX, are also expressed, potential coding regions have been subcloned and sequenced. The SX3 gene is shown to contain region closely homologous to all six exons of DP. A 1 bp deletion in the 2 exon, also observed for the SX4 allele, causes a translation frameshift, suggesting that SX is a pseudogene. However, all the other exons, as well as their splice sites and the putative promoter region, appear to be intact.     

Cloning and expression in Saccharomyces cerevisiae of a β-amylase gene from the oomycete Saprolegnia ferax

Genomic DNA and cDNA encoding the -amylase from the oomycete, Saprolegnia ferax, were cloned into Saccharomyces cerevisiae and analyzed. The Spl. ferax -amylase gene consisted of a 1350 bp open reading frame, encoding a protein of 450 amino acids with a calculated mass of 49353 Da, and was not interrupted by any intron. The deduced amino acid sequence of the -amylase gene had 42% similarity to the -amylase of Arabidopsis thaliana. The -amylase gene was expressed in Sacc. cerevisiae and its product was secreted into the culture medium.     

Characterization of two beta-tubulin genes from Geotrichum candidum.

Summary The -tubulin genes G1 and G2 from the phytopathogenic hemiascomycete Geotrichum candidum were found to be highly diverged in amino acid sequence from those of other filamentous fungi. G1 and G2 were also divergent from each other, with the coding regions sharing only 66% nucleotide sequence homology and 64% amino acid identity. However, the proteins shared 82% similarity and only 25 of the 161 non-identical amino acid substitutions were non-conservative. The organization of G1 is similar to other fungal -tubulin genes, but G2 has several unusual features; it has 2 amino acid additions in the N-terminal 40 residues and must employ an uncommon 5 splice junction sequence in preference to an overlapping perfect consensus. The amino acid change found to confer benomyl resistance in Neurospora crassa was also present in G2. G1 has four introns which are located similarly to those of -tubulin genes in other fungi. G2, however, has a single intron in a unique location. Translational fusions employing the 5 non-coding regions of the two Geotrichum -tubulin genes were made with the hygromycin phosphotransferase gene and shown to function in Schizosaccharomyces pombe and Trichoderma hamatum. However, G. candidum could not be transformed with these or other tested plasmids commonly used for fungal transformation.     

Isolation of a rat immune response gene identical to an alleged mouse A class II β-chain pseudogene

A human HLA-DQ -chain cDNA was used as a probe to identify and isolate a rat major histocompatibility antigen -chain gene from a genomic library constructed in the vector Charon 28 using Wistar rat DNA (RT1 ^u). The isolated exon of the rat gene (RT1.B ₂) encoding a -chain second domain was found to share 93% nucleotide homology with a mouse A ₂ exon. Although the genomic organization of this gene is consistent with the hypothesis that it represents a pseudogene, the remarkable preservation of a specific sequence favors the view that this class II antigen -chain gene has retained its coding function.     

The genetic defect in the various types of human β-galactosidase deficiency

Summary Gene localization studies revealed the presence of two structural -galactosidase (GAL) loci on the human chromosomes 3 and 22 (de Wit et al., 1979). To determine the function of these genes, proliferating hybrid cell lines were isolated following fusion of fibroblasts from two different patients with a GAL deficiency and Chinese hamster cells. The hybrids were analyzed electrophoretically and immunologically.Fibroblasts from a patient with an adult type of GAL deficiency associated with a neuraminidase deficiency were used for the first fusion. No evidence for a structural GAL mutation was found in these hybrids. The absence of a structural GAL mutation is consistent with a primary defect in neuraminidase in this adult patient.Fibroblasts from a patient with the infantile type 1 G_M1-gangliosidosis were used for the second fusion. It is concluded that the human determinants present in the isolated hybrid lines occur in heteropolymeric man-Chinese hamster molecules. The heteropolymeric isoenzyme in (+3–22) hybrids is very labile and is sensitive to neuraminidase treatment. Therefore it is concluded that the infantile type 1 patient is mutated in the structural GAL gene on chromosome 3. Because this patient has a primary defect in G_M1-GAL, the GAL gene on chromosome 3 is apparently a G _M1-GAL gene. Interaction of the two GAL loci results in an additional band of GAL activity on electrophoresis. This suggests that the gene on chromosome 22 is also a structural G _M1-GAL gene.     

Binding of kynurenine to catecholamine

Summary Papiliochrome II is a pale yellow pigment of butterflies and consists of one molecule each ofL-kynurenine and N--alanyldopamine (NBAD). The aromatic amino nitrogen of kynurenine is bonded to the-carbon of NBAD. There are isomers IIa and IIb which show opposite circular dichroism. The-alanine contents of IIa and IIb were determined and the molar ratio of IIa to IIb has proved to be 1.17. The IIa and IIb were decomposed toL-kynurenine and N--alanylnorepinephrine (NBANE) by being heated in water at 80°C for 30 min. In both IIa and IIb, circular dichroism of the NBANE showed the same positive peak at 280 nm. The NBANE were further decomposed to-alanine and norepinephrine (NE) by being heated in 1 N HC1 at 100°C for 2 hr. The NE was submitted to enantioseparation and has proved to be a racemic mixture in both cases of IIa and IIb. These results are discussed in the light of the enzymic synthesis of IIa and IIb.     

A 7.1 kbp beta-myosin heavy chain promoter,efficient for green fluorescent protein expression,probably induces lethality when overexpressing a mutated transforming growth factor-beta type II receptor in transgenic mice

The roles of transforming growth factor-beta (TGF) in heart or skeletal muscle development and physiology are still the subject of controversies. Our aim was to block, in transgenic mice, the TGF signalling pathway by a dominant negative mutant of the TGF type II receptor fused to the enhanced green fluorescent protein (TRII-KR-EGFP) under the control of a 7.1 kbp mouse beta-myosin heavy chain (MHC) promoter to investigate the roles of TGF in the heart and slow skeletal muscles. First, we generated two transgenic lines overexpressing EGFP under the control of the 7.1 kbp MHC promoter. In embryos, EGFP was detectable as early as 7.5 days post coitum. In embryos, newborns and adults, EGFP was expressed mainly in the cardiac ventricles and in slow skeletal muscles. EGFP expression was intense in the bladder but weak in the intestines. In contrast to the endogenous MHC promoter, the activity of the 7.1 kbp MHC promoter in the transgene was not repressed after birth and remained high in adult transgenic mice. We obtained two founders with the transgene comprising the TRII-KR-EGFP sequence under the control of the 7.1 kbp MHC promoter. These founders were generated at a very low frequency and expressed barely detectable levels of TRII-KR-EGFP mRNA. Our failure to obtain transgenic lines overexpressing the dominant negative receptor suggests that the blocking of the TGF signalling pathway in the heart and slow skeletal muscles could be embryonically lethal. To conclude, the 7.1 kbp MHC promoter directs high levels of transgene expression in the cardiac ventricles and in slow skeletal muscles of the mouse. Analysis of the consequences of the blocking of the TGF signalling pathway in the heart will require the use of tissue specific means of conditional gene invalidation.     

The tryptic peptide composition of the beta chains of hemoglobins A and B of the domestic cat (Felis catus)

Summary The tryptic peptides from the ^A and ^B chains of cat hemoglobins A and B have been isolated and the amino acid compositions determined. Differences between the two chains were found in two peptides,T-1 (GlySer) andT-14 (AsnSer and LysArg). The GlySer and LysArg substitutions are placed at-1 and-144 respectively from earlier work, and the third substitution, AsnSer at-139 is suggested from this work. In addition, the presence of a blocked amino terminus in ^B has been confirmed. Tentative sequences constructed by homology with known-chain structures suggest the occurrence of substitutions at _{1 1} contacts in ^A and ^B that may be functionally significant. There are at least 18 differences in amino acid composition between cat ^A and dog-chains and 22 differences between cat ^A and normal adult human-chains.     

Variations in the rate of synthesis of beta and beta'' RNA polymerase polypeptides under the influence of certain factors.

Summary In merodiploid cells containing a double dose of structural genes of RNA polymerase subunits-rpoBand rpoC-the rate of and subunits synthesis is 2 times higher than in haploidcells. Missence mutation rpoC1 (tsX) alters polypeptide and inducesthe and subunits synthesis at increased rate, particularly at a nonpermissive temperature. When rpoBCoperon carrying mutation rpoC1 is duplicated no dosage effect is observed. In the rpoC ⁺/rpoC1 heterodiploid the rpoC1 mutation does not significantly accelerate RNA polymerase subunits synthesis i.e. is recessive with respect to rpoC ⁺ Rifampicin causes 6-fold stimulation of RNA polymerase subunits synthesis in a sensitive wild-type strain. The rpoC1 mutation itself accelerates the synthesis of these subunits 3-fold. In the presence of rifampicin the mutant strain produces 13–22-fold faster as compared to wild-type strain without the drug. Thus, the effects of rifampicin and the mutation are multiplied suggesting that these factors act independently. Similar data have also been obtained with rifampicin-treated cells of rpoB22 (ts22) amber-mutant.After UV-irradiation of cells and synthesis is depressed much stronger than the total protein synthesis. Infection with a transducing phage rif ^d-47 which carries rpoB gene provokes a higher rate of synthesis. When pre-irradiated cells (500 erg/mm²) are infected with this phage, the rate of synthesis grows 20-fold compared to irradiated, non-infected cells and 6.5-fold compared to intact cells.The data are discussed in terms of the possible regulatory mechanisms of RNA polymerase subunit synthesis.     

Degradation of barley β-glucan by endoglucanase C of Clostridium thermocellum

Summary Endoglucanase C encoded by the celC gene of Clostridium thermocellum has been purified to homogeneity from a recombinant Escherichia coli strain. It was found that this enzyme is highly efficient in degrading glucans with alternating -1,4- and -1,3-linkages but lacks activity on unmodified cellulosic substrates. The properties of endoglucanase C were compared to those of Bacillus subtilis -glucanase, an enzyme used in the brewing industry for -glucan degradation. Both enzyme cause a rapid decrease of the viscosity of barley -glucan as a result of internal chain cleavage. Endoglucanase C hydrolyses non-specifically -1,3- and -1,4-bonds adjacent to unsubstituted or 4-O-substituted cellobiose units. Due to its lower pH optimum and increased thermostability endoglucanase C compares favourably with B. subtilis -glucanase and seems suitable for use in the mashing process of beer brewing.     

The β-tubulin gene family evolution in theDrosophila montium subgroup of themelanogaster species group

The 1-, 2-, and 3-tubulin genes have been mapped by in situ hybridization on the polytene chromosomes of 11 selected species (15 strains) belonging to theDrosophila montium subgroup. Although the hybridization pattern among the strains of the same species does not differ, this pattern is significantly different among the species. The -tubulin genes in themontium subgroup seem to be organized in a cluster, or in a semi-cluster, or are completely dispersed. The clustered arrangement is found in the North-Oriental sibling speciesD. auraria, D. triauraria, andD. quadraria. The semi-clustered arrangement, wherein the 1 and 2 genes are located at the same locus while 3 is at a different one, appears in the South-Oriental speciesD. bicomuta, D. serrata, andD. birchii, as well as in the Afrotropical speciesD. diplacantha andD. seguyi. The complete separation of the genes is observed in the Indian speciesD. kikkawai andD. jambulina and in the Afrotropical speciesD. vulcana. Based on the above results, a possible mode of evolution of the -tubulin genes in the montium subgroup is attempted. In addition, phylogenetic relationships among themontium species are discussed. Correspondence to: Z.G. Scouras     

Degradation of β-lactam antibiotics by polyacrylamide-entrapped β-lactamase-producingE. coli cells

Summary Cells of a -lactamase producingE. coli strain were immobilized with acrylamide and lyophilized. The gel particles containing the entrapped cells were used like an immobilized enzyme to study the inactivation of -lactam antibiotics. The substrate profile of the -lactamase was determined and the action of -lactamase inhibitors studied.     

O-Glycosylhydrolases of Marine Filamentous Fungi: β-1,3-Glucanases of Trichoderma aureviride

The ability to produce extracellular O-glycosylhydrolases was studied in 14 strains of marine filamentous fungi sampled from the bottom sediments of the South China Sea. The following activities were detected in the culture liquids of the fungi: N-acetyl--D-glucosaminidase, -D-glucosidase, -D-galactosidase, -1,3-glucanase, amylase, and pustulanase. -1,3-Glucanases were isolated by ultrafiltration, hydrophobic interaction chromatography, and ion exchange chromatography, and their properties were studied. Data on products of enzymatic digestion of laminaran, absence of transglycosylation activity, and the pattern of action of natural inhibitors confirmed that -1,3-glucanase belonged to the exo type. Inhibitor analysis demonstrated the role of a thiol group and tryptophan and tyrosine residues in the catalytic activity.     

Location by fluorescence microscopy of glycosidases and a xylanase in the anaerobic gut fungi Caecomyces communis,Neocallimastix frontalis,and Piromyces rhizinflata

-d-Glucosidase, -d-fucosidase -d-xylosidase, and -cellobiopyranosidase activities in Caecomyces communis, Neocallimastix frontalis, and Piromyces rhizinflata, located with fluorescent conjugates, occur throughout the whole thallus as from zoospore germination and disappear before sporulation. -d-Galactosidase and -l-arabinopyranosidase activities are low or nonexistent. A xylanase, detected by indirect immunofluorescence, was observed at the surface of the vegetative cells, vesicles, or rhizoids. Cross-reactions prove the existence of analogies in structure among the enzymes of these anaerobic gut fungi.     

Purification and characterization of α(2-6)-sialyltransferase from human liver

A Gal1-4GlcNAc (2-6)-sialyltransferase from human liver was purified 34 340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at –20°C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of theN-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Gal1-4GlcNAc (2-6)-sialyltransferase from rat liver.Abbreviations FPLC fast protein liquid chromatography - NeuAc 5-N-acetyl-d-neuraminic acid - 9-amino-NeuAc 5-acetamido-9-amino-3,5,9-trideoxy-d-glycero-2-nonulosonic acid - 9-acetamido-NeuAc 5,9-diacetamido-3,5,9-trideoxy-d-glycero--d-2-nonulosonic acid - 9-benzamido-NeuAc 5-acetamido-9-benzamido-3,5,9-trideoxy-d-glycero--d-galacto-2-nonulosonic acid - 9-fluoresceinyl-NeuAc 9-fluoresceinylthioureido-NeuAc - 5-formyl-Neu 5-formyl--d-neuraminic acid - 5-aminoacetyl-Neu 5-aminoacetyl--d-neuraminic acid - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - G_M1 Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-ceramide - ST sialyltransferase - DTE 1,4-dithioerythritol Enzyme: Gal1-4GlcNAc (2-6)-sialyltransferase, EC 2.4.99.1.     

Synthesis of Aminoethyl Glycosides of the Ganglioside GM1 and Asialo-GM1 Oligosaccharide Chains

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of asialo-GM₁ ganglioside in 72% overall yield. Selective 3-O-glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,6-di-O-benzyl--D-galactopyranosyl)--D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero--D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)oate]-(2 3)-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM₃ ganglioside, in 79% yield. Its 4-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl) 1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-{[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)onate]-(2 3)}-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)-[-D-Neu5Ac-(2 3)]--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of GM₁ ganglioside.