Biosynthesis of P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate in calf pancreas microsomes

Calf pancreas microsomes incubated with UDP-N-acetyl-D-[14C] glucosamine in the presence of Mn2+ incorporated radioactivity into P1-2-acetamido-2-deoxy-D-glucopyranosyl P2-dolichyl pyrophosphate and P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate. The formation of both glycolipids was enhanced to the same extent by exogenous dolichyl phosphate. Labeled P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate was formed from synthetic P1-2-acetamido-2-deoxy-alpha-D-glucopyranosyl P2-dolichyl pyrophosphate and from prelabeled pancreatic P1-2-acetamido-2-deoxy-alpha-D-glucopyranosyl P2-dolichyl pyrophosphate without the addition of divalent cation. Upon thin layer chromatography, it had the same mobility as synthetic P1-di-N-acetyl-alpha-chitobiosyl P2-dolichyl pyrophosphate recently synthesized by Warren et al. (Warren, C. D., Herscovics, A., and Jeanloz, R. W. (1977) Carbohydr. Res., in press), but was different from the synthetic compound prepared by Wedgwood et al. (Wedgwood, J. F., Warren, C. D., Jeanloz, R. W., and Strominger, J. L. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 5022-5026).     

Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus.

Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.     

Effect of the conformation of concanavalin A on its affinity for manganous ion

The stoichiometry of Mn2+ binding to concanavalin A at pH 6.4-7 which had been established in two independent studies [J.A. Sophianopoulos, A.J. Sophianopoulos, and W.C. MacMahon (1983) Arch. Biochem. Biophys. 223, 350-359; D.J. Christie, G.R. Munske, and J.A. Magnuson (1979) Biochemistry 18, 4638-4644] was challenged [C.F. Brewer, R.D. Brown, III, and S.H. Koenig (1983) Biochemistry 22, 3691-3702] on grounds of possible experimental errors. Additional evidence is presented in this study in support of the previous finding that at pH 6.4 only one Mn2+ binds per concanavalin A monomer of Mr 25,550. Also, evidence is presented showing that the results of Sophianopoulos et al. could not have been due to contamination by Ca2+. A comparison is made of the results in the three studies cited above which indicates that the concanavalin A used by Brewer et al. had decreased affinity for Mn2+ and it contained an appreciable fraction of concanavalin A incompetent of binding saccharides.     

Relation Between the Chemical Structure and the Biological Activity of Hydroxystilbenes Against Botrytis cinerea

Abstract A Spanish strain of pepper mild mottle virus (PMMV-S) (A lonso et al. 1989) can be differentiated from the Italian PMMV (W etter et al. 1984) by the responses of Capsicum spp. with resistance genes to tobamoviruses, by radioimmunoassay and by the electrophoretic mobility of their viral particles. Moreover, the analysis of the electrophoretic mobility of the viral particles in agarose gels and that of the viral coat proteins in polyacrylamide-urea gels are reliable and rapid techniques for distinguishing PMMV isolates from other members of the tobamovirus group and thus can be used for diagnostic purposes. These results support the proposal of grouping these pepper viruses, including the "pepper strains" of TMV, as a new tobamovirus subgroup (PARES 1985, BETTI et al. 1987, WETTER et al. 1987, ALONSO et al. 1989).     

竹亚科刚竹属植物的修订(Ⅴ)

在模式材料、实物照片或采集物观察比较以及实地考察的基础上,结合原始描述,对竹亚科Bambusoideae刚竹属Phyllostachys Sieb.et Zucc.中最后一批存疑或悬而未决的拉丁双名进行了考订。分别将彭县刚竹P.sapida并入石绿竹P.arcana,P.balansae并入桂竹P.bambusoides,金竹仔P.subulata并入寿竹P.bambusoides f.shouzhu,广州刚竹P.cantoniensis并入水竹P.heteroclada,大节刚竹P.lofushanensis和刺芒刚竹P.aristata均并入笔笋竹P.nidularia f.basipilis,贵州刚竹P.guizhouensis并入毛金竹P.nigra var.henonis,台湾石竹P.lithophila并入刚竹P.sulphurea var.viridis,均作为异名处理;讨论了产于越南的3个种的归属问题,推测P.caobangensis可能与桂竹P.bambusoides为同种,而P.baccanensis和P.vietbacensis可能均与假毛竹P.kwangsiensis为同种。     

Overexpressed pp60c-src can induce focus formation without complete transformation of NIH 3T3 cells.

NIH 3T3 cells were transfected with plasmids containing Moloney murine leukemia virus long terminal repeats and either chicken c-src or v-src genes. In contrast with the effects observed after transfection with plasmids containing c-src and avian retrovirus or simian virus 40 promoter-enhancers (H. Hanafusa, H. Iba, T. Takeya, and F. R. Cross, p. 1-8, in G. F. Vande Woude, A. J. Levine, W. C. Topp, and J. D. Watson, ed., Cancer Cells, vol. 2, 1984; H. Iba, T. Takeya, F. R. Cross, T. Hanafusa, and H. Hanafusa, Proc. Natl. Acad. Sci. U.S.A. 81:4424-4428, 1984; R. C. Parker, R. Swanstrom, H. E. Varmus, and J. M. Bishop, p. 19-26, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984; R. C. Parker, H. E. Varmus, and J. M. Bishop, Cell 37:131-139, 1984; D. Shalloway, P. M. Coussens, and P. Yaciuk, p. 9-17, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984; D. Shalloway, P. M. Coussens, and P. Yaciuk, Proc. Natl. Acad. Sci. U.S.A. 81:7071-7075; and K. C. Wilhelmsen, W. G. Tarpley, and H. M. Temin, p. 303-308, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984), we found that both types of Moloney murine leukemia virus long terminal repeat-src expression plasmids induced focus formation, although c-src induced only 1% as many foci as v-src. The focus-selected c-src overexpressed cells had altered morphology and limited growth in soft agarose but were not tumorigenic in vivo. Cleveland digests, comparative in vitro kinase assays, secondary transfections, and immunoprecipitations indicated that focus formation was caused by rare transfection events that resulted in very high-level pp60c-src expression rather than by mutations of the transfected c-src genes. These results suggest that pp60v-src induced transformation is not a completely spurious activity which is unrelated to the function of pp60c-src but that it represents a perturbation of already existent molecular control processes involving pp60c-src.     

REVIEWS

Book reviewed in this article:
A Course in Biology . By J. J. B aker and G. E. A llen .
Papers on Plant Systematics . By R. O rnduff .
Die Entstehung der Arten und hoheren Kategorien . By H. L amprecht .
Flora of Iraq , Vol. 9. Gramineae . By N. L. B or . (Ed. by C. C. T ownsend and E van G uest , with the collaboration of A li A l -R awi ).
Wild Elowers of North Carolina . By W illiam S. J ustice and C. R itchie B ell .
The Lichen Genera Cetrelia and Platismatia ( Parmeliaceae ). By W. L. C ulberson and C. F. C ulberson .
Essays on Form in Plants . By C. W. W ardlaw .
Crop Responses to Water at Different Stages of Growth . By P. J. S alter and J. E. G oode .
Protons, Electrons, Phosphorylation and Active Transport . By R. N. R obertson .
Encyclopedia of Plant Physiology, XVIII, Sexuality: Reproduction; Alternation of Generations . General editor, W. R uhland ; sub-editor for this volume, H. F. L inskens .
Annual Review of Plant Physiology , Vol. 19. 1968. By L. M achlis (Ed.).
Biophysical Technique as Applied to Cell Biology . By J. C hayen and E. E. D enby .
The Measurement of Environmental Factors in Terrestrial Ecology . British Ecological Society Symposium No. 8. Ed. by R. M. W adsworth et al .
Guide to the Check Sheet for IBP Areas . IBP Handbook No. 4. By G. E. P eterken .     

Molecular cloning and expression of human tumor necrosis factor and comparison with mouse tumor necrosis factor

U-937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA. Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system. TNF mRNA was enriched by gradient centrifugation and this size-fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153. A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF cDNA clone [Fransen, L., Mueller, R., Marmenout, A., Tavernier, J., Van der Heyden, J., Kawashima, E., Chollet, A., Tizard, R., Van Heuverswyn, H., Van Vliet, A., Ruysschaert, M. R. & Fiers, W. (1985) Nucleic Acids Res. 13, 4417-4429] as a probe. The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al. [Pennica, D., Nedwin, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palladino, M. A., Kohr, W. J., Aggarwal, B. B. & Goeddel, D. V. (1984) Nature (Lond.) 312, 724-729]. The 157-amino-acid-long mature sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF. In contrast to mouse TNF, it contains no potential N-glycosylation site. When compared to other cytokines, like IFN-beta, IFN-gamma, or IL-2, there is a remarkably high preference for G X C pairs in the third-letter positions. Expression of the TNF cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF. A human genomic clone was also identified and sequenced; it was found to be in good agreement with the one recently published by Shirai et al. [Shirai, T., Yamaguchi, H., Ito, H., Todd, C. W. & Wallace, R. B. (1985) Nature (Lond.) 313, 803-806], except for some differences in the introns and 5'-untranslated region.     

The corrected nucleotide sequences of the TaqI restriction and modification enzymes reveal a thirteen-codon overlap.

The nucleotide sequence of the genes encoding methyltransferase TaqI (M.TaqI) and restriction endonuclease TaqI (R.TaqI) with the recognition sequence, TCGA, were analyzed in clones isolated from independent libraries. The genes, originally reported as 363 and 236 codons long [Slatko et al., Nucleic Acids Res. 15 (1987) 9781-9796] were redetermined as 421 and 263 codons long, respectively. The C terminus of the taqIM gene overlaps the N terminus of the taqIR gene by 13 codons, as observed with the isoschizomeric TthHB8I restriction-modification system [Barany et al., Gene 112 (1992) 13-20]. Removal of the overlapping codons did not interfere with in vivo M.TaqI activity. We postulate the overlap plays a role in regulating taqIR expression.     

REVIEWS

Book Reviewed in this article:
Studies in the Vegetational History of the British Isles: Essays in Honour of Harry Godwin. Edited by D. W alker and R. G. W est
Introduction to British Lichens. BY U. K. D uncan assisted by P. W. J ames .
Nightshades-The Paradoxical Plants. By C harles B. H eiser , JR.
Hummingbirds and their Flowers. By K aren A. G rant and V erne G rant.
The Biogenesis of Starch Granules in Higher Plants. By N. P. B adenhuizen.
The Mathematics of Heredity. By G ustave M alécot . Translated by D. M. Y ermanos.
British Fungus Flora. Agarics and Boleti: Introduction. By D. M. H enderson , P. D. O rton and R. W atling .
La Forêt. By C. J acquiot.
Relevé Methodique de la Végétation et du Milieu. By M ichel G odron et al; Editor L. E mberger.
World Pollen Flora . Ed. by G. E rdtman.
Marine Algae of Dominica. By W. R andolph T aylor and C. F. R hyne .     

竹亚科刚竹属植物的修订（V）

在模式材料、实物照片或采集物观察比较以及实地考察的基础上,结合原始描述,对竹亚科Bambuso-ideae刚竹属Phyllostachys Sieb．et Zucc．中最后一批存疑或悬而未决的拉丁双名进行了考订。分别将彭县刚竹P.sapida并入石绿能P.arcana．P.balansae并入桂讯P．bambusoides,金销仔P．subulata并入寿讯Pbambusoidesf．shouzhu,广州刚竹P．cantoniensis并入水竹Pheteroclada,大节刚竹P．lomsnanensis瞎和刺芒刚竹Paristata均并人笔笋竹Pnidulariaf．basipilis,贵州刚竹P．guizhouensis并入毛金竹P．nigra var．henonb,台湾石竹Pfithophila并入刚竹P.sulphureavar．viridis,均作为异名处理;讨论了产于越南的3个种的归属问题,推测P．caobangensis可能与桂竹P．bambusoides为同种,而P．baccanensis和户．vietbacensis可能均与假毛竹尸．kwangsiensis为同种。     

Skeletal muscle sodium channel is affected by an epileptogenic beta1 subunit mutation

The syndrome of generalized epilepsy with febrile seizures plus type 1 (GEFS+) has been associated to the gene SCN1B coding for the sodium channel beta1 subunit (Wallace, R. H. et al. (1998) Nature Genetics 19, 366-370). In patients, a mutation of the cysteine 121 to trpyptophane (C121W) would cause a lack of modulatory activity of the beta1 subunit on sodium channels expressed in the brain, rendering neurons hyperexcitable. We have confirmed that the normal beta1-modulation of type-IIA adult brain alpha subunits (BIIA) expressed in frog oocytes is defective in C121W. We observed that the mixture of wild-type and mutant beta1 subunits is less effective than wild-type alone, suggesting that the mutant beta1 subunit does bind the alpha subunit. However, we also observed a similar lack of modulation by C121W of the in adult skeletal muscle alpha subunit (SkM1). This finding is in contrast with the simple idea that the mutational effect observed in the oocyte expression system is the principal physiopathological correlate of GEFS+, because no skeletal muscle symptoms have been reported in GEFS+ patients. We conclude that the manifestation of the pathological phenotype is conditioned by the presence of susceptibility genes and/or that the frog oocyte expression system is inadequate for the study of the mutant beta1 subunit physiopathology.     

Genes for two herbicide-inducible cytochromes P-450 from Streptomyces griseolus.

Streptomyces griseolus ATCC 11796 contains two inducible, herbicide-metabolizing cytochromes P-450 previously designated P-450SU1 and P-450SU2 (P-450CVA1 and P-450CVB1, respectively, using nomenclature of Nebert et al. [D. W. Nebert, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, DNA 6:1-11, 1987]). Using antibodies directed against cytochrome P-450SU1, its N-terminal amino acid sequence, and amino acid composition, we cloned the suaC gene encoding cytochrome P-450SU1. Similar information about the cytochrome P-450SU2 protein confirmed that a gene cloned by cross-hybridization to the suaC gene was the subC gene encoding cytochrome P-450SU2. The suaC and subC genes were expressed in Escherichia coli, DNA for both genes was sequenced, and the deduced amino acid sequences were compared with that of the well-characterized cytochrome P-450CAM from Pseudomonas putida. Both cytochromes P-450SU1 and P-450SU2 contain several regions of strong similarity with the amino acid sequence of P-450CAM, primarily in regions of the protein responsible for attachment and coordination of the heme prosthetic group.     

Prediction of treatment response using gene expression profiles

This paper concerns prediction of clinical outcome from gene expression profiles using work in a different area, nonlinear system identification. In particular, the approach can predict long-term treatment response from data of a landmark article by Golub et al. (Golub, T. R.; Slonim, D. K.; Tamayo, P.; Huard, C.; Gaasenbeek, M.; Mesirov, J. P. et al. Science 1999, 286, 531-537) that has not previously been achieved with these data. The present paper shows that, for these data, gene expression profiles taken at time of diagnosis of acute myeloid leukemia contain information predictive of eventual response to chemotherapy. This was not evident in previous work; indeed, the Golub et al. article did not find a set of genes strongly correlated with clinical outcome. However, the present approach can accurately predict outcome class of gene expression profiles even when the genes do not have large differences in expression levels between the classes.     

Genetic screen for cloned release factor genes.

Nonsense suppression reflects competition between a nonsense suppressor tRNA and a translational release factor. This provides a simple way to screen for release factor genes cloned into a multicopy plasmid. We have confirmed that the expected competition occurs with the gene for release factor 2, cloned by C.T. Caskey et al. (C.T. Caskey, W.C. Forrester, W. Tate, and C.D. Ward, J. Bacteriol. 158:365-368, 1984), and used it to clone the gene for release factor 1.     

Effects of substitution of tryptophan 412 in the substrate activation pathway of yeast pyruvate decarboxylase.

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at W412, located on the putative substrate activation pathway and linking E91 on the alpha domain with W412 on the gamma domain of the enzyme. While C221 on the beta domain is the residue at which substrate activation is triggered [Baburina, I., et al. (1994) Biochemistry 33, 5630-5635; Baburina, I., et al. (1996) Biochemistry 35, 10249-10255], that information, via the substrate bound at C221, is transmitted to H92 on the alpha domain, across the domain divide from C221 [Baburina, I., et al. (1998) Biochemistry 37, 1235-1244; Baburina, I., et al. (1998) Biochemistry 37, 1245-1255], thence to E91 on the alpha domain [Li, H., and Jordan, F. (1999) Biochemistry 38, 9992-10003], and then on to W412 on the gamma domain and to the active site thiamin diphosphate located at the interface of the alpha and gamma domains [Arjunan, D., et al. (1996) J. Mol. Biol. 256, 590-600]. Substitution at W412 with F and A was carried out, resulting in active enzymes with specific activities about 4- and 10-fold lower than that of the wild-type enzyme. Even though W412 interacts with E91 and H115 via a main chain hydrogen bond donor and acceptor, respectively, there is clear evidence for the importance of the indole side chain of W412 from a variety of experiments: thermostability, fluorescence quenching, and the binding constants of the thiamin diphosphate, and circular dichroism spectroscopy, in addition to conventional steady-state kinetic measurements. While the substrate activation is still prominent in the W412F variant, its level is very much reduced in the W412A variant, signaling that the size of the side chain is also important in positioning the amino acids surrounding the active center to achieve substrate activation. The fluorescence studies demonstrate that W412 is a relatively minor contributor to the well-documented fluorescence of apopyruvate decarboxylase in its native state. The information about the W412 variants provides strong additional support for the putative substrate activation pathway from C221 --> H92 --> E91 --> W412 --> G413 --> thiamin diphosphate. The accumulating evidence for the central role of the beta domain in stabilizing the overall structure is summarized.     

Book Reviews

Book reviewed in this article:
Microbiological Applications of Gas Chromatography (1981). D.B. Drucker.
Introduction to Modern Mycology (1980). Volume 7 in the series Basic Microbiology. J.W. Deacon.
Current Topics in Microbiology and Immunology (1980). Volume 90. Edited by W. Arber et al.
Mould Growth in Buildings (1981). Proceedings of a joint BRE/Paint Research Association Seminar held at the Building Research Establishment
The Biochemistry and Pharmacology of Antibacterial Agents (1981). R.A.D. Williams & Z.L. Kruk.
Sexual Interactions in Eukaryotic Microbes (1981). Edited by D.H. O'Day & P.A. Horgen.
Microbiology of Human Skin (1981). W.C. Noble, 2nd Edition.
Antibiotics Volume IV. Biosynthesis (1981). Edited by J.W. Corcoran.
Food Microbiology: A Framework for the Future (1980). A.N. Sharpe.     

Evaluation of a pour-plate system with a rabbit plasma-bovine fibrinogen agar for the enumeration of Staphylococcus aureus in food

Investigations were carried out concerning the selectivity and productivity of rabbit plasma fibrinogen (RPF) agar according to Beckers et al. (H. J. Beckers, F. M. van Leusden, W. M. Hogeboom, and E. H. M. Delfgou-van Asch. 1980. De Ware(n)-Chemicus, 10: 125-130). Its selectivity was compared with pork plasma fibrinogen (PPF) medium according to Hauschild et al. (A. H. W. Hauschild, C. E. Park, and R. Hilsheimer. 1979. Can. J. Microbiol. 25: 1052-1057) and its productivity was compared with PPF medium and Baird-Parker's egg yolk tellurite glycine pyruvate (ETGP) agar. In total 139 samples of naturally contaminated foodstuffs were examined. RPF agar scored higher than ETGP agar; although only small (mean value of differences 0.09 log units), the differences were statistically significant. While no significant differences in sensitivity between RPF agar and PPF medium were encountered, RPF agar was statistically more selective than PPF medium. It is concluded that RPF agar is very suitable for the enumeration of Staphyloccus aureus in foods.