Construction of intra-domain chimeras of aminoacyl-tRNA synthetases

To explore new approaches to enzyme engineering, intra-domain chimeras of two aminoacyl-tRNA synthetases were constructed. Connections were made within the nucleotide folds of these enzymes at sites earlier shown either to be dispensable for activity or able to accommodate oligopeptide insertions. (R.M. Starzyk, T.A. Webster and P. Schimmel, Science 237, 1614 (1987); R.M. Starzyk, J.J. Burbaum and P. Schimmel, Biochemistry, in press). Based on the known structure of one synthetase and structural modeling of the other, the locations of the connection sites allow the possibility of functional "compound" ATP and tRNA binding sites. Of five chimeric genes which were constructed, three direct synthesis of polypeptides that accumulate in vivo. These stable hybrids provide prototypes to which mutagenesis procedures may be applied to produce enzymatically active chimeric synthetases.     

Regulation of the biosynthesis of aminoacyl-tRNA synthetases and of tRNA in Escherichia coli. IV. Mutants with increased levels of leucyl- or seryl-tRNA synthetase.

Summary Spontaneous revertants of a temperature-sensitive Escherichia coli strain harboring a thermolabile leucyl-tRNA synthetase and seryl-tRNA synthetase were selected for growth at 40°C. Among these, strains were found with increased levels of both thermolabile synthetases. Two distinct genetic loci were found responsible for enzyme overproduction. leuR, located near xyl, causes elevated levels of leucyl-tRNA synthetase; while serR, located near leu, causes elevated levels of seryl-tRNA synthetase.The preceding paper in this series is by R. LaRossa, J. Mao, K.B. Low and D. Söll. J. Mol. Biol. 117, 1049 (1977)     

1H, 15N and 13C resonance assignments and secondary structure of group II phospholipase A2 from Agkistrodon piscivorus piscivorus: Presence of an amino-terminal helix in solution

Summary ¹H, ¹⁵N and ¹³C resonance assignments are presented for the group II phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus. The secondary structure of the enzyme has been inferred from an analysis of coupling constants, interproton distances, chemical shifts, and kinetics of amide exchange. Overall, the secondary structure of this PLA2 is similar to the crystal structure of the homologous group II human nonpancreatic secretory phospholipase [Scott, D.L., White, S.P., Browning, J.L., Rosa, J.J., Gelb, M.H. and Sigler, P.B. (1991) Science, 254, 1007–1010]. In the group I enzyme from porcine pancreas, the amino-terminal helix becomes fully ordered in the ternary complex of enzyme, lipid micelles and inhibitor. The formation of this helix is thought to be important for the increase in activity of phospholipases on aggregated substrates [Van den Berg, B., Tessari, M., Boelens, R., Dijkman, R., De Haas, G.H., Kaptein, R. and Verheij, H.M. (1995) Nature Struct. Biol., 2, 402–406]. However, the group II enzyme from Agkistrodon piscivorus piscivorus possesses a defined and well-positioned aminoterminal helix in the absence of substrate. Therefore, there is a clear difference between the conformations of group I and group II enzymes in solution. These conformational differences suggest that formation of the amino-terminal helix is a necessary, but not sufficient, step in interfacial activation of phospholipases.Abbreviations PLA2 phospholipase A2 - App-D49 phospholipase from Agkistrodon piscivorus piscivorus - NOE nuclear Overhauser effect     

Aminoacyl-tRNA synthetase-catalyzed cleavage of the glycosidic bond of 5-halogenated uridines.

Because of previous data suggesting that aminoacyl-tRNA synthetases make a transient Michael adduct with a specific uridine residue in the tRNA structure, (Schoemaker, H.J.P., and Schimmel, P.R. (1977) Biochemistry 16, 5454-5460) attempts were made to find simple model systems in which this reaction might be studied in more detail. In the course of these investigations, it was found that Escherichia coli Ile-tRNA synthetase catalyzes cleavage of the glycosidic bond of 5-bromouridine. At pH 7.5, ambient temperatures, the turnover number is roughly 5/h. 5-Fluoro-, 5-chloro-, and 5-iodouridine are also cleaved in an analogous way by Ile-tRNA synthetase. In the case of uridine, conversion of uridine to uracil and ribose was also detected, but with a smaller turnover number. Three other E. coli and one mammalian aminoacyl-tRNA synthetases were also examined and all were found to catalyze glycosidic bond cleavage of 5-bromouridine. The data indicate that, in general, synthetases have a catalytic center that shows an unusual reactivity for uridine.     

A New Synthesis of 9-(2-Deoxy-2-fluoro-β-D-arabinofuranosyl)guanine (araF-G)

Abstract

Interesting and very promising antisense properties of 2′-deoxy-2′-fluoroarabinonucleic acids ((a) Wilds, C.J.; Damha, M.J. 2′-Deoxy-2′-fluoroarabinonucleosides and oligonucleotides (2′F-ANA): synthesis and physicochemical studies. Nucl. Acids Res. 2000, 28, 3625–3635; (b) Viazovkina, E.; Mangos, M.; Elzagheid, M.I.; Damha, M.J. Current Protocols in Nucleic Acid Chemistry 2002, 4.15.1–4.15.21) (2′F-ANA) has encouraged our research group to optimize the synthetic procedures for 2′-deoxy-2′-fluoro-β-D-arabinonucleosides (araF-N). The synthesis of araF-U, araF-T, araF-A and araF-C is straightforward, (Tann, C.H.; Brodfuehrer, P.R.; Brundidge, S.P.; Sapino, C., Jr. Howell H.G. Fluorocarbohydrates in synthesis. An efficient synthesis of 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodouracil (β-FIAU) and 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)thymine (β-FMAU). J. Org. Chem. 1985, 50, 3644–3647; Howell, H.G.; Brodfuehrer, P.R.; Brundidge, S.P.; Benigni, D.A.; Sapino, C., Jr. Antiviral nucleosides. A stereospecific, total synthesis of 2′-fluoro-2′-deoxy-β-D-arabinofuranosyl nucleosides. J. Org. Chem. 1988, 53, 85–88; Maruyama, T.; Takamatsu, S.; Kozai, S.; Satoh, Y.; Izana, K. Synthesis of 9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine bearing a selectively removable protecting group. Chem. Pharm. Bull. 1999, 47, 966–970) however, the synthesis of the guanine analogue is more complicated and affords poor to moderate yields of araF-G (4) ((a) Elzagheid, M.I.; Viazovkina, E.; Masad, M.J. Synthesis of protected 2′-deoxy-2′-fluoro-β-D-arabinonucleosides. Synthesis of 2′-fluoroarabino nucleoside phosphoramidites and their use in the synthesis of 2′F-ANA. Current Protocols in Nucleic Acid Chemistry 2002, 1.7.1–1.7.19; (b) Tennila, T.; Azhayeva, E.; Vepsalainen, J.; Laatikainen, R.; Azhayev, A.; Mikhailopulo, I. Oligonucleotides containing 9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-adenine and -guanine: synthesis, hybridization and antisense properties. Nucleosides, Nucleotides and Nucl. Acids 2000, 19, 1861–1884). Here we describe an efficient synthesis of araF-G (4) that involves coupling of 2-deoxy-2-fluoro-3,5-di-O-benzoyl-α-D- arabinofuranosyl bromide (1) with 2-chlorohypoxanthine (2) to afford 2-chloro-β-araF-I (3) in 52% yield. Nucleoside (3) was transformed into araF-G (4) by treatment with methanolic ammonia (150°C, 6 h) in 67% yield.     

脓肿分枝杆菌MBT结构鉴定与比较演化分析

【背景】作为临床最常见的非结核条件致病分枝杆菌,脓肿分枝杆菌(Mycobacteroides abscessus)因其天然、多耐药等特性成为目前临床治疗的一大挑战。作为分枝杆菌限制性营养元素——铁摄取的关键系统,分枝杆菌素(mycobactin,MBT)、羧基分枝杆菌素(carboxymycobactin,cMBT)与病原分枝杆菌的毒力、耐药等密切相关。【目的】丰富分枝杆菌MBT、cMBT结构数据,探究MBT在致病分枝杆菌起源过程中的演化规律。【方法】在MALDI-TOF-MS与FT-MS/MS解析脓肿MBT、cMBT结构的基础上,进一步开展其活性分析与生物合成基因簇比较基因组分析。【结果】虽然脓肿分枝杆菌MBT、cMBT母核修饰模式与海洋分枝杆菌最相似,R1、R2、R3、R5等位置的修饰完全相同,而且脂肪酸链均位于R4位置;但脂肪酸链长度不同[C10-17 (MBT)、C4-8 (cMBT)],为新结构。Fe-cMBT不仅以浓度依赖方式促进脓肿分枝杆菌生长,而且利用效率显著高于FeCl₃,相关结果表明MBT-cMBT是脓肿分枝杆菌高效获取铁元素的关键系统。与MBT结构结果一致,mbt-1基因簇共线性分析及mbt-1、mbt-2系统发育分析结果均表明脓肿分枝杆菌与海洋分枝杆菌(M.marinum)亲缘关系最近,而非结核分枝杆菌(M.tuberculosis)或耻垢分枝杆菌(M.smegmatis) (基于16S rRNA基因序列分析)。进一步分析发现,M.marinum、M.tuberculosis、M.bovis等病原分枝杆菌脂肪酸链长度变化范围仅4 C,而M.abscessus、M.fortuitum、M.avium和M.smegmatis等条件致病与非致病菌的脂肪酸链长度变化范围为7-11 C,暗示MBT同系物脂肪酸链长度变化范围与分枝杆菌不同生活方式、环境之间可能存在关联。【结论】作为获取铁元素的关键系统,具有独特结构的脓肿分枝杆菌MBT-cMBT在致病、耐药等方面的作用及起源、演化规律值得深入研究。     

MTH187 from Methanobacterium thermoautotrophicum has three HEAT-like Repeats

With the completion of genome sequencing projects, there are a large number of proteins for which we have little or no functional information. Since protein function is closely related to three-dimensional conformation, structural proteomics is one avenue where the role of proteins with unknown function can be investigated. In the present structural project, the structure of MTH187 has been determined by solution-state NMR spectroscopy. This protein of 12.4 kDa is one of the 424 non-membrane proteins that were cloned and purified for the structural proteomic project of Methanobacterium thermoautotrophicum [Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Gerstein, M., Arrowsmith, C.H. and Edwards, A.M. (2000) Prog. Biophys. Mol. Biol., 73, 339–345]. Methanobacterium thermoautotrophicum is a thermophilic archaeon that grows optimally at 65 °C. A particular characteristic of this microorganism is its ability to generate methane from carbon dioxide and hydrogen [Smith, D.R., Doucette-Stamm, L.A., Deloughery, C., Lee, H., Dubois, J., Aldredge, T., Bashirzadeh, R., Blakely, D., Cook, R., Gilbert, K., Harrison, D., Hoang, L., Keagle, P., Lumm, W., Pothier, B., Qiu, D., Spadafora, R., Vicaire, R., Wang, Y., Wierzbowski, J., Gibson, R., Jiwani, N., Caruso, A., Bush, D., Reeve, J. N. et al. (1997) J. Bacteriol., 179, 7135–7155].Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. Structure data have been deposited at PDB (1TE4) and NMR data at BMRB (5629).Olivier Julien and Isabelle Gignac - Both authors contributed equally to this work.     

Evidence for the early divergence of tryptophanyl- and tyrosyl-tRNA synthetases

Each amino acid is attached to its cognate tRNA by a distinct aminoacyl-tRNA synthetase (aaRS). The conventional evolutionary view is that the modern complement of synthetases existed prior to the divergence of eubacteria and eukaryotes. Thus comparisons of prokaryotic and eukaryotic aminoacyl-tRNA synthetases of the same type (charging specificity) should show greater sequence similarities than comparisons between synthetases of different types—and this is almost always so. However, a recent study [Ribas de Pouplana L, Furgier M, Quinn CL, Schimmel P (1996) Proc Natl Acad Sci USA 93:166–170] suggested that tryptophanyl- (TrpRS) and tyrosyl-tRNA (TyrRS) synthetases of the Eucarya (eukaryotes) are more similar to each other than either is to counterparts in the Bacteria (eubacteria). Here, we reexamine the evolutionary relationships of TyrRS and TrpRS using a broader range of taxa, including new sequence data from the Archaea (archaebacteria) as well as species of Eucarya and Bacteria. Our results differ from those of Ribas de Pouplana et al.: All phylogenetic methods support the separate monophyly of TrpRS and TyrRS. We attribute this result to the inclusion of the archaeal data which might serve to reduce long branch effects possibly associated with eukaryotic TrpRS and TyrRS sequences. Furthermore, reciprocally rooted phylogenies of TrpRS and TyrRS sequences confirm the closer evolutionary relationship of Archaea to eukaryotes by placing the root of the universal tree in the Bacteria. Received: 7 December 1996 / Accepted: 11 February 1997     

Synthesis of 2′,3′-Dideoxy-2′-fluoro-3′-thioarabinothymidine and Its 3′-Phosphoramidite Derivative

Abstract

An efficient method for the synthesis of 5′-O-monomethoxytrityl-2′,3′-dideoxy-2′-fluoro-3′-thioarabinothymidine [^5′-MMTaraF-T_3′SH, (5)] and its 3′-phosphoramidite derivative (6) suitable for automated incorporation into oligonucleotides, is demonstrated. A key step in the synthesis involves reaction of 5′-O-MMT-2,3′-O-anhydrothymidine (4) (Eleuteri, A.; Reese, C.B.; Song, Q., J. Chem. Soc. Perkin Trans. 1 1996, 2237 pp.) with sodium thioacetate to give ^5′-MMTaraF-T_3′SAc (5) (Elzagheid, M.I.; Mattila, K.; Oivanen, M.; Jones, B.C.N.M.; Cosstick, Lönnberg, H. Eur. J. Org. Chem. 2000, 1987–1991). This nucleoside was then converted to its corresponding phosphoramidite derivative, 6, as described previously ((a) Sun, S.; Yoshida, A.; Piccirilli, J.A. RNA, 1997, 3, 1352–1363; (b) Matulic-Adamic, J.; Beigelman, L. Helvetica Chemica Acta 1999, 82, 2141–2150; (c) Fettes, K.J.; O’Neil, I.; Roberts, S.M.; Cosstick, R. Nucleosides, Nucleotides and Nucl. Acids 2001, 20, 1351–1354).     

A novel plasmid-mediated DNA restriction-modification system in E. coli

R plasmids from 101 clinical isolates were transferred to E. coli J62 by conjugation and tested for the presence of R plasmid-mediated restriction-modification DNA systems. Thirty R plasmids were found to inhibit phage λ. vir development. Ten plasmids determined restriction modification system; nine of them proved identical with R.M. EcoRII. One transconjugant, E. coli J62 pLG74, was shown to have a restriction-modification system different from all the known R plasmid-mediated systems. Site-specific endonuclease has been isolated from E. coli J62 pLG74 which differed from all the known restriction endonucleases in the number of cleavage sites on phages λ, φX 174, virus SV40, plasmid pBR322 DNA molecules.     

Reviews of publications

Book reviewed in this article: Reproduction of Marine Invertebrates. Vol. VI. Echinoderms and Lophophorates, edited by A. C. Giese, J. S. Pearse and V. B. Pearse. Pacific Grove, California, U.S.A. The Butterflies of Kenya and their Natural History, by T. B. Larsen. The Land and Freshwater Molluscs of Suffolk, by I. J. Killeen. Ipswich, U.K. Flycatcher. Memoirs of an Amateur Entomologist by K. A. Spencer. Ecosystems of the World 24. Intertidal and Littoral Ecosystems, edited by A. C. Mathieson and P. H. Nienhuis. Revision of the Northeast Atlantic and Mediterranean Stylasteridae (Cnidaria: Hydrozoa), by Helmut Zibrowius and Stephen D. Cairns. Paris: Mémoires du Musée Nationale d'Histoire Naturellee, 1992. Animals and Disease. An Introduction to the History of Comparative Medicine, by L. Wilkinson. The Inevitable Bond. Examining Scientist-Animal Interactions, edited by H. Davis and D. Balfour. Martin Lister's English Spiders (1678), edited by J. Parker and B. Harley (translated by M. Davies and B. Harley)). The Ecology of Fishes on Coral Reefs, edited by P. F. Sale. San Diego, USA: The Lepidoptera: Form, Function and Diversity, by M. J. Scoble. Bats. A Community Perspective, by J. S. Findley. The Insects of Australia. A textbook for students and research workers, Commonwealth Scientific and Industrial Research Organisation. Second Edition. Botanical monkeys, by E. J. H. Corner. The Biology of Millipedes, by S. P. Hopkin and H. J. Read. Mammals in the Palaearctic Desert: Status and Trends in the Sahara-Gobian Region, edited by J. A. McNeely and V. M. Neronov. Coelenterate Biology: Recent Research on Cnidaria and Ctenophora, edited by R. B. Williams, P. F. S. Cornelius, R. G. Hughes and E. A. Robson. Dordrecht, The Netherlands: Kluwer Academic Publishers, 1991. Animal Behaviour, 4th ed., by A. Manning and M. S. Dawkins. Weevils, Naturalists' Handbook 16, by M. G. Morris. Insects on Cabbage and Oilseed Rape. Naturalists' Handbooks 18, by W. D. J. Kirk.     

Isolation of Antibiotics Turbomycin A and B from a Metagenomic Library of Soil Microbial DNA

To access the genetic and biochemical potential of soil microorganisms by culture-independent methods, a 24,546-member library in Escherichia coli with DNA extracted directly from soil had previously been constructed (M. R. Rondon, P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A. MacNeil, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman, Appl. Environ. Microbiol. 66:2541-2547, 2000). Three clones, P57G4, P89C8, and P214D2, produced colonies with a dark brown melanin-like color. We fractionated the culture supernatant of P57G4 to identify the pigmented compound or compounds. Methanol extracts of the acid precipitate from the culture supernatant contained a red and an orange pigment. Structural analysis revealed that these were triaryl cations, designated turbomycin A and turbomycin B, respectively; both exhibited broad-spectrum antibiotic activity against gram-negative and gram-positive organisms. Mutagenesis, subcloning, and sequence analysis of the 25-kb insert in P57G4 demonstrated that a single open reading frame was necessary and sufficient to confer production of the brown, orange, and red pigments on E. coli; the predicted product of this sequence shares extensive sequence similarity with members of the 4-hydroxyphenylpyruvate dioxygenase (4HPPD) family of enzymes. Another member of the same family of genes, lly, which is required for production of the hemolytic pigment in Legionella pneumophila, also conferred production of turbomycin A and B on E. coli. We further demonstrated that turbomycin A and turbomycin B are produced from the interaction of indole, normally secreted by E. coli, with homogentisic acid synthesized by the 4HPPD gene products. The results demonstrate successful heterologous expression of DNA extracted directly from soil as a means to access previously uncharacterized small organic compounds, serving as an example of a chimeric pathway for the generation of novel chemical structures.     

Mathematical modeling of capillary formation and development in tumor angiogenesis: penetration into the stroma

The purpose of this paper is to present a mathematical model for the tumor vascularization theory of tumor growth proposed by Judah Folkman in the early 1970s and subsequently established experimentally by him and his coworkers [Ausprunk, D. H. and J. Folkman (1977) Migration and proliferation of endothelial cells in performed and newly formed blood vessels during tumor angiogenesis, Microvasc Res., 14, 53–65; Brem, S., B. A. Preis, ScD. Langer, B. A. Brem and J. Folkman (1997) Inhibition of neovascularization by an extract derived from vitreous Am. J. Opthalmol., 84, 323–328; Folkman, J. (1976) The vascularization of tumors, Sci. Am., 234, 58–64; Gimbrone, M. A. Jr, R. S. Cotran, S. B. Leapman and J. Folkman (1974) Tumor growth and neovascularization: an experimental model using the rabbit cornea, J. Nat. Cancer Inst., 52, 413–419]. In the simplest version of this model, an avascular tumor secretes a tumor growth factor (TGF) which is transported across an extracellular matrix (ECM) to a neighboring vasculature where it stimulates endothelial cells to produce a protease that acts as a catalyst to degrade the fibronectin of the capillary wall and the ECM. The endothelial cells then move up the TGF gradient back to the tumor, proliferating and forming a new capillary network. In the model presented here, we include two mechanisms for the action of angiostatin. In the first mechanism, substantiated experimentally, the angiostatin acts as a protease inhibitor. A second mechanism for the production of protease inhibitor from angiostatin by endothelial cells is proposed to be of Michaelis-Menten type. Mathematically, this mechanism includes the former as a subcase. Our model is different from other attempts to model the process of tumor angiogenesis in that it focuses (1) on the biochemistry of the process at the level of the cell; (2) the movement of the cells is based on the theory of reinforced random walks; (3) standard transport equations for the diffusion of molecular species in porous media. One consequence of our numerical simulations is that we obtain very good computational agreement with the time of the onset of vascularization and the rate of capillary tip growth observed in rabbit cornea experiments [Ausprunk, D. H. and J. Folkman (1977) Migration and proliferation of endothelial cells in performed and newly formed blood vessels during tumor angiogenesis, Microvasc Res., 14, 73–65; Brem, S., B. A. Preis, ScD. Langer, B. A. Brem and J. Folkman (1997) Inhibition of neovascularization by an extract derived from vitreous Am. J. Opthalmol., 84, 323–328; Folkman, J. (1976) The vascularization of tumors, Sci. Am., 234, 58–64; Gimbrone, M. A. Jr, R. S. Cotran, S. B. Leapman and J. Folkman (1974) Tumor growth and neovascularization: An experimental model using the rabbit cornea, J. Nat. Cancer Inst., 52, 413–419]. Furthermore, our numerical experiments agree with the observation that the tip of a growing capillary accelerates as it approaches the tumor [Folkman, J. (1976) The vascularization of tumors, Sci. Am., 234, 58–64]. An erratum to this article is available at .     

Gene Pool Structure of Eastern Ukrainians as Inferred from the Y-Chromosome Haplogroups

Y chromosomes from representative sample of Eastern Ukrainians (94 individuals) were analyzed for composition and frequencies of haplogroups, defined by 11 biallelic loci located in non-recombining part of the chromosome (SRY1532, YAP, 92R7, DYF155S2, 12f2, Tat, M9, M17, M25,M89, andM56). In the Ukrainian gene, pool six haplogroups were revealed: E, F (including G and I), J, N3, P, and R1a1. These haplogroups were earlier detected in a study of Y-chromosome diversity on the territory of Europe as a whole. The major haplogroup in the Ukrainian gene pool, haplogroup R1a1 (earlier designated HG3), accounted for about 44% of all Y chromosomes in the sample examined. This haplogroup is thought to mark the migration patterns of the early Indo-Europeans and is associated with the distribution of the Kurgan archaeological culture. The second major haplogroup is haplogroup F (21.3%), which is a combination of the lineages differing by the time of appearance. Haplogroup P found with the frequency of 9.6%, represents the genetic contribution of the population originating from the ancient autochthonous population of Europe. Haplogroups J and E (11.7 and 4.2%, respectively) mark the migration patterns of the Middle-Eastern agriculturists during the Neolithic. The presence of the N3 lineage (9.6%) is likely explained by a contribution of the assimilated Finno–Ugric tribes. The data on the composition and frequencies of Y-chromosome haplogroups in the sample studied substantially supplement the existing picture of the male lineage distribution in the Eastern Slav population.     

Book Reviews

A DEPARTMENT DEVOTED TO ABSTRACTS OF BOOKS AND PAPERS FROM OTHER JOURNALS DEALING WITH STAINS AND MICROSCOPIC TECHNIC IN GENERAL

CONN, H. J., DARROW, MARY A., and EMMEL, VICTOR M. Staining Procedures, 2nd Ed. 289 pages, 9 × 6 inches, perforated; spiral plastic binding, with cardbooard covers. The Williams & Wilkins Company, Baltimore 2, Maryland. $5.00. 1960.

GURR, EDWARD. Encyclopaedia of Microscopic Stains. 498 pages, 10 × 6 inches. The Williams & Wilkins Co., Baltimore 2, Maryland, exclusive U. S. agents. $18.50. 1960.

ELECTRON MICROSCOPY AMELUNXEN, F., and THEMANN, H. Zur Fixation mit Kaliumpermanganat. Mikroscopie, 14, 276-83. 1960.

PHOTOMICROGRAPHY MIGNANI, G., MARCHETTI, P. G., and HUSSL, B. Die Anwendung der mikroradiographischen Technik zum Studium des Knochengewebes. Mikroskopie, 14, 131-43. 1959.

DYES AND THEIR BIOLOGICAL USES KAUFMAN, L., and WEAVER, R. H. Use of neutral red fluorescence for the identification of colonies of Clostridia. J. Bact., 79, 292-4. 1960.

ANIMAL MICROTECHNIC LOUIS, C. J., and WHITE, J. Fluorescein-globulin staining of cells in tissue cultures. Lab. Invest., 9, 273-82. 1960.

McFARLANE, J. E., and KENNARD, C. P. Further observations on water absorption by the eggs of Acheta domesticus (L). Canad. J. Zool., 38, 77-85. 1960.

PROVENZA, D. V., and CHENG, T. C. A simplified parlodion method for sectioning teeth; with notes on the decalcification of teeth. Trans. Am. Micr. Soc., 79, 103-4. 1960.

PLANT MICROTECHNIC DEVIDÉ, Z., and WRISCHER, M., Versuche über gasblasenfreie Plexiglas Einbettung von pflanzlichen Objecten für Ultramikrotomie. Mikroskopie, 14, 337-42. 1960.

MICROORGANISMS BORZANI, W. Measurement of gram-positiveness. J. Bact., 79, 431-3. 1960.

DEIBEL, R. H., and EVANS, J. B. Modified benzidine test for the detection of cytochrome-containing respiratory systems in microorganisms. J. Bact., 79, 356-60. 1960.

SAHAB, K. Method for demonstrating capsules of enterobacteriaceae. J. Bact., 79, 198-202. 1960.

WEIBULL, C. Counting procedures for phase contrast microscopy. J. Bact., 79, 155. 1960.

HISTOCHEMISTRY BAKER, J. R. II, HEW, H., and FISHMAN, W. H. The use of chloral hydrate formaldehyde fixative solution in enzyme histochemistry. J. Histochem. Cytochem., 6, 244-50. 1958.

FISCHER, R., and ZALESCHUK, J. A semi-micro method for the determination of crystal violet sorbed to biological material. J. Histochem. Cytochem., 6, 237-43. 1958.

TAKEUCHI, T. Histochemical demonstration of branching enzyme (amylo-1,4 £ 1,6-transglucosidase), in animal tissues. J. Histochem. Cytochem., 6, 208-16. 1958.

ZUGIBE, F. T., KOPACZYK, K. C., CAPE, W. E., and LAST, J. H. A new Carbowax method for routinely performing lipid, hematoxylin and eosin and elastic staining techniques on adjacent freeze-dried or formalin-fixed sections. J. Histochem. Cytochem., 6, 133-8. 1958.     

铜绿假单胞菌二鸟苷酸环化酶SiaD突变体的功能研究

【目的】铜绿假单胞菌(Pseudomonas aerugionsa)二鸟苷酸环化酶SiaD调控着铜绿假单胞菌的生物被膜形成等表型。在研究过表达siaD对生物被膜的调控作用时发现,与野生型siaD基因回补菌株相比,一株回补菌株的生物被膜产量显著升高。本文的目的即是探究该菌株生物被膜产量升高的原因,并对该菌株的其他表型进行研究。【方法】通过测序确定突变位点;利用生物被膜定性和定量实验对发生点突变的菌株表型进行分析;利用Western blotting实验检测SiaD^R119M蛋白表达水平;利用GST-pulldown实验检测SiaC蛋白与SiaD^R119M蛋白在体外的结合能力;针对siaD^R119M点突变基因进行融合蛋白表达载体的构建,表达并纯化该蛋白,利用高效液相色谱检测SiaD^R119M的酶活;为了进一步研究c-di-GMP与细菌运动能力的关系,对细菌的运动能力进行检测。【结果】测序比对结果显示,序列的第119个氨基酸发生了突变,由精氨酸突变成了甲硫氨酸。生物被膜定性和定量实验显示,与野生型siaD...     

The Conserved Motif in Hydrophilic Loop 2/3 and Loop 8/9 of the Lactose Permease of Escherichia coli. Analysis of Suppressor Mutations

The major facilitator superfamily (MFS) of transport proteins, which includes the lactose permease of Escherichia coli, contains a conserved motif G-X-X-X-D/E-R/K-X-G-R/K-R/K in the loops that connect transmembrane segments 2 and 3, and transmembrane segments 8 and 9. In three previous studies (Jessen-Marshall, A.E., & Brooker, R.J. 1996. J. Biol. Chem. 271:1400–1404; Jessen-Marshall, A.E., Parker, N., & Brooker, R.J. 1997. J. Bacteriol. 179:2616–2622; and Pazdernik, N., Cain, S.M., & Brooker, R.J. 1997. J. Biol. Chem. 272:26110–26116), suppressor mutations at twenty different sites were identified which restore function to mutant permeases that have deleterious mutations in the conserved loop 2/3 or loop 8/9 motif. In the current study, several of these second-site suppressor mutations have been separated from the original mutation in the conserved motif. The loop 2/3 suppressors were then coupled to a loop 8/9 mutation (P280L) and the loop 8/9 suppressors were coupled to a loop 2/3 mutation (i.e., G64S) to determine if the suppressors could restore function only to a loop 2/3 mutation, a loop 8/9 mutation, or both. The single parent mutations changing the first position in loop 2/3 (i.e., G64S) and loop 8/9 (i.e., P280L) had less than 4% lactose transport activity. Interestingly, most of the suppressors were very inhibitory when separated from the parent mutation. Two suppressors, A50T and G370V, restored substantial transport activity when individually coupled to the mutation in loop 2/3 and also when coupled to the corresponding mutation in loop 8/9. In other words, these suppressors could alleviate a defect imposed by mutations in either half of the permease. From a kinetic analysis, these suppressors were shown to exert their effects by increasing the V _max values for lactose transport compared with the single G64S and P280L strains. These results are discussed within the context of our model in which the two halves of the lactose permease interact at a rotationally symmetrical interface, and that lactose transport is mediated by conformational changes at the interface. Received: 18 November 1999/Revised: 11 April 2000     

Calcium,magnesium, and phosphorus content of hair from two populations of rhesus monkeys

The use of hair for the assessment of human mineral nutritional status is controversial, yet has potential because of the ease of collection and storage, and the high concentration of minerals in hair. This study generated baseline values for hair macromineral content in the most commonly used primate model for human nutrition and disease, the rhesus monkey. Hair digests from monkeys in Maryland (n=98) and Puerto Rico (n=208) were analyzed by inductively coupled plasma atomic emission spectrometry (ICP-AES). Significant differences existed between the two populations for hair calcium (Ca), magnesium (Mg), and phosphorus (P) (P<0.01,P<0.001,P<0.02). Male hair had higher concentrations of Mg in specimens from the Puerto Rican population (P<0.001), whereas hair phosphorus was higher in female hair (P<0.02). There were no significant differences attributable to pregnancy status. Differences in hair content attributable to categorical age were found for Ca and Mg (P<0.001,P<0.01). Significant location differences paralleled dietary differences. These values therefore can be considered reference ranges for hair Ca, Mg, and P for free-ranging and caged rhesus monkeys. Preliminary reports of portions of these data were presented at the 69th Annual Meeting of the Federation of American Societies for Experimental Biology, April 1982, New Orleans, LA (B. M. Marriott, J. C. Smith, R. Jacobs, A. O. Jones, M. J. Kessler, and R. Rawlins,Fed. Proc. 41:770 [1982]) and in a symposium at the 6th Annual Meeting of the American Society of Primatologists, 1983, East Lansing, MI (B. Marriott, J. C. Smith, R. Jacobs, A. O. Lee Jones, R. Rawlins, and M. J. Kessler,Am. J. Primatol. 4(3):157 [1983]; B. Marriott, J. C. Smith, Jr., R. M. Jacobs, A. O. Lee Jones, R. Rawlins, and M. J. Kessler, inThe Cayo Santiago Macaques, R. Rawlins and M. J. Kessler, eds., SUNY Press, Albany, NY, pp. 219–231 [1986]).