Incompatibility groups and the classification of fi - resistance factors

Incompatibility between R factors has been reported by several authors, and four incompatibility groups have already been described by Datta and Hedges among Rfi(-) factors. The stability of 12 plasmids in pairs was studied after 116 crosses, and five new groups were found, designated 5, 6, 7, 8, and 9. Each plasmid studied belongs to one single group. Incompatibility between plasmids in pairs is a clear-cut phenomenon, is easy to observe, and can provide a reliable method for recognizing and classifying resistance factors, and for tracing their spread among bacterial species.     

6-(Levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl groups as novel base-labile groups for 5'-hydroxy protection in solid-phase oligonucleotide synthesis

The 6-(levulinyloxymethyl)-3-methoxy-2-nitrobenzoyl (LMMoNBz) and 2-(levulinyloxymethyl)-5-methoxy-4-nitrobenzoyl (LMMpNBz) groups were developed as novel base-labile groups for 5'-hydroxy protection in solid-phase oligonucleotide synthesis. A comparative study of the utility of LMMoNBz, LMMpNBz, and 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) groups is described.     

Phase transition properties of 1,2- and 1,3-diacylphosphatidylethanolamines with modified head groups

The phase transition properties of dilute aqueous suspensions of "nonhydrated" (i.e., lipid suspensions which had not been heated above room temperature or above the main phase transition temperature of the fully hydrated lipid, whichever was lower) and hydrated 1,2(alpha)- and 1,3(beta)-dipalmitoylphosphatidylethanolamines with modified head groups have been determined by high-sensitivity differential scanning calorimetry at a scan rate of 0.1 K min-1. In both the 1,2 and 1,3 series, the head-group modifications of the phosphoethanolamine moiety included N-methyl, N,N-dimethyl, and N,N,N-trimethyl (phosphocholine). In the 1,2 series, additional modifications were dinitrophenyl, trinitrophenyl, N-(dinitrophenyl)aminocaproyl, N-(trinitrophenyl)aminocaproyl, and N-4-nitro-2,1,3-benzoxadiazole. Also included in this study were 1,2-dihexadecylphosphatidylethanolamine and the corresponding N-methyl-substituted lipid. In general, increasing bulkiness of the head-group substituent caused increasing lowering of the transition temperature, the most extreme cases among the hydrated lipids being the 45 degrees C lowering produced by the N-(dinitrophenyl)aminocaproyl substitution and its trinitrophenyl analogue in the 1,2 series. No simple trend is evident in the changes produced in the calorimetric enthalpy of transitions.     

Synthesis of beta-D-GlcA-(1----3)-beta-D-Gal disaccharides with 4- and 6-sulfate groups and 4,6-disulfate groups.

The sodium salts of the 6-sulfate 7, the 4-sulfate 10, and the 4,6-disulfate 12 of benzyl 3-O-(beta-D-glucopyranosyl uronate)-beta-D-galactopyranoside (5) have been synthesized. Methyl (2,3,4-tri-O-acetyl-1-bromo-1-deoxy-alpha-d-glucopyran)uronate (1) was coupled with benzyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-galactopyranoside (2) to yield 3. The benzylidene acetal of 3 was hydrolyzed to give benzyl 2-O-benzoyl-3-O-[methyl (2,3,4-tri-O-acetyl-beta-D-glucopyranosyl)uronate]-beta-D-galactopyra noside (4). Compound 4 was utilized as a key intermediate to prepare the sulfated disaccharides 7,10, and 12. Direct sulfation of 4 with sulfur trioxide-trimethylamine for 2 days yielded the 6-sulfate 6. The 4,6-disulfate 11 was accessible by running the reaction under the same conditions for 14 days. The 4-sulfate 9 was obtained after protecting the 6-OH group of 4 by reaction with benzoyl imidazole to give the 6-benzoate 8, followed by sulfation under vigorous conditions. Treatment of the protected compounds 4, 6, 9, and 11 with aqueous sodium hydroxide in tetrahydrofuran gave the unprotected 5, 7, 10, and 12, respectively.     

Affinity labelling to - SH groups in adenosine - triphosphate - phosphoribosyl transferase with the dinitrophenyl group from S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate.

Adenosine-triphosphate-phosphoribosyl transferase from Escherichia coli reacts with S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate. In this reaction the dinitrophenyl group becomes attached to the enzyme, while the nucleotide is split off. Most aliphatic high and low-molecular-weight-SH compounds react with the thioether in the opposite way, i.e. bind the nucleotide and split off dinitrothiophenol. It appears that the dinitrophenyl moiety of the thioether interacts with the enzyme in a specific way, and that this interaction activates the bond between the dinitrophenyl group and the sulfur atom. In support of this it was found that dinitrophenol inhibits the transferase reaction with half maximal effect at 0.4 mM. The inhibition is competitive with ATP. Dinitrophenol also competes with ATP in binding studies.     

2,2-Bis(ethoxycarbonyl)- and 2-(alkylaminocarbonyl)-2-cyano-substituted 3-(pivaloyloxy)propyl groups as biodegradable phosphate protections of oligonucleotides

Oligonucleotides bearing biodegradable phosphate protecting groups have been synthesized on a solid support. For this purpose, two dimeric building blocks, viz. 5'-O-(4,4'-dimethoxytrityl)-(R(P),S(P))-O(P)-[2,2-bis(ethoxycarbonyl)-3-(pivaloyloxy)propyl]-P-thiothymidylyl-(3',5')-thymidine 3'-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] (1) and 5'-O-(4,4'-dimethoxytrityl)-(R(P),S(P))-O(P)-[2-cyano-2-(2-phenylethylaminocarbonyl)-3-(pivaloyloxy)propyl]thymidylyl-(3',5')-thymidine 3'-(H-phosphonate) (2), were prepared. Phosphoramidite 1 was incorporated into an phosphorothioate oligothymidylate sequence on a base-labile hydroquinone-O,O'-diacetic acid linker (Q-linker) and on a photolabile 4-alkoxy-5-methoxy-2-nitrobenzyl carbonate linker (11). H-Phosphonate 2 was, in turn, incorporated into an oligothymidylate sequence only on the photolabile linker. Kinetics of the removal of the protecting groups by porcine liver esterase and subsequent retro aldol condensation/phosphate elimination were then studied. While the pro-oligonucleotide that contained only one phosphate protection gave the deprotected phosphorothioate oligonucleotide in a quantitative yield, the enzymatic step was markedly decelerated upon increasing the number of protection groups, and hence chain cleavage started to compete.     

Investigations into the preparation of groups 13-15 N-heterocyclic carbene analogues

Attempts have been made to prepare a variety of groups 13-15 N-heterocyclic carbenoid systems. The work in group 13 led to two structurally characterized, paramagnetic gallium(III) heterocycles, [I₂Ga{[N(R)C(Me)]₂}], or C₆H₃(C₆H₄Bu^t-4)₂-2,6. Reduction of the former gave the anionic gallium(I) heterocyclic complex, [K(tmeda)][:Ga{[N(Ar)C(Me)]₂}], which was oxidatively coupled, affording the structurally characterized digallane(4), [Ga{[N(Ar)C(Me)]₂}]₂. From group 14, the new germanium(IV) heterocycle, [Cl₂Ge{[N(Ar)C(H)]₂}], and the N-heterocyclic germylene, [:Ge{[N(Ar)C(H)]₂}], have been prepared and fully characterized. Attempts to prepare N-heterocyclic silylene, phosphenium and arsenium compounds were unsuccessful and led instead to the silyl, phosphino and arsino-substituted ene-amines, [(Cl_nE)₂{μ-[N(Ar)C(H)]₂}], E = Si, n = 3; E = P or As, n = 2.     

Regeneration of native bacteriorhodopsin structure following acetylation of epsilon-amino groups of Lys-30, -40, and -41

The chymotryptic fragment of bacteriorhodopsin, C-2 (residues 1-71), has been acetylated completely at its three lysines (residues 30, 40, and 41) by treatment with acetic anhydride. The triacetylated C-2 fragment is able to reassociate with fragment C-1 (residues 72-248) and the complex binds all-trans-retinal to form a native bacteriorhodopsin-like chromophore, which is essentially identical with that formed from fragments C-2 and C-1. Further, the kinetics and pH dependence of chromophore regeneration and the proton pumping of the reconstituted triacetylated C-2 and C-1 complex are indistinguishable from that of the unmodified C-2 and C-1 complex. However, the extent of regeneration of the chromophore from triacetylated C-2 and C-1 is less than that from fragments C-2 and C-1, suggesting that the acetylated C-2 fragment is less stable than unacetylated C-2 in the reconstitution medium. We conclude that the amino groups in Lys-30, -40, and -41 do not contribute to the stabilization of the folded bacteriorhodopsin structure and are not required for proton translocation.     

Surface-assisted delivery of fluorescent groups to hGST A1-1 and a lysine mutant

Human glutathione transferase (hGST) A1-1 and a lysine mutant (A216K) can both be rapidly and site-specifically acylated on Y9 and K216, respectively, using a range of thiolesters of glutathione (GS-thiolesters) as modifying reagents. The present investigation was aimed at developing a method with which to deliver a fluorescent acyl group from a solid support under conditions compatible with standard protein purification schemes. A number of fluorescent GS-thiolesters with modified peptide backbones were therefore prepared and tested for reactivity toward hGST A1-1 and the A216K mutant. Substitutions at the alpha-NH2 part of the glutathione backbone were not tolerated by the proteins. However, two fluorescent reagents that carry a biotin moiety at the C-terminal part of glutathione were found through MALDI-MS experiments to react in solution with Y9 of the wild-type protein and one reagent with K216 of A216K. The reaction can take place in the presence of glutathione and even in a crude E. coli lysate of cells expressing A216K. Delivery of the fluorescent group to Y9 or K216 was possible using NeutrAvidin (NA) beads that had been preincubated with biotinylated reagent. Alternatively, excess reagent can be removed by a brief incubation with NA beads. We have thus now developed a system for protein labeling with easy removal of excess and used up low-molecular weight reagent. This strategy can conceivably be utilized in future protein purification and labeling experiments.     

Inactivation of D-(-)-beta-hydroxybutyrate dehydrogenase by modifiers of carboxyl and histidyl groups

Data presented in this paper suggest that D-(-)-beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains an essential carboxyl group and an essential histidyl residue at or near the active site. Lactate and malate dehydrogenases, which catalyze reactions analogous to that catalyzed by BDH, also contain an aspartyl and a histidyl residue at the active site [Birktoft, J.J., & Banaszak, L.J. (1983) J. Biol. Chem. 258, 472-482]. In addition, all three enzymes contain an essential arginyl residue, apparently concerned with electrostatic interaction with their respective carboxylic acid substrates, and promote ternary adduct formation involving the enzyme, NAD, and sulfite.     

Violent and accidental mortality among four immigrant groups in Canada, 1970-1972.

For most people immigration to a new country such as Canada entails a positive move and an improvement in life. The many challenges associated with resettlement may, however, lead to insurmountable difficulties, stresses and conflict for a significant number of newcomers. The mortality experience of immigrants, as reflected in cause-of-death statistics, may provide indication of the extent of stress and conflict in their migration experience. This situation is most clearly exhibited in mortality from suicide, homicide, and motor vehicle accidents. In this study, hypotheses concerning immigrant mortality in Canada are developed and tested with a log-linear model for rates pertaining to rare events. Overall, the results give support for the importance of country-of-origin effects in explaining suicide propensities, but not for homicide and motor vehicle accidents mortality. Income discrepancies are a significant determinant of variability in death rates overall, but discrepancies between the immigrants in this study and the Canadian-born are not of much significance. The strongest net effect on the cause-specific death rate is associated with group membership. This effect likely reflects a number of residual unmeasured sources of variation including the influence of the immigrant ethnic community as a source of social support, and the potential confounding effects of migration selectivity.     

Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10-23

The RNA phosphodiester bond cleavage activity of a series of 16 thio-deoxyribozymes 10-23, containing a P-stereorandom single phosphorothioate linkage in predetermined positions of the catalytic core from P1 to P16, was evaluated under single-turnover conditions in the presence of either 3 mM Mg(2+) or 3 mM Mn(2+). A metal-specificity switch approach permitted the identification of nonbridging phosphate oxygens (proR(P) or proS(P)) located at seven positions of the core (P2, P4 and P9-13) involved in direct coordination with a divalent metal ion(s). By contrast, phosphorothioates at positions P3, P6, P7 and P14-16 displayed no functional relevance in the deoxyribozyme-mediated catalysis. Interestingly, phosphorothioate modifications at positions P1 or P8 enhanced the catalytic efficiency of the enzyme. Among the tested deoxyribozymes, thio-substitution at position P5 had the largest deleterious effect on the catalytic rate in the presence of Mg(2+), and this was reversed in the presence of Mn(2+). Further experiments with thio-deoxyribozymes of stereodefined P-chirality suggested direct involvement of both oxygens of the P5 phosphate and the proR(P) oxygen at P9 in the metal ion coordination. In addition, it was found that the oxygen atom at C6 of G(6) contributes to metal ion binding and that this interaction is essential for 10-23 deoxyribozyme catalytic activity.