Adjacent cysteine residues as a redox switch.

Oxidation of adjacent cysteine residues into a cystine forms a strained eight-membered ring. This motif was tested as the basis for an enzyme with an artificial redox switch. Adjacent cysteine residues were introduced into two different structural contexts in ribonuclease A (RNase A) by site-directed mutagenesis to produce the A5C/A6C and S15C/S16C variants. Ala5 and Ala6 are located in an alpha-helix, whereas Ser15 and Ser16 are located in a surface loop. Only A5C/A6C RNase A had the desired property. The catalytic activity of this variant decreases by 70% upon oxidation. The new disulfide bond also decreases the conformational stability of the A5C/A6C variant. Reduction with dithiothreitol restores full enzymatic activity. Thus, the insertion of adjacent cysteine residues in a proper context can be used to modulate enzymatic activity.     

Inosine.adenine base pairs in a B-DNA duplex.

The structure of the synthetic deoxydodecamer d(C-G-C-I-A-A-T-T-A-G-C-G) has been determined by single crystal X-ray diffraction techniques at 2.5A resolution. The refinement converged with a crystallographic residual, R = 0.19 and the location of 64 solvent molecules. The sequence crystallises as a B-DNA helix with 10 Watson-Crick base-pairs (4 A.T. and 6 G.C) and 2 inosine.adenine (I.A) pairs. The present work shows that in the purine.purine base-pairs the adenine adopts syn orientation with respect to the furanose moiety while the inosine is in the trans (anti) orientation. Two hydrogen bonds link the I.A. base-pair, one between N-1(I) and N-7(A), the other between O-6(I) and N-6(A). This bulky purine.purine base-pair is incorporated in the double helix at two positions with little distortion of either local or global conformation. The pairing observed in this study is presented as a model for I.A base-pairs in RNA codon-anticodon interactions and may help explain the thermodynamic stability of inosine containing base-pairs. Conformational parameters and base stacking interactions are presented and where appropriate compared with those of the native compound, d(C-G-C-G-A-A-T-T-C-G-C-G) and with other studies of oligonucleotides containing purine.purine base-pairs.     

Crystallographic studies on damaged DNAs. I. An N(6)-methoxyadenine residue forms a Watson-Crick pair with a cytosine residue in a B-DNA duplex

Oxyamines such as hydroxylamine and methoxylamine disturb DNA replication and act as potent mutagens, causing nucleotide transition from one purine to another or one pyrimidine to another. In order to investigate mismatch base-pairing in DNA damaged with oxyamines, a dodecamer with the sequence d(CGCGmo(6)AATCCGCG), where mo(6) A is 2'-deoxy-N(6)-methoxyadenosine, was synthesized and its crystal structure determined. No significant conformational changes are found between the present dodecamer and the original undamaged B-form dodecamer. Electron density maps clearly show that the mo(6)A residue forms a base-pair with a 2'-deoxycytidine residue through hydrogen bonds similar to a Watson-Crick G.C base-pair. For these hydrogen bonds to be made, N(6)-methoxyadenine must chemically take the imino form. The methoxylation thus enables the adenine base to mimic a guanine base. As a result, misincorporation of 2'-deoxycytidine instead of thymidine, or 2'-deoxyadenosine instead of 2'-deoxyguanosine, can occur in DNA replication.     

Microbiological transformations of nabilone, a synthetic cannabinoid.

A screening program was conducted to find microorganisms that modify the synthetic cannabinoid nabilone. After purification, the products from three cultures were analyzed by spectral methods to determine their chemical structures. An optically active 9S-hydroxy-6aR,10aR-trans cannabinoid was isolated from a culture of an unidentified soil bacterium designated A24007. From Bacillus cereus cultures were isolated a 9S,6'-dihydroxy-6aR,10aR-trans cannabinoid, a 9S-hydroxy-6'-keto-6aR,10aR-trans cannabinoid, a 9-keto-6'-hydroxy-6aS,10aS-trans cannabinoid, and a 6',9-diketo-6aS,10aS-trans cannabinoid. All of these products were optically active, as was a 9S-hydroxy-6aS,10AS-trans cannabinoid also isolated from B. cereus cultures. A series of acidic products were isolated from cultures of Nocardia salmonicolor. All of these products contained a carboxylic acid group at the terminal end of three-position alkyl side chains having varying numbers of carbon atoms. Two of the acidic products contained a 9-keto group, whereas all other carboxylic acid products were 9-hydroxy cannabinoids. The array of products obtained from incubation of nabilone indicates the usefulness of microbial transformations in the preparation of new cannabinoids.     

Characterization of a zwf mutant of Synechococcus sp. strain PCC 7942.

A mutant of the cyanobacterium Synechococcus sp. strain PCC 7942 carrying a disrupted gene encoding glucose-6-phosphate dehydrogenase (zwf) produced no detectable glucose-6-phosphate dehydrogenase as assessed by enzyme assay and Western blot (immunoblot) analysis. This mutant exhibited significantly impaired dark viability.     

Role of Ly-6 in lymphocyte activation. I. Characterization of a monoclonal antibody to a nonpolymorphic Ly-6 specificity

In studies with alloantisera and monoclonal antibodies (mAb) a number of antigenic determinants have been defined that are the products of the Ly-6 locus on murine chromosome 2 and that are expressed primarily on B and T lymphoid cells. It remains controversial whether these antigenic determinants are encoded by a single gene or a multigene complex. We have characterized a new rat mAb, D7, which recognizes a cell surface antigen whose expression on nonactivated peripheral lymphocytes varies from strain to strain. The phenotype of the staining profile, i.e., high or low percentage of D7-positive cells, mapped to the Ly-6 locus as assayed by strain distribution studies, RI lines, and Ly-6 congenic strains. The binding of D7 to Ly-6.1-positive strains could be inhibited by mAb directed to the Ly-6E.1 specificity, whereas D7 could inhibit the binding of mAb specific for Ly-6A.2 to cells from Ly-6.2-positive strains. Coprecipitation studies followed by Western blot analysis confirmed that D7 reacts with both Ly-6E.1- and Ly-6A.2-bearing molecules. The most likely explanation for these findings is that Ly-6A.2 and Ly-6E.1 represent allelic specificities. Further dissection of the complexity of the Ly-6 antigen system and determination of its possible functional importance in lymphocyte activation should be greatly facilitated by the availability of xenogeneic mAb that recognize framework determinants on multiple Ly-6 products.     

Identification of HHR23A as a substrate for E6-associated protein-mediated ubiquitination.

The human papilloma virus E6-associated protein (E6AP) functions as a ubiquitin protein ligase (E3) in the E6-mediated ubiquitination of p53. E6AP is also an E3 in the absence of E6, but its normal cellular substrates have not yet been identified. Here we report the identification of HHR23A, one of the human homologues of the yeast DNA repair protein Rad23, as an E6-independent target of E6AP. HHR23A binds E6AP and is ubiquitinated in vitro in an E6AP-dependent manner. Ubiquitinated forms of endogenous HHR23A are detectable in mammalian cells. Overexpression of wild-type E6AP in vivo enhances the ubiquitination of HHR23A, whereas a dominant negative E6AP mutant inhibits HHR23A ubiquitination. Although HHR23A is a stable protein in non-synchronized cells, its levels are regulated in a cell cycle-dependent manner, with specific degradation occurring during S phase. The S phase degradation of HHR23A could be blocked in vivo by dominant negative E6AP, providing direct evidence for the involvement of E6AP in the regulation of HHR23A. Consistent with a role of the HHR23 proteins in DNA repair, UV-induced DNA damage inhibited HHR23A degradation. Although the precise role of HHR23 proteins in DNA repair and cell cycle progression remains to be elucidated, our data suggest that E6AP-mediated ubiquitination of HHR23A may have important implications in DNA repair and cell cycle progression.     

The significance of the homozygous CYP2A6 deletion on nicotine metabolism: a new genotyping method of CYP2A6 using a single PCR-RFLP.

A convenient and specific CYP2A6 genotyping method was developed in this study. This method consisting of a single PCR-RFLP is capable of resolving the genotype into either CYP2A6*1 (wild type), CYP2A6*2, or CYP2A6*3. Among 252 Japanese persons genotyped, 241 were genotyped as the wild type, 1 as an unknown variant, and none as either CYP2A6*2 or CYP2A6*3. A homozygous deletion was found in the 10 remaining subjects. To clarify the metabolic significance of this deletion in the whole human body, urinary cotinine, the principal metabolite of nicotine, was analyzed subsequent to smoking. Cumulated urinary cotinine excretion in the homozygously CYP2A6-deleted individuals was about one-seventh compared to the control group (wild type). This study provides a firm experimental basis for correlating genotypic characterization of CYP2A6 with phenotypic expression of nicotine metabolism.     

DNA sequence specificity of a naphthylquinoline triple helix-binding ligand.

We have examined the effect of a naphthylquinoline triplex-binding ligand on the formation of intermolecular triplexes on DNA fragments containing the target sites A6G6xC6T6 and G6A6xT6C6. The ligand enhances the binding of T6C2, but not T2C6, to A6G6xC6T6 suggesting that it has a greater effect on TxAT than C+xGC triplets. The complex with T6C2 is only stable below pH 6.0, confirming the requirement for protonation of the third strand cytosines. Antiparallel triplexes with GT-containing oligonucleotides are also stabilised by the ligand. The complex between G5T5 and A6G6xC6T6 is stabilised by lower ligand concentrations than that between T5G5 and G6A6xC6T6. The ligand does not promote the interaction with GT-containing oligonucleotides which have been designed to bind in a parallel orientation. Although the formation of antiparallel triplexes is pH independent, we find that the ligand has a greater stabilising effect at lower pH, suggesting that the active species is protonated. The ligand does not promote the binding of antiparallel GA-containing oligonucleotides at pH 7.5 but induces the interaction between A5G5 and G6A6xT6C6 at pH 5.5. Ethidium bromide does not promote the formation of any of these triplexes and destabilises the interaction of acridine-linked pyrimidine-containing third strands with these target sites.     

Synthesis of a novel conformationally restricted Val-Phe dipeptidomimetic.

A method for the synthesis of (3(R,S),6S,11b(R,S))-1,3,4,6,7,11b-hexahydro-4-oxo-3-phthalimidopyrido[2,1-a]isoquinoline-6-carboxylic acid 2 as a new conformationally restricted dipeptidomimetic of Val-Phe is reported. It involved cyclisation via an intramolecular electrophilic addition at the reactive bridgehead carbon. This new scaffold can be used as a building block in the preparation of libraries of peptidomimetics.     

Crystallization and preliminary X-ray analysis of a fungal endoglucanase I.

An endoglucanase I (EG1) from a fungal source (Humicola insolens) has been crystallized in a number of forms suitable for X-ray diffraction analysis. Four crystal forms have been grown from various precipitants using vapour phase diffusion methods in hanging drops. Three of these crystal forms diffract to beyond 2.5 A resolution. Two forms, obtained from ammonium sulphate at pH 5.4, or 8.0, grow as tetragonal bipyramids in space group P4(1)22 or P4(3)22, with approximate cell dimensions a = b = 102 A, c = 282 A. The other crystal forms were grown from polyethylene glycol 8000 at pH 8.0. One grows as monoclinic plates, space group P2(1), with cell dimensions a = 66.9 A, b = 75.2 A, c = 86.9 A and beta = 102.9 degrees and the other as long hexagonal rods in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 119 A, c = 83 A.     

Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism.

Three point mutations R335S, L336V and V476L, distinguish the sequence of a cytochrome P450 CYP6A2 variant assumed to be responsible for 1,1,1-trichloro-2,2-bis-(4'-chlorophenyl)ethane (DDT) resistance in the RDDT(R) strain of Drosophila melanogaster. To determine the impact of each mutation on the function of CYP6A2, the wild-type enzyme (CYP6A2wt) of Cyp6a2 was expressed in Escherichia coli as well as three variants carrying a single mutation, the double mutant CYP6A2vSV and the triple mutant CYP6A2vSVL. All CYP6A2 variants were less stable than the CYP6A2wt protein. Two activities enhanced in the RDDT(R) strain were measured with all recombinant proteins, namely testosterone hydroxylation and DDT metabolism. Testosterone was hydroxylated at the 2beta position with little quantitative variation among the variants. In contrast, metabolism of DDT was strongly affected by the mutations. The CYP6A2vSVL enzyme had an enhanced metabolism of DDT, producing dicofol, dichlorodiphenyldichloroethane and dichlorodiphenyl acetic acid. The apparent affinity of the enzymes CYP6A2wt and CYP6A2vSVL for DDT and testosterone was not significantly different as revealed by the type I difference spectra. Sequence alignments with CYP102A1 provided clues to the positions of the amino acids mutated in CYP6A2. These mutations were found spatially clustered in the vicinity of the distal end of helix I relative to the substrate recognition valley. Thus this area, including helix J, is important for the structure and activity of CYP6A2. Furthermore, we show here that point mutations in a cytochrome P450 can have a prominent role in insecticide resistance.     

Deprotection of methylphosphonate oligonucleotides using a novel one-pot procedure.

Deprotection of methylphosphonate oligonucleotides with ethylenediamine was evaluated in a model system. Methylphosphonate sequences of the form 5'-TTTNNTTT, where N was either N4-bz-dC, N4-ibu-dC, N2-ibu-O6-DPC-dG, N2-ibu-dG, N6-bz-dA, or T, were used to determine the extent of modifications that occur during deprotection. Up to 15% of N4-bz-dC was found to transaminate at the C4 position when treated with ethylenediamine. A similar displacement reaction with ethylenediamine was observed at the O6 position of N2-ibu-O6-DPC-dG, and to a much lesser extent of N2-ibu-dG. Side reactions were not observed when oligonucleotides containing N4-ibu-dC, N6-bz-dA, or T were treated with ethylenediamine. A novel method of deprotecting methylphosphonate oligonucleotides was developed from these studies. The method incorporates a brief treatment with dilute ammonia for 30 minutes followed by addition of ethylenediamine for 6 hours at room temperature to complete deprotection in a one-pot format. The solution is then diluted and neutralized to stop the reaction and prepare the crude product for chromatographic purification. This method was used to successfully deprotect a series of oligonucleotides at the 1, 100, and 150 mumole scales. These deprotection results were compared to a commonly used two-step method and found to be superior in yield of product by as much as 250%.     

Enzymatic cleavage of a bacterial chromosome at a transposon-inserted rare site.

The sequential use of the methylase M.Xbal (5'.TCTAGm6A) and the methylation-dependent endonuclease Dpnl (5'-Gm6A decreases TC) results in cleavage at 5'.TCTAGA decreases TCTAGA. This recognition sequence was introduced into a transposon derived from the Mu bacteriophage and transposed into the genome of the bacterium Salmonella typhimurium. M.Xbal methylation was provided in vivo by a plasmid containing the M.Xbal gene and the S. typhimurium genome was cleaved to completion by Dpnl at one or more sites, depending on the number of transposon insertions. The resulting genomic fragments were resolved by pulsed-field electrophoresis. The potential use of single M.Xbal/Dpnl cleavage sites as reference positions to map rare restriction sites is discussed.     

Crystal structure of a viral cyclin, a positive regulator of cyclin-dependent kinase 6.

BACKGROUND: Cyclin-dependent kinases (CDKs) have a central role in cell-cycle control and are activated by complex formation with positive regulatory proteins called cyclins and by phosphorylation. The overexpression and mutation of cyclins and CDKs has been associated with tumorigenesis and oncogenesis. A virus-encoded cyclin (v-cyclin) from herpesvirus saimiri has been shown to exhibit highest sequence homology to type D cyclins and specifically activates CDK6 of host cells to a very high degree. RESULTS: We have determined the first X-ray structure of a v-cyclin to 3.0 A resolution. The structure of the core domains is very similar to those of cyclin A and cyclin H from human cells. To understand the structural basis for the v-cyclin specificity for CDK6 and the insensitivity of the complex to inhibitors of the p21 and INK4 families, a v-cyclin-CDK2 model was built on the basis of the known structures of human cyclin A in complex with CDK2 and the CDK inhibitor p27(Kip1). CONCLUSIONS: Although many critical interactions between cyclin A and CDK2 would be conserved in a v-cyclin-CDK2 complex, some appear sterically or electrostatically unfavorable due to shifts in the backbone conformation or sidechain differences and may contribute to v-cyclin selectivity for CDK6. The insensitivity of v-cyclin-CDK6 complexes to inhibitors of the p21 family is probably due to structural changes in v-cyclin that lead to a flatter surface area offering fewer potential contacts with the protein inhibitor. In addition, sequence changes in v-cyclin eliminate hydrogen-bonding partners for atoms of the p27(Kip1) inhibitor. This structure provides the first model for interactions between v-cyclins and host cell-cycle proteins; these interactions may be important for virus survival as well as oncogenic transformation of host cells.     

Unique DNA repair properties of a xeroderma pigmentosum revertant.

A group A xeroderma pigmentosum revertant with normal sensitivity was created by chemical mutagenesis. It repaired (6-4) photoproducts normally but not pyrimidine dimers and had near normal levels of repair replication, sister chromatid exchange, and mutagenesis from UV light. The rate of UV-induced mutation in a shuttle vector, however, was as high as the rate in the parental xeroderma pigmentosum cell line.     

Conformational analysis of a ribopentanucleoside tetraphosphate in aqueous solution. A two-dimensional NMR study at 500 MHz.

The 30 ribose proton resonances of the pentaribonucleoside tetraphosphate m6(2)AUm6(2)AUm6(2)A have been assigned unequivocally by means of spin-echo-correlated spectroscopy, 2D J-resolved spectroscopy and Nuclear Overhauser difference spectroscopy, carried out at 500 MHz. A detailed comparison of the conformational properties of the title compound with its constituent fragments m6(2)AUm6(2)AU, m6(2)AUm6(2)A, m6(2)AU and the relevant monomers is given. Chemical shift data indicate the existence of a doubly "bulged out" conformer, in which the two interior U-fragments are not involved in regular nearest neighbour stacking interactions. The coupling constants of the ribose-ring are interpreted in terms of the N/S equilibrium, and population distributions along the backbone angles beta and gamma are presented. The combined data suggest a strong similarity between the 5'-terminal triplets in m6(2)AUm6(2)AUm6(2)A, m6(2)AUm6(2)AU and m6(2)AUm6(2)A2.     

Efficient propagation of human herpesvirus 6B in a T-cell line derived from a patient with adult T-cell leukemia/lymphoma.

The efficient propagation of the OK strain of the B variant of human herpesvirus 6 (HHV-6B) was demonstrated in a line of T cells, TaY, established from the peripheral blood lymphocytes of a patient with adult T-cell leukemia/lymphoma (ATL). Growth of TaY cells depends on the presence of IL-2 and the cells harbor HTLV-I genomes. A severe cytopathic effect (CPE) was observed in many HHV-6B(OK)-infected TaY cells one week after infection. The release of virus from HHV-6B(OK)-infected TaY cells [TaY(OK)] was first detected after three days and increased rapidly for up to seven days after infection, as demonstrated by PCR. The titer of HHV-6B(OK) in the supernatant was comparable to the value of 10(3.5) TCID50/ml obtained with PHA-activated cord blood lymphocytes (CBL) that had been infected with HHV-6B(OK). The replication of the virus was shown to depend to a considerable extent on cell viability. Electron microscopy revealed many herpesvirus-type capsid- and enveloped-viruses in the nuclei and cytoplasm of degenerated cells in TaY(OK) cultures. The U1102 strain of HHV-6A and the Z29 strain of HHV-6B also infected TaY cells productively, as detected by PCR and an immunofluorescence test. These results suggest that the activation of CD4+ T lymphocytes with mitogens such as PHA or IL-2 and the expression of some cellular gene or the HTLV-I gene might be essential for efficient propagation of HHV-6B. TaY cells should play an important role in future investigations of cell-virus interactions and genetic variations or cell tropism of HHV-6 isolates since no cell line that shows propagation of both HHV-6A and HHV-6B has been reported to date.