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PubMed2000

Characterization of the interaction of zopiclone with gamma-aminobutyric acid type A receptors.

Zopiclone is a cyclopyrrolone that is used clinically as a hypnotic. Although this drug is known to interact with neuronal gamma-aminobutyric acid type A receptors, its binding site(s) within the receptor oligomer has been reported to be distinct from that of the classical benzodiazepines. After photoaffinity labeling with flunitrazepam, receptors in rat cerebellar membranes showed differentially reduced affinity for flunitrazepam and zopiclone by 50- and 3-fold, respectively. Because histidine 101 of the alpha-subunit is a major site of photolabeling, we have made specific substitutions of this residue and studied the consequences on the binding properties of zopiclone and diazepam using recombinant alpha1beta2gamma2-receptors transiently expressed in tsA201 cells. Both compounds showed similar binding profiles with receptors containing mutated alpha-subunits, suggesting a similar interaction with the residue at position 101. At alpha1beta2gamma3-receptors, flunitrazepam affinity was dramatically decreased by approximately 36-fold, whereas the affinity for zopiclone was decreased only 3-fold, suggesting a differential contribution of the gamma-subunit to the binding pocket. Additionally, we used electrophysiological techniques to examine the contribution of the gamma-subunit isoform in the receptor oligomer to ligand recognition using recombinant receptors expressed in Xenopus oocytes. Both compounds are agonists at alpha1beta2gamma2- and alpha1beta2gamma3-receptors, with flunitrazepam being more potent but less efficacious. In summary, these data suggest that histidine 101 of the alpha1-subunit plays a similar role in ligand recognition for zopiclone, diazepam, and flunitrazepam.

Amino Acid Substitution↗
PubMed1996

Analysis of early region 3 mutants of mouse adenovirus type 1.

Early region 3 (E3) of mouse adenovirus type 1 has the potential to produce three proteins which have identical amino termini but unique carboxy-terminal sequences. Three recombinant deletion viruses were constructed so that each could produce only one of the three E3 proteins. A fourth mutant that should produce no E3 proteins was also constructed. These recombinants were able to grow in mouse 3T6 cells and produced wild-type levels of viral mRNAs and proteins except for those specifically deleted by the mutations. Early mRNA production from the mutant viruses was analyzed by reverse transcriptase PCR and confirmed that each deletion mutant would be able to produce only one of the three E3 proteins. Late mRNA production was analyzed by Northern (RNA) blotting and found to be similar in wild-type and mutant viruses. Capsid morphology was unaltered in the mutant viruses as seen by electron microscopy. Immunoprecipitation of E3 proteins from infections of mouse 3T6 cells using an antiserum specific for all three E3 proteins was used to examine the effect of the introduced mutations on protein expression. Two mutants produced only one class of E3 protein as predicted from their specific mutations and mRNA expression profiles. One mutant virus failed to produce any detectable E3 proteins. The predicted E3-null mutant was found to be leaky and could produce low levels of E3 proteins. Outbred Swiss mice were infected with the E3 mutant viruses to determine if the E3 proteins have an effect on the pathogenicity of the virus in mice. All of the mutants showed decreased pathogenicity as determined by increased 50% lethal doses, indicating that the proteins of the E3 region are important determinants of the pathogenesis of mouse adenovirus in its natural host.

Adenoviridae↗
PubMed2005

Robo2 is required for establishment of a precise glomerular map in the zebrafish olfactory system.

Olfactory sensory neurons (OSNs) expressing a given odorant receptor project their axons to specific glomeruli, creating a topographic odor map in the olfactory bulb (OB). The mechanisms underlying axonal pathfinding of OSNs to their precise targets are not fully understood. Here, we demonstrate that Robo2/Slit signaling functions to guide nascent olfactory axons to the OB primordium in zebrafish. robo2 is transiently expressed in the olfactory placode during the initial phase of olfactory axon pathfinding. In the robo2 mutant, astray (ast), early growing olfactory axons misroute ventromedially or posteriorly, and often penetrate into the diencephalon without reaching the OB primordium. Four zebrafish Slit homologs are expressed in regions adjacent to the olfactory axon trajectory, consistent with their role as repulsive ligands for Robo2. Masking of endogenous Slit gradients by ubiquitous misexpression of Slit2 in transgenic fish causes posterior pathfinding errors that resemble the ast phenotype. We also found that the spatial arrangement of glomeruli in OB is perturbed in ast adults, suggesting an essential role for the initial olfactory axon scaffold in determining a topographic glomerular map. These data provide functional evidence for Robo2/Slit signaling in the establishment of olfactory neural circuitry in zebrafish.

Animals↗
PubMed2004

"Virostatics" as a potential new class of HIV drugs.

The combination of three or more antiretroviral drugs is referred to as highly active antiretroviral therapy (HAART) and constitutes the standard of care for HIV-1 patients in industrialized nations. Although HAART is usually effective in reducing viral load and re-constituting CD4 counts, latent virus reservoirs persist, and as many as 60 years therapy [1, 2] may be required to eradicate the virus. Meanwhile, patients are likely to experience drug related toxicity and may have to change therapy due to the emergence of drug resistant strains. For these reasons, the search for different therapeutic approaches continues. A new concept of antiviral/cytostatic ("virostatics") drugs has been proposed within the context of HAART to restrict virus target populations (CD4(+) T lymphocytes), target viral reservoirs, and possibly restore immune functions, by reducing excess immune activation, a fundamental component of HIV/AIDS pathogenesis. These virostatics include drugs such as hydroxyurea, mycophenolic acid, leflunomide and rapamycin, which are currently used for other therapeutic indications; and other experimental drugs, which are not for human use. They utilize multiple novel mechanisms of action to impede HIV by targeting host cellular proteins that are not susceptible to mutation. Therefore, their resistance profile appears to be quite favorable. Since many of these drugs act by inhibiting the synthesis of deoxynucleotides, essential for HIV reverse transcription, they favor the incorporation of nucleoside analogues into viral DNA, thus synergizing with the antiviral activity of currently used nucleoside reverse transcriptase inhibitors (NRTI). The rationale for the use of virostatics in HIV/AIDS, their mechanism of action, and ongoing preclinical and clinical research will be reviewed.

Animals↗
PubMed2006

Investigating the molecular basis of drug action and response: chemocentric genomics and proteomics.

The genomics and proteomics sciences have fundamentally changed the ways in which drug targets are being identified, characterized and validated. Here we review how genomics and proteomics research is improving our understanding of genetic determinants of drug susceptibility and response and, conversely, how organic small molecules mediate their pharmacological effects by modulating genome and proteome activities. We also examine the effect this improved understanding has on the drug discovery and development process.

Animals↗
PubMed2005

Fluoroquinolone-resistant Escherichia coli, Indonesia.

In a recent, population-based survey of 3,996 persons in Indonesia, fluoroquinolone (FQ)-resistant Escherichia coli was prevalent in the fecal flora of 6% of patients at hospital admission and 23% of patients at discharge, but not among healthy relatives or patients visiting primary healthcare centers (2%). Molecular typing showed extensive genetic diversity with only limited clonality among isolates. This finding suggests that independent selection of resistant mutants occurs frequently. FQ-resistant isolates exhibited a higher rate of spontaneous mutation, but sparser virulence profiles, than FQ-susceptible isolates from the same population. The resistant isolates belonged predominantly to phylogenetic groups A (57%) and B1 (22%) but also to the moderately virulent group D (20%). Hypervirulent strains from the B2 cluster were underrepresented (1%). Because FQ-resistant E. coli can cause disease, especially nosocomial infections in immunocompromised patients, spread of such strains must be stopped.

Anti-Infective Agents↗
PubMed2002

[Non alcoholic steatohepatitis: a multifactorial, frequent, paucysymptomatic liver disease with a fibrotic outcome].

BACKGROUND: The aim of this study was to evaluate the morphological, clinical and biochemical characteristics of non alcoholic steatohepatitis to understand its pathogenesis. PATIENTS AND METHODS: From January 1993 to June 2000, 44 patients were selected on histological criteria. Alcohol intake, blood pressure, weight, glycaemia, lipid, immune, iron profiles hemochromatosis (HFE) gene mutations were analyzed. Patients were re-examined thereafter or in June 2000. RESULTS: Twenty one women and 10 men were included (mean age=54). Nineteen patients were asymptomatic (61.3%). Patients often presented with an increase in alanine aminotransferase. This was correlated with steatosis (P=0.008). Hypertension, excess weight, abnormal serum glucose levels and dyslipidaemia were respectively observed in 10 (32.2%), 24 (77.4%), 16 (51.6%) and 18 (58.1%) patients. Thirteen of these patients (41.9%) presented abnormal autoantibodies titers without autoimmune hepatitis; 18 (58.1%) presented an iron overload. A mutation of the HFE gene was detected in 14 of 25 patients (51.6%). Liver iron concentrations were not correlated to the extent of fibrosis extension or with mutations. CONCLUSION: Increased alanine aminotransferase levels usually revealed non alcoholic steatohepatitis. A high prevalence of autoantibodies, iron overload and mutation of the HFE gene were detected. Non alcoholic steatohepatitis should be diagnosed because it can be associated with cirrhosis.

Alanine Transaminase↗
PubMed2002

Pyrosequencing as an alternative to single-strand conformation polymorphism analysis for detection of N-ras mutations in human melanoma metastases.

BACKGROUND: Mutations in codons 12, 13, and 61 of the N-ras gene are common alterations in cutaneous malignant melanoma. We evaluated pyrosequencing, a simple and rapid method used mainly for single-nucleotide polymorphism analysis, as a possible alternative to single-strand conformation polymorphism (SSCP) analysis and sequencing of N-ras. METHODS: We evaluated the sensitivity and accuracy of the pyrosequencing method for identification of mutations in N-ras codons 12, 13, and 61. Nucleotide dispensation orders were created to produce distinct pyrogram peak profiles for the most frequent mutations in codon 61 and codons 12 and 13, respectively. RESULTS: The detection limits for the two most common codon 61 mutations found in malignant melanoma, which code for Arg and Lys, were 30% and 15%, respectively. To evaluate the pyrosequencing method on clinical samples, we performed a parallel analysis of 82 melanoma metastases using SSCP analysis and pyrosequencing. All mutations detected by SSCP analysis and confirmed by sequencing were also correctly identified by pyrosequencing. Codon 61 mutations were identified in 26 of the 82 samples (32%), whereas no mutations were found in codons 12 and 13. Four types of codon 61 mutations, Arg (17%), Lys (10%), Leu (4%), and His (1%), were identified. CONCLUSION: Pyrosequencing is an attractive alternative to SSCP analysis for N-ras mutation detection in malignant melanoma tumor samples because it displays the same sensitivity and accuracy as SSCP analysis and is simple and rapid.

Codon↗
PubMed2002

[Present and future prospects of individualized medical services by genetic tests].

With progress of human genome projects many gene mutations and their expression profiles have been much identified. Laboratory tests have become essential to achieve proper individualized medical services. In particular single nucleotide polymorphisms (SNPs) and gene expression profiles are of high interests. As one of these ongoing mmodels, we here present the SNPs of human Protein 1' also called as human Clara cell 10-kd protein (CC10). We initially found six different SNPs (A-908G, G38A, G118A, C1225T, G1226A, C4777G). For laboratory use multiplex PCR was developed to determine frequencies in normal control. The -908G and 38A genotypes were more frequent in sarcoidosis than those in controls. The SNPs of in the human protein 1 gene may be the genetic susceptibility factors for Sarcoidosis.

Gene Expression Profiling↗
PubMedDate not supplied

P53-induced gene 11 (PIG11) involved in arsenic trioxide-induced apoptosis in human gastric cancer MGC-803 cells.

Arsenic trioxide (As2O3) has been used as an effective chemotherapy agent for some human cancer, such as acute promyelocytic leukemia. We have demonstrated that low level of As2O3 relatively selectively inhibited growth of the solid tumor MGC-803 cells by triggering apoptosis. In this study, we found PIG11, a p53-induced gene, was upregulated markedly by As2O3 using the technique of differential display reverse transcriptase PCR (DDRT-PCR). Addition of anti-PIG11 phosphorothioated oligonucleotide (5'-GGC CGC CAT CTT CTC CTC-3') before As2O3 treatment, abolished the transient increase in PIG11 gene expression. Furthermore, it significantly inhibited the As2O3-induced apoptosis of MGC-803 cells, but had no effect in addition of missense (5'-GAG GAG AAG ATG GCG GCC-3') phosphorothioated oligonucleotides. These results suggest that PIG11, as a downstream target of p53, is involved in apoptosis of MGC-803 cells.

Apoptosis↗
PubMed1992

Activation of the Ha-, Ki-, and N-ras genes in chemically induced liver tumors from CD-1 mice.

We compared the profile of ras gene mutations in spontaneous CD-1 mouse liver tumors with that found in liver tumors that were induced by a single i.p. injection of either 7,12-dimethylbenz(a)anthracene (DMBA), 4-aminoazobenzene, N-hydroxy-2-acetylaminofluorene, or N-nitrosodiethylamine. By direct sequencing of polymerase chain reaction-amplified tumor DNA, the carcinogen-induced tumors were found to have much higher frequencies of ras gene activation than spontaneous tumors. Furthermore, each carcinogen caused specific types of ras mutations not detected in spontaneous tumors, including several novel mutations not previously associated with either the carcinogen or mouse hepatocarcinogenesis. For example, the model compound DMBA is known to cause predominantly A to T transversions in Ha-ras codon 61 in mouse skin and mammary tumors, consistent with the ability of DMBA to form bulky adducts with adenosine. Our results demonstrate that the predominant mutation caused by DMBA in mouse liver tumors is a G to C transversion in Ki-ras codon 13 (DMBA is also known to form guanosine adducts), illustrating the influence of both chemical- and tissue-specific factors in determining the type of ras gene mutations in a tumor. 4-Aminoazobenzene and N-hydroxy-2-acetylaminofluorene also caused the Ki-ras codon 13 mutation. In addition, we found that N-nitrosodiethylamine, 4-aminoazobenzene, and N-hydroxy-2-acetylaminofluorene all caused G to T transversions in the N-ras gene (codons 12 or 13). This is the first demonstration of N-ras mutations in mouse liver tumors, establishing a role for the N-ras gene in mouse liver carcinogenesis. Finally, comparison of the ras mutations detected in the direct tumor analysis with those detected after NIH3T3 cell transfection indicates that spontaneous ras mutations (in Ha-ras codon 61) are often present in only a small fraction of the tumor cells, raising the possibility that they may sometimes occur as a late event in CD-1 mouse hepatocarcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

2-DG induced modulation of chromosomal DNA profile, cell survival, mutagenesis and gene conversion in X-irradiated yeast.

Effects of post-irradiation modulation in presence of 2-deoxy-D-glucose and yeast extract, on chromosomal DNA profile, cell survival, reverse mutation (ILV+) and gene conversion (TRP+), were studied in X-irradiated stationary phase yeast cells (diploid strain D7 of Saccharomyces cerevisiae). The damage and repair in chromosomal DNA bands, resolved by using contour clamped homogeneous electric pulsed-field gel electrophoresis (PFGE) technique, was estimated by calculating intensity ratio, rho n (rho n I(n)/I(t); where I(n) is the intensity of nth band in a lane and I(t) is the sum of intensities of all bands and the well in the lane). The data indicate linear correlation between relative compactness (tau) of a chromosome [chromosome size (Kb)/length of synaptonemal complex (microns)] and DNA damage and repair. The chromosome repair kinetics were biphasic, showing initial decrease followed by an increase in rho n. Variations were observed among different chromosomes with respect to DNA damage, repair and post-irradiation repair modulation. 2-DG inhibited both components of chromosomal DNA repair and also repair of potentially lethal damage but enhanced frequencies of mutants. Relatively the effects on revertants were greater in cells irradiated with lower doses (50 Gy) of X-rays and post-irradiation incubation in presence of phosphate buffer having 2-DG (50 mM) and glucose (10 mM). Yeast extract increased frequencies of revertants and convertants thus promoting error-prone DNA repair. Yeast extract in combination with 2-DG showed complex effects on chromosomal DNA repair and enhanced mutagenesis further.

Cell Survival↗
PubMed2005

Mimicking natural evolution in metallo-beta-lactamases through second-shell ligand mutations.

Metallo-beta-lactamases (MBLs) represent the latest generation of beta-lactamases. The structural diversity and broad substrate profile of MBLs allow them to confer resistance to most beta-lactam antibiotics. To explore the evolutionary potential of these enzymes, we have subjected the Bacillus cereus MBL (BcII) to a directed evolution scheme, which resulted in an increased hydrolytic efficiency toward cephalexin. A systematic study of the hydrolytic profile, substrate binding, and active-site features of the evolved lactamase reveal that directed evolution has shaped the active site by means of remote mutations to better hydrolyze cephalosporins with small, uncharged C-3 substituents. One of these mutations is found in related enzymes from pathogenic bacteria and is responsible for the increase in that enzyme's hydrolytic profile. The mutations lowered the activation energy of the rate-limiting step rather than improved the affinity of the enzyme toward these substrates. The following conclusions can be made: (i) MBLs are able to expand their substrate spectrum without sacrificing their inherent hydrolytic capabilities; (ii) directed evolution is able to mimic mutations that occur in nature; (iii) the metal-ligand strength is tuned by second-shell mutations, thereby influencing the catalytic efficiency; and (iv) changes in the position of the second Zn(II) ion in MBLs affect the substrate positioning in the active site. Overall, these results show that the evolution of enzymatic catalysis can take place by remote mutations controlling reactivity.

Amino Acid Substitution↗
PubMed2002

Assessing the relative importance of the biophysical properties of amino acid substitutions associated with human genetic disease.

The inclusion of a mutation in a pathology-based database such as the Human Gene Mutation Database (HGMD) is a two-stage process: first, the mutation must occur at the DNA level, then it must cause a clinically detectable disease state. The likelihood of the latter step, termed the relative clinical observation likelihood (RCOL), can be regarded as a function of the structural/functional consequences of a mutation at the protein level. Following this paradigm, we modeled in silico all amino acid replacements that could potentially have arisen from an inherited single base pair substitution in five human genes encoding arylsulphatase A (ARSA), antithrombin III (SERPINC1), protein C (PROC), phenylalanine hydroxylase (PAH), and transthyretin (TTR). These proteins were chosen on the basis of 1) the availability of a crystallographic structure, and 2) a sufficiently large number of amino acid replacements being logged in HGMD. A total of 9,795 possible mutant structures were modeled and 20 different biophysical parameters assessed. Together with the HGMD-derived spectra of clinically detected mutations, these data allowed maximum likelihood estimation of RCOL profiles for the 20 parameters studied. Nine parameters (including energy difference between wild-type and mutant structures, accessibility of the mutated residue, and distance from the binding/active site) exhibited statistically significant variability in their RCOL profiles, indicating that mutation-associated changes affected protein function. As yet, however, a biological meaning could only be attributed to the RCOL profiles of solvent accessibility and, for three proteins, local energy change, disturbed geometry, and distance from the active center. The limited ability of the biophysical properties of mutations to explain clinical consequences is probably due to our current lack of understanding as to which amino acid residues are critical for protein folding. However, since the proteins examined here were unrelated, and our findings consistent, it may nevertheless prove possible to extrapolate to other proteins whose dysfunction underlies inherited disease.

Amino Acid Substitution↗
PubMed2000

The phenotype of mutations of the base-pair C2658.G2663 that closes the tetraloop in the sarcin/ricin domain of Escherichia coli 23 S ribosomal RNA.

The sarcin/ricin domain (SRD) in Escherichia coli 23 S rRNA is a part of the site for the association of elongation factors with ribosomes and for that reason is critical for the binding of aminoacyl-tRNA and for translocation during the reiterative elongation reactions of protein synthesis. The SRD has a GAGA tetraloop that is shut off by a Watson-Crick C2658 x G2663 pair. The contribution of this pair to the function of the ribosome has been evaluated by constructing mutations in the nucleotides and determining their phenotype. Constitutive expression of a plasmid-encoded rrnB operon with a G2663C transversion mutation that disrupts the Watson-Crick pair was lethal. Double transversion mutations, C2658G x G2663C and C2658A x G2663U, that reverse the polarity of the pyrimidine and the purine but restore the potential to form a canonical pair, were also lethal. Induction of transcription of 23 S rRNA with the same mutations, but encoded in a plasmid with a lambdaP(L) promoter and expressed at a lower level, retarded growth. The sedimentation profiles of ribosomes with transversion mutations in C2658 and/or G2663 are altered; the ratio of 50 S subunits to 30 S particles is changed and polysomes are reduced. Ribosomes with a G2663C, a C2658G x G2663C, or a C2658A x G2663U mutation in 23 S rRNA were not active in protein synthesis, indeed, they appeared to inhibit the activity of ribosomes with wild-type 23 S rRNA. Transversion mutations in the analogs of C2658 and G2663 decreased binding of EF-G to SRD oligoribonucleotides; the same mutations in 23 S rRNA decreased binding of the factor to intact ribosomes. The most severe phenotype, in growth, in protein synthesis, and in the binding of EF-G, was associated with a C2658G x G2663C mutation; it is surprising that this was more severe than an analogous C2658A x G2663U mutation. A double transition mutation, C2658U x G2663A, which is not known to have occurred in nature, had no effect on the growth of cells or on the function of ribosomes. The lethal phenotype of transversion mutations in C2658 and G2663 appears to derive from a loss of the capacity of ribosomes to bind EF-G and by indirection the EF-Tu ternary complex.

Bacterial Proteins↗