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At least 991 records · Page 55Linked to original sources
PubMed2001

Aminoglycoside resistance with homogeneous and heterogeneous populations of antibiotic-resistant ribosomes.

Aminoglycosides bind to rRNA in the small subunit of the bacterial ribosome. Mutations in the decoding region of 16S rRNA confer resistance to specific subsets of aminoglycoside antibiotics. The two major classes of 2-deoxystreptamine aminoglycosides are the 4,5- and the 4,6-disubstituted antibiotics. Antibiotics of the 4,5-disubstituted class include neomycin, paromomycin, and ribostamycin. Gentamicins and kanamycins belong to the 4,6-disubstituted class of aminoglycosides. Structural studies indicated the potential importance of position 1406 (Escherichia coli numbering) in the binding of ring III of the 4,6-disubstituted class of aminoglycosides to 16S rRNA. We have introduced a U1406-to-A mutation in a plasmid-encoded copy of E. coli 16S rRNA which has been expressed either in a mixture with wild-type ribosomes or in a strain in which all rRNA is transcribed from the plasmid-encoded rrn operon. High-level resistance to many of the 4,6-disubstituted aminoglycosides is observed only when all the rRNA contains the U1406-to-A mutation. In contrast to the partial dominance of resistance observed with other mutations in the decoding region, there is a dominance of sensitivity with the 1406A mutation. Chemical footprinting experiments indicate that resistance arises from a reduced affinity of the antibiotic for the rRNA target. These results demonstrate that although position 1406 is an important determinant in the binding and action of the 4,6-disubstituted aminoglycosides, other rRNA mutations that perturb the binding of ring I of both classes of 2-deoxystreptamine aminoglycosides confer higher levels of resistance as well as a partial dominance of resistance.

Adenosine↗
PubMed2000

The T9176G mutation of human mtDNA gives a fully assembled but inactive ATP synthase when modeled in Escherichia coli.

A new mutation in human F(1)F(0) ATPase6, T9176G, which changes Leu 217 to an Arg, has been described in two siblings with Leigh syndrome [Carrozzo et al. (2000) Neurology, in press]. This mutation was modeled in Escherichia coli by changing Leu 259 (the equivalent residue) to Arg and the properties of the altered ECF(1)F(0) were compared to those of previously characterized ATPase6 mutants also modeled in the E. coli enzyme. The L259R change produced a fully assembled ECF(1)F(0) which had no significant ATP hydrolysis, ATP synthesis or proton pumping functions. This is very different from previously described human ATPase6 mutations. The presence of Arg at position 259 in subunit a did not make membranes permeable to protons. We conclude that the mutation inhibits functioning by blocking the rotary motor action of the enzyme.

Adenosine Triphosphatases↗
PubMed2004

Does KCNE5 play a role in long QT syndrome?

BACKGROUND: Long QT syndrome [LQTS] is a congenital cardiac disease characterised by prolonged QTC-time, syncopes and sudden cardiac death. LQTS is caused by mutations in genes coding for ion channels involved in the action potential. KCNE5 codes for a novel beta-subunit of the ion channel conducting the delayed rectifier repolarizing current IKs. As KCNE5 is expressed in the human heart and suppresses the IKs current in heterologous systems, it is a candidate gene that may be mutated in LQTS families where no causative mutations in known LQTS associated genes have been found. We examined whether this was the case. METHODS: Genomic DNA from LQTS patients [n=88] and normal controls [n=90] was screened for mutations in KCNE5 by endonuclease-enhanced single strand conformation polymorphism analysis [EE-SSCP], and DNA sequencing of aberrant conformers. Mutations in other LQTS associated ion channels were excluded by SSCP. RESULTS: No mutations were found in the coding region of the KCNE5 gene in LQTS patients. One polymorphism, a T-to-C transition at nucleotide 97, causing an amino acid polymorphism P33S, was present in 16 persons, nine heterozygotes and seven homozygotes. The T-allele frequency was 0.13 in LQTS patients and 0.10 in controls.

Action Potentials↗
PubMed1984

Formation of termination-resistant transcription complex at phage lambda nut locus: effects of altered translation and a ribosomal mutation.

Transcription antitermination by lambda N gene product is affected in a mutant Escherichia coli with altered ribosomal protein S10, caused by the nusE71 mutation. To study the role of translation in antitermination, we have fused the phage nutR locus, the site of action of N, with the lac regulatory region. We have monitored N action by measuring galactokinase, whose synthesis depends on suppression of terminators located between nutR and the galK cistron. We show that a deletion removing potential ribosome binding signals and AUG codons from the upstream region of nut site does not affect N action. Moreover, the lack of translation upstream of nutR does not overcome the antitermination defect caused by nusE mutation. When the upstream region is translated, however, N action is impaired if translation terminates 19 base pairs upstream of nutR . Termination of translation at further upstream sites, such as 23 or 97 base pairs upstream, does not interfere with N action. Our results suggest that the S10 ribosomal protein is required for N action without involving translation. These results also suggest that the nut site RNA itself plays an important role in the formation of a termination-resistant transcription complex.

Bacteriophage lambda↗
PubMed1999

Mutations and altered expression of the human cancer genes: what they tell us about causes.

To understand the causes of cancer, it is necessary to elucidate the molecular basis and environmental factors that influence the carcinogenesis process. Cancers are progressive diseases characterized by the accumulation of defects in many different genes. The patterns of mutation of some genes identified in tumours suggest a direct action of chemicals binding to and altering DNA. Other cancer-associated genes may be altered as a consequence of endogenous mutagens, germ-line mutations, spontaneous mutations that occur during cell replication or increased genetic instability in precancerous cells. Recent advances in molecular biology and genetics have provided new tools and concepts for studying the causes of cancer. We know that cancers are caused by a combination of environmental and genetic factors, and the discovery of the molecular alterations that occur at various stages in different tumours is increasing our understanding of these causes. Thus, we are now beginning to discover which genes are involved, how they function normally and in tumour tissues and why cancers develop after a series of genetic and epigenetic changes in certain cells. As data from studies on cancer-associated genes have accrued, the categories of genes and molecular pathways that have been found to play a role in carcinogenesis have also increased. Genes involved in development and other normal cellular processes have been implicated in cancer. These include genes involved in signal transduction, cell cycle control, DNA repair, cell growth and differentiation (growth factors and growth factor receptors), transcriptional regulation, senescence and apoptosis. Genes involved in angiogenesis, immune regulation, cellular responses to stress, motility, adhesion and invasion are also involved, but less is known about their relationship to carcinogenesis, and these processes are not discussed in this review. The diverse nature of these categories of cancer-related genes indicates the variety of processes that must be disrupted in order for tumours to develop. Many of the genes have several functional domains, and the functions of some have only recently been proposed. In this review, we describe some of the major classes of genes implicated in human cancers and some of the major findings on genetic alterations and dysfunction in human tumours. Comparisons are made with certain rodent models.

Animals↗
PubMed1994

inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis.

Isoniazid (isonicotinic acid hydrazide, INH) is one of the most widely used antituberculosis drugs, yet its precise target of action on Mycobacterium tuberculosis is unknown. A missense mutation within the mycobacterial inhA gene was shown to confer resistance to both INH and ethionamide (ETH) in M. smegmatis and in M. bovis. The wild-type inhA gene also conferred INH and ETH resistance when transferred on a multicopy plasmid vector to M. smegmatis and M. bovis BCG. The InhA protein shows significant sequence conservation with the Escherichia coli enzyme EnvM, and cell-free assays indicate that it may be involved in mycolic acid biosynthesis. These results suggest that InhA is likely a primary target of action for INH and ETH.

Amino Acid Sequence↗
PubMed1999

Role of FSH in male gonadal function.

The production of male gametes depends on the concerted action of the two gonadotropins FSH and LH on the testis. The action of LH is mediated through the production of testosterone by the Leydig cells. Since male germ cells possess neither FSH nor androgen receptors, the action of FSH and testosterone occurs through the Sertoli cells. Although the precise function of these two hormones remains elusive, the existing evidence suggest that both FSH and testosterone are able to stimulate all phases of spermatogenesis. In the male FSH is required for the determination of Sertoli cell number, and for induction and maintenance of normal sperm production. The crucial role of FSH in male gonadal function has been clearly illustrated by the discovery of a patient with an activating mutation of the FSH receptor. This patient had been hypophysectomized because of a pituitary tumor and, under testosterone substitution was unexpectedly fertile in spite of undetectable serum gonadotropin levels and had fathered three children. In this patient we could demonstrate a heterozygous activating mutation of the FSH receptor which resulted in cAMP production independent of FSH stimulation. This finding represents the first description of an activating mutation of the FSH receptor and demonstrates that FSH alone maintains spermatogenesis in man. On the other hand, the effects of the lack of FSH action are unclear. Among five men with a homozygous inactivating mutation of the FSH receptor only one was infertile and spermatogenesis was variably affected in the others. However, serum inhibin B values in these men were not completely suppressed and serum FSH levels were only moderately elevated, indicating the possibility that FSH receptor function was not completely abolished by the mutation. Elimination of FSH action is a prerequisite to suppress completely spermatogenesis for contraceptive purposes, while administration of both LH and FSH is necessary to induce sperm production in patients with hypogonadotropic hypogonadism. Experimental immunization of male monkeys against FSH markedly reduced germ cell proliferation and even induced infertility. At the cellular level, FSH stimulates the cAMP-dependent activation of protein kinase A in Sertoli cells, but the molecular mechanism of FSH action is poorly understood. In the primate, the gonadotropin withdrawal achieved by administration of a GnRH antagonist leads to a premeiotic arrest of germ cell proliferation, probably due to inhibition of the mitotic division of A-pale spermatogonia. Therefore, FSH might be the prime inducer of spermatogonial proliferation, while the successive maturation process could proceed independently of FSH. In summary, clinical and experimental evidence support the concept of an irreplaceble role of FSH in the primate. Only the combination of FSH and testosterone, however, supports a qualitatively and quantitatively fully normal spermatogenesis.

Animals↗
PubMed1975

Allelic negative complementation at the Abruptex locus of Drosophila melanogaster.

The mutations of the Abruptex locus in Drosophila melanogaster fall into three categories. There are recessive lethal alleles and viable alleles. The latter can be divided into suppressors and nonsuppressors of Notch mutations. The recessive lethals are lethal in heterozygous combination with Notch. As a rule the recessive lethals are lethal also in heterozygous combination with the viable alleles. Heterozygous combinations of certain viable alleles are also lethal. In such heterozygotes, one heteroallele is a suppressor of Notch and the other is a nonsuppressor. Other heterozygous combinations of viable alleles are viable and have an Abruptex phenotype. The insertion of the wild allele of the Abruptex locus as an extra dose (carried by a duplication) into the chromosomal complement of the fly fully restores the viability of the otherwise lethal heterozygotes if two viable alleles are involved. The extra wild allele also restores the viability of heterozygotes in which a lethal and a suppressor allele are present. If, however, a lethal and a nonsuppressor are involved, the wild allele only partly restores the viability, and the effect of the wild allele is weakest if two lethal alleles are involved. It seems likely that of the viable alleles the suppressors of Notch are hypermorphic and the nonsuppressors are hypomorphic. The lethal alleles share properties of both types, and are possibly antimorphic mutations. It is suggested that the locus is responsible for a single function which, however, consists of two components. The hypermorphic mutations are defects of the one component and the hypomorphic mutations of the other. In heterozygotes their cumulative action leads to decreased viability. The lethal alleles are supposed to be defects of the function as a whole. The function controlled by the locus might be a regulative function.

Alleles↗
PubMed1998

Secreted beta-amyloid precursor protein counteracts the proapoptotic action of mutant presenilin-1 by activation of NF-kappaB and stabilization of calcium homeostasis.

Mutations in the presenilin-1 (PS-1) gene account for approximately 50% of the cases of autosomal dominant, early onset, inherited forms of Alzheimer's disease (AD). PS-1 is an integral membrane protein expressed in neurons and is localized primarily in the endoplasmic reticulum (ER). PS-1 mutations may promote neuronal degeneration by altering the processing of the beta-amyloid precursor protein (APP) and/or by engaging apoptotic pathways. Alternative processing of APP in AD may increase production of neurotoxic amyloid beta-peptide (Abeta) and reduce production of the neuroprotective alpha-secretase-derived form of APP (sAPPalpha). In differentiated PC12 cells expressing an AD-linked PS-1 mutation (L286V), sAPPalpha activated the transcription factor NF-kappaB and prevented apoptosis induced by Abeta. Treatment of cells with kappaB decoy DNA blocked the antiapoptotic action of sAPPalpha, demonstrating the requirement for NF-kappaB activation in the cytoprotective action of sAPPalpha. Cells expressing mutant PS-1 exhibited an aberrant pattern of NF-kappaB activity following exposure to Abeta, which was characterized by enhanced early activation of NF-kappaB followed by a prolonged depression of activity. Blockade of NF-kappaB activity in cells expressing mutant PS-1 by kappaB decoy DNA was associated with enhanced Abeta-induced increases of [Ca2+]i and mitochondrial dysfunction. Treatment of cells with sAPPalpha stabilized [Ca2+]i and mitochondrial function and suppressed oxidative stress by a mechanism involving activation of NF-kappaB. Blockade of ER calcium release prevented (and stimulation of ER calcium release by thapsigargin induced) apoptosis in cells expressing mutant PS-1, suggesting a pivotal role for ER calcium release in the proapoptotic action of mutant PS-1. Finally, a role for NF-kappaB in preventing apoptosis induced by ER calcium release was demonstrated by data showing that sAPPalpha prevents thapsigargin-induced apoptosis, an effect blocked by kappaB decoy DNA. We conclude that sAPPalpha stabilizes cellular calcium homeostasis and protects neural cells against the proapoptotic action of mutant PS-1 by a mechanism involving activation of NF-kappaB. The data further suggest that PS-1 mutations result in aberrant NF-kappaB regulation that may render neurons vulnerable to apoptosis.

Amyloid beta-Peptides↗
PubMed1979

Measurement of mutagenesis in mammalian cells.

A method using mammalian cells in vitro for detection and quantitation of mutagenic actions that appears to be useful for screening for carcinogenesis and genetic damage by environmental agents is presented. The method involves use of stable human--Chinese hamster ovary hybrid cells that have retained a single human chromosome not necessary for cell reproduction. Forward mutations are detected in genes necessary for production of specific human cell surface antigens. Such mutants form colonies in the presence of specific antisera and complement that destroy the unmutagenized cells. Use of the method is illustrated for the action of x-irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, and caffeine. The method appears to be unique in that it permits assessment of lesions that cause loss of all or most of the chromosome as well as various localized gene mutations. The former action is particularly important because of the major involvement of chromosomal lesions in an extremely important class of human genetic disease.

Amniotic Fluid↗
PubMed1995

A frame-shift mutation of the androgen receptor gene in a patient with receptor-negative complete testicular feminization: comparison with a single base substitution in a receptor-reduced incomplete form.

Mutations of the androgen receptor that impair the action of androgens result in abnormal male sexual development. We studied the structure of the androgen receptor gene in a patient with the receptor-negative form of complete testicular feminization and another patient with a receptor-reduced form of incomplete testicular feminization. In the subject with complete testicular feminization, the deletion of a single nucleotide occurred at nucleotide number 1893 at exon 2. The subsequent frame-shift mutation changes the sense of codon 622 from cysteine to a translational stop signal. Codon 622 is exon 3, so the mutation predicts the synthesis of a truncated receptor that lacks the entire androgen-binding domain. Analysis of a subject with incomplete testicular feminization revealed a single substitution (CGT --> CAT) at nucleotide 2675 of exon 7, resulting in the conversion of an arginine at amino acid 840 to a histidine. This mutation in the androgen-binding domain may impair, but not remove, the androgen binding to its receptor. These results suggest that the phenotypes in our subjects are due to the mutations, and that single amino acid substitution and premature termination codon can cause variably severe functional abnormalities.

Adult↗
PubMed2004

Non-classical regulation of estrogen receptor-alpha by ICI182,780.

Estrogen receptor-alpha (ER alpha) regulates transcription through a number of molecular mechanisms. Two mechanisms by which ER alpha acts directly in the nucleus have emerged: (1) in classical ER alpha action, estrogen-bound receptor binds estrogen response elements (ERE) and regulates promoters by recruiting coactivators or corepressors to DNA; (2) non-classical action is not dependent on ER alpha binding to EREs; its mechanism is not as clearly defined as classical action. In many instances, non-classical action is mediated by tethering of ER alpha to other DNA-binding proteins, facilitating recruitment of coregulators to transcription regulatory sequences. In some cell types, non-classical stimulation can be enhanced by antagonists and repressed by agonists of ER alpha. Here, we show that non-classical action of ER alpha in 293 cells occurs in a wide range of enhancers and enhancer binding proteins. ER alpha stimulates AP-1 elements, cyclic AMP response elements (CRE), and serum response elements (SRE) in the presence of the antiestrogen ICI182,780. Further, in the presence of ICI182,780, ER alpha stimulates activation domains of Jun, ATF-2, Elk, and CRE-binding protein (CREB). Non-classical ER alpha regulation described here does not appear to be sensitive to point mutations which affect classical and tethered ER alpha action; moreover, in our experiments, non-classical action is uniquely sensitive to nuclear transport inhibition by leptomycin B. Because ICI182,780 appears to affect multiple and diverse transcriptional systems, our results are likely explained by ER alpha-dependent modulation of common components of the transcriptional machinery and may not be completely explained by tethering of ER alpha to specific transcription factors.

Cell Line↗
PubMed2003

Characterization of disease-associated mutations affecting an exonic splicing enhancer and two cryptic splice sites in exon 13 of the cystic fibrosis transmembrane conductance regulator gene.

Sequences in exons can play an important role in constitutive and regulated pre-mRNA splicing. Since exonic splicing regulatory sequences are generally poorly conserved and their mechanism of action is not well understood, the consequence of exonic mutations on splicing can only be determined empirically. In this study, we have investigated the consequence of two cystic fibrosis (CF) disease-causing mutations, E656X and 2108delA, on the function of a putative exonic splicing enhancer (ESE) in exon 13 of the CFTR gene. We have also determined whether five other CF mutations D648V, D651N, G654S, E664X and T665S located near this putative ESE could lead to aberrant splicing of exon 13. Using minigene constructs, we have demonstrated that the E656X and 2108delA mutations could indeed cause aberrant splicing in a predicted manner, supporting a role for the putative ESE sequence in pre-mRNA splicing. In addition, we have shown that D648V, E664X and T665S mutations could cause aberrant splicing of exon 13 by improving the polypyrimidine tracts of two cryptic 3' splice sites. We also provide evidence that the relative levels of two splicing factors, hTra2alpha and SF2/ASF, could alter the effect on splicing of some of the exon 13 disease mutations. Taken together, our results suggest that the severity of CF disease could be modulated by changes in the fidelity of CFTR pre-mRNA splicing.

Animals↗
PubMed1993

[The effect of inhibitors on induced mutagenesis].

The mutagenic effect induced by mitomycin C (MC) 20 mg/ml in early, middle and late G1-phase of Crepis capillaris seeds was modified by DNA synthesis inhibitors--caffeine 5 mM, hydroxyurea (HU) 5 x 10(-3) M, 1-beta-D-arabinofuranosylcytosine (araC) 1 x 10(-3) M and aphidicolin (APC) 0.5 mg/ml in the DNA post-synthesis phase (G2). It was shown that under the action of MC in early G1 and the inhibitors in the G2 the total number of aberrations for fixations is lower in all the cases than under the action of MC only. The action of MC in the middle phase results in the differential effect of the inhibitors. HU and APC were shown to potentiate the effect induced by MC and ara-C to reduce it. In both cases, caffeine at the dose 5 mM had small effect on DNA damages induced by MC in early and middle G1. Under the action of caffeine and HU in the G2-phase the crosslinks induced by MC in the late G1 result in aberrations of chromosomal type, illegal for the S-dependent mutagenesis. On the whole, the process of mutation formation turned out to be sensibilized. The specificity of mutagenesis under the action of MC suggests the appearance of mutations of the chromosomal type after the DNA synthesis in G2 phase.

Aphidicolin↗
PubMed2003

A mutation in transmembrane domain II of the 5-hydroxytryptamine(3A) receptor stabilizes channel opening and alters alcohol modulatory actions.

Mutant 5-hydroxytryptamine (5-HT)3A receptors, in which changes were made at Ile294, position 16', of the second transmembrane domain, were assessed for alterations in macroscopic response kinetics and modulation by alcohols. Function of heterologously expressed receptors was measured in Xenopus oocytes in the two-electrode voltage clamp configuration and in human embryonic kidney 293 cells using whole cell patch-clamp electrophysiological recordings with rapid drug application. Compared with the wild-type receptor, a decrease in the 5-HT EC50 value in the Ile294Thr mutant was observed, whereas an increase in the 5-HT EC50 value in the Ile294Leu mutant was measured. Ile294Thr receptors showed a marked reduction in the extent of desensitization. Ethanol and 2,2,2-trichloroethanol (TCEt) enhanced 5-HT-mediated currents in wild-type and Ile294Leu receptors, but inhibited or had little stimulatory effect in the Ile294Thr mutant. Kinetic analysis revealed that in the presence of TCEt, the slope of activation was unchanged in the Ile294Thr mutant and increased in the wild-type receptor. Alcohol cutoff was altered with wild-type = heptanol and Ile294Leu = hexanol. Kinetic changes in the Ile294Thr mutant that favor the open channel state, as well as reduction in the rate of channel activation in the presence of TCEt, likely underlie this mutant's altered response to n-chain alcohols.

Cells, Cultured↗