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PubMed1996

Luteinizing hormone receptor mutations and sex differentiation.

Mutations in the luteinizing hormone (LH) receptor gene have been found in patients with abnormalities in their sexual differentiation. In this paper we review results obtained in the studies of these LH receptor mutations. Activating and inactivating mutations are discussed with respect to the mechanism of action of LH/human chorionic gonadotrophin but also in light of their impact of the present knowledge of the physiology of sex differentiation and gonadal function.

Disorders of Sex Development↗

PubMed1992

gyrB mutations which confer coumarin resistance also affect DNA supercoiling and ATP hydrolysis by Escherichia coli DNA gyrase.

Coumarins are inhibitors of the ATP hydrolysis and DNA supercoiling reactions catalysed by DNA gyrase. Their target is the B subunit of gyrase (GyrB), encoded by the gyrB gene. The exact mode and site of action of the drugs is unknown. We have identified four mutations conferring coumarin resistance to Escherichia coli: Arg-136 to Cys, His or Ser and Gly-164 to Val. In vitro, the ATPase and supercoiling activities of the mutant GyrB proteins are reduced relative to the wild-type enzyme and show resistance to the coumarin antibiotics. Significant differences in the susceptibility of mutant GyrB proteins to inhibition by either chlorobiocin and novobiocin or coumermycin have been found, suggesting wider contacts between coumermycin and GyrB. We discuss the significance of Arg-136 and Gly-164 in relation to the notion that coumarin drugs act as competitive inhibitors of the ATPase reaction.

Adenosine Triphosphate↗

PubMed2006

Light-induced depolarization of neurons using a modified Shaker K(+) channel and a molecular photoswitch.

To trigger action potentials in neurons, most investigators use electrical or chemical stimulation. Here we describe an optical stimulation method based on semi-synthetic light-activated ion channels. These SPARK (synthetic photoisomerizable azobenzene-regulated K(+)) channels consist of a synthetic azobenzene-containing photoswitch and a genetically modified Shaker K(+) channel protein. SPARK channels with a wild-type selectivity filter elicit hyperpolarization and suppress action potential firing when activated by 390 nm light. A mutation in the pore converts the K(+)-selective Shaker channel into a nonselective cation channel. Activation of this modified channel with the same wavelength of light elicits depolarization of the membrane potential. Expression of these depolarizing SPARK channels in neurons allows light to rapidly and reversibly trigger action potential firing. Hence, hyper- and depolarizing SPARK channels provide a means for eliciting opposite effects on neurons in response to the same light stimulus.

Action Potentials↗

PubMed2004

Dysregulated IP3 signaling in cortical neurons of knock-in mice expressing an Alzheimer's-linked mutation in presenilin1 results in exaggerated Ca2+ signals and altered membrane excitability.

Disruptions in intracellular Ca2+ signaling are proposed to underlie the pathophysiology of Alzheimer's disease (AD), and it has recently been shown that AD-linked mutations in the presenilin 1 gene (PS1) enhance inositol triphosphate (IP3)-mediated Ca2+ liberation in nonexcitable cells. However, little is known of these actions in neurons, which are the principal locus of AD pathology. We therefore sought to determine how PS1 mutations affect Ca2+ signals and their subsequent downstream effector functions in cortical neurons. Using whole-cell patch-clamp recording, flash photolysis, and two-photon imaging in brain slices from 4-5-week-old mice, we show that IP3-evoked Ca2+ responses are more than threefold greater in PS1(M146V) knock-in mice relative to age-matched nontransgenic controls. Electrical excitability is thereby reduced via enhanced Ca2+ activation of K+ conductances. Action potential-evoked Ca2+ signals were unchanged, indicating that PS1(M146V) mutations specifically disrupt intracellular Ca2+ liberation rather than reduce cytosolic Ca2+ buffering or clearance. Moreover, IP3 receptor levels are not different in cortical homogenates, further suggesting that the exaggerated cytosolic Ca2+ signals may result from increased store filling and not from increased flux through additional IP3-gated channels. Even in young animals, PS1 mutations have profound effects on neuronal Ca2+ and electrical signaling: cumulatively, these disruptions may contribute to the long-term pathophysiology of AD.

Action Potentials↗

PubMed2003

Herpes simplex virus resistance to antiviral drugs.

Herpes simplex virus (HSV) infections are efficiently treated with antiviral drugs such as acyclovir (ACV). However, resistance has been reported, mainly among immunocompromised patients (prevalence around 5%) and particularly allogeneic bone marrow transplant patients (prevalence reaching 30%). Resistance to ACV is associated with mutations on one of the two viral enzymes involved in the ACV mechanism of action: thymidine kinase (TK) and DNA polymerase. In 95% of the cases, ACV resistance is associated with a mutation in the TK gene as this enzyme is not essential for viral replication, unlike viral DNA polymerase, which is rarely involved in resistance. Strains resistant to ACV are almost always cross-resistant to other TK-dependent drugs such as penciclovir and famciclovir. Resistant infections can be managed by foscarnet or cidofovir but both are more toxic than ACV. These drugs also inhibit viral DNA polymerase but they are active on most ACV-resistant HSV as they do not depend on TK; nevertheless virus resistant to ACV because of a mutation in the DNA polymerase may be cross-resistant to these molecules. Published data on genetic characterization of resistant clinical isolates point out hot spots in viral TK and DNA polymerase genes. TK mutations associated with resistance are either insertion or deletion (codons 92 and 146 of TK gene) or substitution (codon 176-177, 336 of TK gene). DNA polymerase mutations are mainly located in conserved sites of the enzyme. A high level of gene polymorphism has also been reported for these genes, especially for TK. These results are useful for the development of rapid genotypic assays for the detection of mutations associated with resistance.

Acyclovir↗

PubMed1998

[Radiosensitivity of Saccharomyces yeasts and srm genes: effects of srm1 and srm5 mutations].

The presence in the cell genotype of srm1 and srm5 (cdc28-srm) mutations decreasing the spontaneous rho- mutability was shown to have no effect on the rates of spontaneous nuclear gene mutations and gamma-ray-induced mitotic recombination. Mutation cdc28-srm exerts a marked effect on cell sensitivity to the lethal action of ionizing radiation and on the appearance of homoplasmic segregants generated from heteroplasmic diploids. Additive interactions between mutations cdc28-srm and each of the rad6 and rad52 mutations were revealed by an analysis of double mutants with respect to their sensitivity to radiation. Mutation rad9 was epistatic with mutation cdc28-srm. These data agree with the idea that the p34CDC28 gene product is a target for the RAD9-dependent feedback control operating at the cell cycle checkpoints (checkpoint control) and ensuring an additional amount of time for premitotic repair of chromosomal DNA damage.

Alleles↗

PubMed1998

Different pathways for protein degradation by the FtsH/HflKC membrane-embedded protease complex: an implication from the interference by a mutant form of a new substrate protein, YccA.

Escherichia coli FtsH (HflB) is a membrane-bound and ATP-dependent zinc-metalloproteinase, which forms a complex with a pair of periplasmically exposed membrane proteins, HflK and HflC. It is the protease that degrades uncomplexed forms of the SecY subunit of protein translocase. Here, we characterized a new class of SecY-stabilizing mutation on the E. coli chromosome. The mutation (yccA11) is an internal deletion within a gene (yccA) known as an open reading frame for a hydrophobic protein with putative seven transmembrane segments. The YccA protein was found to be degraded in an FtsH-dependent manner in vivo and in vitro, whereas the YccA11 mutant protein, lacking eight amino acid residues within the amino-terminal cytoplasmic domain, was refractory to the degradation. The yccA11 mutation exhibited partial dominance when overexpressed. Cross-linking, co-immunoprecipitation, and histidine tagging experiments showed that YccA11 as well as YccA can associate with both the FtsH and the HflKC proteins. Thus, the mutant YccA protein appeared to compete with SecY for recognition by the FtsH proteolytic system and the residues deleted by the yccA mutation are required for the initiation of proteolysis by FtsH. Interestingly, the inhibitory action of YccA11 was mediated by HflKC, since the deletion of hflK-hflC suppressed the yccA11 phenotype. The yccA11 mutation stabilized subunit a of the proton ATPase F0 segment as well, but not the CII protein of bacteriophage lambda or the sigma 32 protein. From these results we suggest that there are at least two pathways for FtsH-dependent protein degradation, only one of which (probably for membrane proteins) is subject to the HflKC-dependent interference by the YccA11 mutant substrate.

ATP-Dependent Proteases↗

PubMed1993

Synergism and antagonism between N-methyl-N'-nitro-N-nitroso-guanidine and 9-aminoacridine in mutation induction in Escherichia coli K12.

When bacteria were treated simultaneously with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and low concentrations of 9-aminoacridine (9AA), the yield of frameshift mutations was much greater than that expected on the basis of independent action of both mutagens. In combination with a high concentration of 9AA, however, MNNG had an antagonistic effect upon the induction of frameshift mutations. There was no synergistic interaction between the two mutagens in bacteria in which the adaptive response to methylating agents had been induced. 9AA not only induced frameshift mutations, but also caused a small increase in reversions of a nonsense (ochre) mutation and, in combination with low MNNG concentrations, it had a small synergistic effect.

Aminacrine↗

PubMed1994

Mechanisms of hormone resistance: lessons from insulin-resistant patients.

Hormones are secreted by endocrine glands and transported to the target cell at which the hormone acts. The hormone binds to its receptor, thereby eliciting various biological responses within the target cell. Examples of disease mechanisms that function at the different stages in the development of the insulin receptor, and result in insulin resistance, are discussed in this review. Antibodies to insulin can impair delivery of the hormone to the target cell, and can desensitize that target cell to insulin action. In recent years, several genetic diseases have been identified that result from mutations in the genes encoding the relevant receptors. Studies of syndromes of insulin resistance provide illustrations of the multiple types of defects in receptor function that can generally cause hormone resistance (12, 13). For example, mutations in the receptor can decrease the number of receptors on the cell surface by inhibiting receptor biosynthesis, impairing receptor transport to the cell surface, or accelerating the rate of receptor degradation. Alternatively, mutations have been identified that decrease the affinity of insulin binding or inhibit receptor tyrosine kinase activity. In recent years, there has been considerable progress toward elucidating post-receptor mechanisms in the biochemical pathways of hormone action. At present, there are a limited number of examples of mutations in genes encoding proteins that function in this part of the pathway, but it seems likely that additional examples will be discovered in the future. It is likely that these insights into biochemical mechanisms of disease will ultimately lead to an improvement in our ability to treat human disease.

Genetic Diseases, Inborn↗

PubMedDate not supplied

Knockout mice challenge our concepts of glucose homeostasis and the pathogenesis of diabetes.

A central component of type 2 diabetes and the metabolic syndrome is insulin resistance. Insulin exerts a multifaceted and highly integrated series of actions via its intracellular signaling systems. Generation of mice carrying null mutations of the genes encoding proteins in the insulin signaling pathway provides a unique approach to determining the role of individual proteins in the molecular mechanism of insulin action and the pathogenesis of insulin resistance and diabetes. The role of the four major insulin receptor substrates (IRS1-4) in insulin and IGF-1 signaling have been examined by creating mice with targeted gene knockouts. Each produces a unique phenotype, indicating the complementary role of these signaling components. Combined heterozygous defects often produce synergistic or epistatic effects, although the final severity of the phenotype depends on the genetic background of the mice. Conditional knockouts of the insulin receptor have also been created using the Cre-lox system. These tissue specific knockouts have provide unique insights into the control of glucose homeostasis and the pathogenesis of type 2 diabetes, and have led to development of new hypotheses about the nature of the insulin action and development of diabetes.

Animals↗

PubMed1979

High-level resistance to streptomycin in Escherichia coli with R1 plasmid and the analysis of genetic determinants.

Some properties of streptomycin-resistant mutants of Escherichia coli were analyzed. In a R+ culture, the phenotype under study may be significantly selected at a frequency of 10(-5) on media with higher streptomycin level. The lrs mutation is present in the cells prior to the action of streptomycin and remains in the cells even after curing of the R1 plasmid. The mapping of the lrs gene by conjugation with a concomitant transfer of chromosome and the R1 plasmid in different Hfr strains of E. coli failed to establish the localization of this gene in the tested chromosome regions. The presence of a cryptic plasmid was detected in cells with the lrs mutation after curing of the R1 plasmid. This plasmid codes neither fertility functions nor chloramphenicol-acetyltransferase, streptomycin-adenyltransferase, or ampicillin-beta-lactamase.

Chromosome Mapping↗

PubMed1990

NS-1 and NS-2 proteins may act synergistically in the cytopathogenicity of parvovirus MVMp.

The interaction of parvovirus minute virus of mice (prototype strain, MVMp) with simian virus 40 (SV40)-transformed human cells (NB-E) was investigated by means of transfection with MVMp molecular clones derived from the infectious recombinant plasmid (pMM984). pMM984 inhibits stable transformation of NB-E cells to geneticin resistance (G418R) upon cotransfection with the selectable pSV2neo plasmid. We show here that this inhibition is not merely caused by a repression of marker gene expression from the SV40 early region promoter in pSV2neo and rather is likely to reflect the cytotoxic action of the parvovirus. Starting from plasmid pMM984, defined mutations were introduced into the genome of MVMp and more particularly into sequences coding for the NS-1 and/or NS-2 nonstructural proteins. In this way we could show that the NS-1 protein is necessary for the inhibition of transformation to G418R and that the NS-2 protein acts synergistically to enhance this effect. Moreover, results obtained with different viral mutants indicate that the inhibitory action of NS-1 on stable transformation can be dissociated from the ability of this protein both to transactivate the parvoviral p39 promoter of the capsid protein-encoding region and to drive parvoviral DNA amplification. Altogether these data point to a probable direct toxicity of MVMp nonstructural proteins for permissive host cells.

Capsid↗

PubMed1999

Mutation screening of the interferon-gamma gene as a candidate gene for multiple sclerosis.

Interferon-gamma (IFN-gamma) plays a crucial role in the regulation of the immune response. Alterations in IFN-gamma production have been found in several diseases including multiple sclerosis (MS). Such alterations could be caused by the action of different factors on cytokine production, or, theoretically, by mutations in the gene. We screened the IFN-gamma gene promoter and part of the first intron, known to contain a c-Rel specific enhancer, for possible mutations by sequencing. We found a C to T substitution in the IFN-gamma promoter at position -333. Screening for this mutation by sequence-specific PCR in 214 MS patients and 164 controls identified two patients, both heterozygous, but no controls with this mutation. No mutations were found in the first intron. The interferon-gamma gene is highly conserved and changes in IFN-gamma expression are probably due to the influence of regulatory factors on gene transcription, rather than gene polymorphisms.

Case-Control Studies↗

PubMed2006

The POLARIS peptide of Arabidopsis regulates auxin transport and root growth via effects on ethylene signaling.

The rate and plane of cell division and anisotropic cell growth are critical for plant development and are regulated by diverse mechanisms involving several hormone signaling pathways. Little is known about peptide signaling in plant growth; however, Arabidopsis thaliana POLARIS (PLS), encoding a 36-amino acid peptide, is required for correct root growth and vascular development. Mutational analysis implicates a role for the peptide in hormone responses, but the basis of PLS action is obscure. Using the Arabidopsis root as a model to study PLS action in plant development, we discovered a link between PLS, ethylene signaling, auxin homeostasis, and microtubule cytoskeleton dynamics. Mutation of PLS results in an enhanced ethylene-response phenotype, defective auxin transport and homeostasis, and altered microtubule sensitivity to inhibitors. These defects, along with the short-root phenotype, are suppressed by genetic and pharmacological inhibition of ethylene action. PLS expression is repressed by ethylene and induced by auxin. Our results suggest a mechanism whereby PLS negatively regulates ethylene responses to modulate cell division and expansion via downstream effects on microtubule cytoskeleton dynamics and auxin signaling, thereby influencing root growth and lateral root development. This mechanism involves a regulatory loop of auxin-ethylene interactions.

Amino Acids, Cyclic↗

PubMed2006

Locking CNGA1 channels in the open and closed state.

With the aim of understanding the relation between structure and gating of CNGA1 channels from bovine rod, an extensive cysteine scanning mutagenesis was performed. Each residue from Phe-375 to Val-424 was mutated into a cysteine one at a time and the modification caused by various sulfhydryl reagents was analyzed. The addition of the mild oxidizing agent copper phenanthroline (CuP) in the open (presence of 1 mM cGMP) or closed state locked the channel in the respective states. A subsequent treatment with the reducing agent DTT restored normal gating fully in the open state and partially in the closed state. This action of CuP was not observed when F380 was mutated into a cysteine in the cysteine-free CNGA1 channel and in the double mutant C314S&F380C. These observations suggest that these effects are mediated by the formation of a disulfide bond (S-S) between F380C and the endogenous Cys-314 in the S5 segment. It can be rationalized by supposing that during gating the S6 segment rotates anticlockwise-when viewed from the extracellular side-by approximately 30 degrees .

Animals↗

PubMed1998

Search for mutations near the alternatively spliced 8-amino-acid exon in the GABAA receptor gamma 2 subunit gene and lack of allelic association with alcoholism among four aboriginal groups and Han Chinese in Taiwan.

The alternatively spliced 8-amino-acid exon for the GABAA receptor gamma2 subunit gene (GABRC2) has been postulated to mediate behavioral actions of alcohol. A rapid search for splice-junction mutations near the 8-amino-acid exon using restriction enzymes which normally recognize sequences near or in the exon gave negative results among 217 alcoholics in four aboriginal groups (Ami, Atayal, Bunun and Paiwan) and Han Chinese in Taiwan. The role of the GABRC2 gene in alcoholism was further assessed by a comparison of allelic frequencies revealed by a NciI RFLP between case and control groups. No significant association of alcohol dependence with GABRC2 alleles was observed. These results suggest that the GABRC2 gene probably does not play an essential role in predisposition to alcoholism in the sample population.

Alcoholism↗

PubMed1996

Gene conversion as a focusing mechanism for correlated mutations: a hypothesis.

Ways of producing complex mutational events without substantially raising the primary mutation rate are explored. If the small amount of DNA that is resynthesised through the action of the mismatch DNA repair system is not subject to further repair, the incidence of double mutations can increase by a factor of 100, while single mutations would increase by only 30%. Such a boost in the incidence of double mutations seems insufficient to meet the needs of higher organisms. For them, an alternative strategy would be to produce complex events by a succession of single mutations occurring in a correlated manner over several sexual generations. It is proposed that gene conversion may fulfill this role. Assuming that the resynthesis of DNA that occurs during gene conversion produces mutations in the conversion tract, one predicts a tendency for close mutations in corresponding sequences in the two homologous chromosomes, to promote, during conversion, further mutations in their vicinity. Semiquantitative calculations suggest that such a mechanism can be quite effective, provided the divergence between two paired chromosomes is around 10(-4) or less. Such a mechanism might constitute an adaptive mutation strategy acting at the population level.

Animals↗

PubMed1999

Convergent pathways for utilization of the amino sugars N-acetylglucosamine, N-acetylmannosamine, and N-acetylneuraminic acid by Escherichia coli.

N-Acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NANA) are good carbon sources for Escherichia coli K-12, whereas N-acetylmannosamine (ManNAc) is metabolized very slowly. The isolation of regulatory mutations which enhanced utilization of ManNAc allowed us to elucidate the pathway of its degradation. ManNAc is transported by the manXYZ-encoded phosphoenolpyruvate-dependent phosphotransferase system (PTS) transporter producing intracellular ManNAc-6-P. This phosphorylated hexosamine is subsequently converted to GlcNAc-6-P, which is further metabolized by the nagBA-encoded deacetylase and deaminase of the GlcNAc-6-P degradation pathway. Two independent mutations are necessary for good growth on ManNAc. One mutation maps to mlc, and mutations in this gene are known to enhance the expression of manXYZ. The second regulatory mutation was mapped to the nanAT operon, which encodes the NANA transporter and NANA lyase. The combined action of the nanAT gene products converts extracellular NANA to intracellular ManNAc. The second regulatory mutation defines an open reading frame (ORF), called yhcK, as the gene for the repressor of the nan operon (nanR). Mutations in the repressor enhance expression of the nanAT genes and, presumably, three distal, previously unidentified genes, yhcJIH. Expression of just one of these downstream ORFs, yhcJ, is necessary for growth on ManNAc in the presence of an mlc mutation. The yhcJ gene appears to encode a ManNAc-6-P-to-GlcNAc-6-P epimerase (nanE). Another putative gene in the nan operon, yhcI, likely encodes ManNAc kinase (nanK), which should phosphorylate the ManNAc liberated from NANA by the NanA protein. Use of NANA as carbon source by E. coli also requires the nagBA gene products. The existence of a ManNAc kinase and epimerase within the nan operon allows us to propose that the pathways for dissimilation of the three amino sugars GlcNAc, ManNAc, and NANA, all converge at the step of GlcNAc-6-P.

Acetylglucosamine↗