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PubMed1999

Dietary restriction protects hippocampal neurons against the death-promoting action of a presenilin-1 mutation.

Alzheimer's disease (AD) is an age-related disorder that involves degeneration of synapses and neurons in brain regions involved in learning and memory processes. Some cases of AD are caused by mutations in presenilin-1 (PS1), an integral membrane protein located in the endoplasmic reticulum. Previous studies have shown that PS1 mutations increase neuronal vulnerability to excitotoxicity and apoptosis. Although dietary restriction (DR) can increase lifespan and reduce the incidence of several age-related diseases in rodents, the possibility that DR can modify the pathogenic actions of mutations that cause AD has not been examined. The vulnerability of hippocampal neurons to excitotoxic injury was increased in PS1 mutant knockin mice. PS1 mutant knockin mice and wild-type mice maintained on a DR regimen for 3 months exhibited reduced excitotoxic damage to hippocampal CA1 and CA3 neurons compared to mice fed ad libitum; the DR regimen completely counteracted the endangering effect of the PS1 mutation. The magnitude of increase in levels of the lipid peroxidation product 4-hydroxynonenal following the excitotoxic insult was lower in DR mice compared to mice fed ad libitum, suggesting that suppression of oxidative stress may be one mechanism underlying the neuroprotective effect of DR. These findings indicate that the neurodegeneration-promoting effect of an AD-linked mutation is subject to modification by diet.

Alzheimer Disease↗
PubMed1993

Differential action of the albino mutation on two components of the rat's uncrossed retinofugal pathway.

The development of the uncrossed retinofugal pathways in normally pigmented and albino rats, aged from embryonic day (E) 14.5 to E18.5, was investigated. DiI was placed into one optic tract and the retinal origin of the uncrossed component, as well as its course in the optic stalk, was studied. The results show that, as in the mouse, the uncrossed retinal projection has two components. The first component is seen at E15.5 in normally pigmented animals. It develops exclusively in the central parts of the retina and is normal in albino littermates. The second component, which arises from the peripheral parts of the ventrotemporal retina, is seen two days later at E17.5 in all animals but is significantly smaller in albinos than in their pigmented littermates. Studies of axons in the optic stalk labelled retrogradely with DiI placed in the optic tract indicate that the uncrossed axons have no preference for any position in the stalk except when they approach the chiasm, where they tend to accumulate at the caudal region of the stalk. The uncrossed axons intermingle with the crossed axons along the entire length of the stalk. In albino embryos, no obvious difference in the prechiasmatic course of uncrossed axons was seen at any age examined. It is concluded that the albino mutation in rats affects the late ventrotemporal component of the uncrossed pathway selectively. It does not act on the early central component. Further, the intermingling of crossed and uncrossed axons in the stalk and the apparently unaffected prechiasmatic course of uncrossed axons in albinos indicate that the albino gene has its primary action in the retina.

Albinism↗
PubMed1977

[Induction of gene mutations and the lethal action of ultraviolet rays in synchronized Chinese hamster cells].

Lethal and mutagenic effects of UV light were studied in two synchronized UV-sensitive Chinese hamster cell clones differing in the degree of sensitivity (CHS1, CHS2). It is shown that the phase of mitosis is most resistant to the lethal effect of UV. The sensitivity of both cell clones increases in the pre-synthetic phase and reaches its maximum during the phase of DNA synthesis. Positive correlation of cell sensitivity to mutagenic and lethal action of UV was observed when studying induced mutability in both cell clones during the phase of DNA synthesis. However, the study of the mutagenic effect of UV on different phases of the synthesis. However, the study of the mutagenic effect of UV on different phases of the cell cycle (M, G1, S) in the less UV-sensitive cell clone has revealed that the maximal mutation yield takes place when cells are irradiated at G1 (CHS1). The discrepancy observed may be due to different probability of the phenotypic detection of pre-mutational lesions, arising at different phases of the cell cycle. It is shown that only one cell generation is necessary for the expression of pre-mutational changes. These data allow to conclude that the increased mutation rate observed at G1 (as compared with S) reveals rather a probability of the expression but not of the occurrence of pre-mutational lesions. It is suggested that the fixation of mutations in the cells studied proceeds during the post-replication repair synthesis.

Animals↗
PubMed1985

Intrinsic growth control in the imaginal primordia of Drosophila, and the autonomous action of a lethal mutation causing overgrowth.

Cell proliferation in Drosophila imaginal discs appears to be regulated by a disc-intrinsic mechanism involving local cell interactions that also control the formation of patterns of differentiation. This growth-control mechanism breaks down in animals homozygous for the mutation lethal (2) giant discs (l(2)gd) which remain as larvae for up to 9 days longer than normal. During this time cell proliferation continues in the imaginal discs as well as in the imaginal rings for the salivary glands, foregut, and hindgut, so that these tissues become greatly overgrown. When wild-type wing discs from mid-third instar larvae were removed and cultured for up to 28 days in wild-type female adult hosts, they grew and terminated growth at a cell number close to that which would be attained in situ by the time of pupariation. On the other hand, wing discs from l(2)gd homozygotes grew rapidly and continuously when cultivated in wild-type hosts, reached an enormous size, and acquired abnormal folding patterns. Overgrowth of mutant imaginal rings also continued during culture of these tissues in wild-type hosts. We conclude that overgrowth in this mutant is due to an autonomous defect in the imaginal primordia, which requires an extended larval period for its expression in situ.

Animals↗
PubMed1995

The biological action of cDNAs from mutated estrogen receptors transfected into breast cancer cells.

While tamoxifen may inhibit breast cancer proliferation, mutations in the estrogen receptor could potentially result in breast cancer cells which can circumvent the tamoxifen blockade. Previously, we identified a mutation at codon 351 in the estrogen receptor from a tamoxifen-stimulated human breast cancer. This receptor was stably transfected into the estrogen receptor-negative human breast cancer cell line MDA-MB-231 (clone 10A). Clones were compared to stably transfected cell lines containing either the wild type or codon 400 mutant estrogen receptor to study the effect of either estradiol or the tamoxifen analogue, fixed-ring 4-hydroxytamoxifen ((fr)4-OH TAM), on cell growth and reporter gene activation. (fr)4-OH TAM reduced the growth rate in cell lines containing mutant estrogen receptors, while the cell line containing the wild type estrogen receptor is minimally influenced by (fr)4-OH TAM. We then needed to show that the ligand-estrogen receptor interaction resulted in estrogen receptor activation. As a ligand-dependent transcription factor, estrogen receptor activation is measured by its ability to stimulate reporter gene (luciferase) transcription when bound to an estrogenic ligand. We found that the wild type estrogen receptor is activated by estradiol but not by the tamoxifen analogue, while the codon 351 estrogen receptor is activated by both (fr)4-OH TAM and estradiol.

Amino Acid Sequence↗
PubMed1995

Dominant-negative action of the jimpy mutation in mice complemented with an autosomal transgene for myelin proteolipid protein.

Mutations in genes encoding membrane proteins have been associated with cell death of unknown cause from invertebrate development to human degenerative diseases. A point mutation in the gene for myelin proteolipid protein (PLP) underlies oligodendrocyte death and dysmyelination in jimpy mice, an accurate model for Pelizaeus-Merzbacher disease. To distinguish the loss of PLP function from other effects of the misfolded protein, we took advantage of the X chromosomal linkage of the gene and have complemented jimpy with a wild-type PLP transgene. In this artificial heterozygous situation, the jimpy mutation emerged as genetically dominant. At the cellular level oligodendrocytes showed little increase in survival although endogenous PLP gene and autosomal transgene were truly coexpressed. In surviving oligodendrocytes, wild-type PLP was functional and immunodetectable in myelin. Moreover, compacted myelin sheaths regained their normal periodicity. This strongly suggests that, despite the presence of functional wild-type PLP, misfolded jimpy PLP is by itself the primary cause of abnormal oligodendrocyte death.

Animals↗
PubMed1989

Scope of action of the immunoglobulin mutator system.

The authors have developed a method to measure the rate of spontaneous mutations taking place in IgH, the gene encoding the immunoglobulin heavy chain. When an amber chain-termination codon mutates to a sense codon, translation of the polypeptide chain will be completed, and mutant cells producing the heavy chain can be detected with a fluorescent labelled antibody. The protocol used is the compartmentalization test which minimizes any effect of selection. In subclones of the pre-B lymphocyte line 18-81, the spontaneous mutation rate in the part of IgH encoding the variable region is somewhat greater than 10(-5) mutations per base pair per generation. This supports the hypothesis that hypermutation is not dependent on cell stimulation by an antigen. In a hybrid between a cell of this line and a myeloma (which represents the terminal stage of the B-cell lineage), the mutation rate was too low to be determined by this test, less than 10(-9). When the same loss to gain procedure system was used with an opal chain-terminating codon in the part of IgH encoding the constant region (C mu), a high rate of reversion by deletion was found. Long (more than one exon) and short (less than one exon) deletions occurred at rates of 1.7 x 10(-5) and 1.4 x 10(-7) per generation, respectively. It is thought that the high rate of deletion is not related to somatic hypermutation but rather to DNA rearrangement during the heavy-chain class switch, which is occurring in these pre-B cell lines. The point mutation rate was too low to be detected above the background of deletion mutants, less than 5 x 10(-8). The immunoglobulin mutator system works weakly, if at all, on two other, nonimmunoglobulin, genes tested: B2m (beta 2 microglobulin) and the gene for ouabain resistance.

Antibody Diversity↗
PubMed2005

Long-QT syndrome-related sodium channel mutations probed by the dynamic action potential clamp technique.

Long-QT3 syndrome (LQT3) is linked to cardiac sodium channel gene (SCN5A) mutations. In this study, we used the 'dynamic action potential clamp' (dAPC) technique to effectively replace the native sodium current (I(Na)) of the Priebe-Beuckelmann human ventricular cell model with wild-type (WT) or mutant I(Na) generated in a human embryonic kidney (HEK)-293 cell that is voltage clamped by the free-running action potential of the ventricular cell. We recorded I(Na) from HEK cells expressing either WT or LQT3-associated Y1795C or A1330P SCN5A at 35 degrees C, and let this current generate and shape the action potential (AP) of subepicardial, mid-myocardial and subendocardial model cells. The HEK cell's endogenous background current was completely removed by a real-time digital subtraction procedure. With WT I(Na), AP duration (APD) was longer than with the original Priebe-Beuckelmann model I(Na), due to a late I(Na) component of approximately 30 pA that could not be revealed with conventional voltage-clamp protocols. With mutant I(Na), this late component was larger ( approximately 100 pA), producing a marked increase in APD ( approximately 70-80 ms at 1 Hz for the subepicardial model cell). The late I(Na) magnitude showed reverse frequency dependence, resulting in a significantly steeper APD-frequency relation in the mutant case. AP prolongation was more pronounced for the mid-myocardial cell type, resulting in increased APD dispersion for each of the mutants. For both mutants, a 2 s pause following rapid (2 Hz) pacing resulted in distorted AP morphology and beat-to-beat fluctuations of I(Na). Our dAPC data directly demonstrate the arrhythmogenic nature of LQT3-associated SCN5A mutations.

Action Potentials↗
PubMed2004

Hepatic nuclear factor 3 and nuclear factor 1 regulate 5-aminolevulinate synthase gene expression and are involved in insulin repression.

Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5'-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3beta or HNF3beta plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3beta is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3beta can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3beta binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.

5-Aminolevulinate Synthetase↗
PubMed1994

Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate.

The effect of protein kinase C (PKC) on rapid N-type inactivation of K+ channels has not been reported previously. We found that PKC specifically eliminates rapid inactivation of a cloned human A-type K+ channel (hKv3.4), converting this channel from a rapidly inactivating A type to a noninactivating delayed rectifier type. Biochemical analysis showed that the N-terminal domain of hKv3.4 is phosphorylated in vitro by PKC, and mutagenesis experiments revealed that two serines within the inactivation gate at the N-terminus are sites of direct PKC action. Moreover, mutating one of these serines to aspartic acid mimics the action of PKC. Serine phosphorylation may thus prevent rapid inactivation by shielding basic residues known to be critical to the function of the inactivation gate. The regulatory mechanism reported here may have substantial effects on signal coding in the nervous system.

Amino Acid Sequence↗
PubMed2000

The long QT syndromes: genetic basis and clinical implications.

It is becoming clear that mutations in the KVLQT1, human "ether-a-go-go" related gene, cardiac voltage-dependent sodium channel gene, minK and MiRP1 genes, respectively, are responsible for the LQT1, LQT2, LQT3, LQT5 and LQT6 variants of the Romano-Ward syndrome, characterized by autosomal dominant transmission and no deafness. The much rarer Jervell-Lange-Nielsen syndrome (with marked QT prolongation and sensorineural deafness) arises when a child inherits mutant KVLQT1 or minK alleles from both parents. In addition, some families are not linked to the known genetic loci. Cardiac voltage-dependent sodium channel gene encodes the cardiac sodium channel, and long QT syndrome (LQTS) mutations prolong action potentials by increasing inward plateau sodium current. The other mutations cause a decrease in net repolarizing current by reducing potassium currents through "dominant negative" or "loss of function" mechanisms. Polymorphic ventricular tachycardia (torsade de pointes) is thought to be initiated by early after-depolarizations in the Purkinje system and maintained by reentry in the myocardium. Clinical presentations vary with the specific gene affected and the specific mutation. Nevertheless, patients with identical mutations can also present differently, and some patients with LQTS mutations may have no manifest baseline phenotype. The question of whether the latter situation is one of high risk for administration of QT prolonging drugs or during myocardial ischemia is under active investigation. More generally, the identification of LQTS genes has provided tremendous new insights for our understanding of normal cardiac electrophysiology and its perturbation in a wide range of conditions associated with sudden death. It seems likely that the approach of applying information from the genetics of uncommon congenital syndromes to the study of common acquired diseases will be an increasingly important one in the next millennium.

Cardiac Pacing, Artificial↗
PubMed2001

Thyrotropin receptor mutations as a tool to understand thyrotropin receptor action.

A large number of mutations have been identified in the thyrotropin (TSH) receptor (TSHR) gene causing human diseases. Toxic thyroid nodules are frequently associated with somatic constitutively activating TSHR mutations. Autosomal dominant non-autoimmune hyperthyroidism is caused by activating TSHR germline mutations. Inactivating germline mutations cause TSH unresponsiveness. Discovery of the different TSHR mutations in various regions of the receptor molecule has led to the identification of important domains for intramolecular TSHR signal transduction. However, despite the functional characterization of the naturally occurring mutations the precise molecular mechanisms of receptor activation including the processes of hormone binding, intramolecular signaling between the different TSHR domains and of G protein coupling are not completely understood. This review discusses the importance of the various receptor domains for TSHR activation identified on the basis of the naturally occurring gain or loss of function mutations and in vitro investigations performed with site-directed mutagenesis, synthetic peptides, or antibodies. Several in vitro studies have provided new insights into structure-function relationships by site-directed mutagenesis in combination with molecular modeling. These in vitro investigations have often been guided by naturally occurring mutations and have provided new insights into intramolecular changes during receptor activation. This has led to progress in understanding the mechanism of TSHR activation.

Animals↗
PubMed2003

Bone morphogenetic proteins and folliculogenesis: lessons from the Booroola mutation.

The Booroola phenotype is associated with a point mutation in the kinase domain of the bone morphogenetic protein receptor 1 B (BMPR1 B), and is characterized by 'precocious' differentiation of ovarian follicles, leading to the production of large numbers of ovulatory follicles that are smaller in diameter than wild-type follicles. These smaller follicles attain differentiation markers, such as expression of mRNA for P450 aromatase and inhibin-betaA subunit, granulosa cell LH receptors and aromatase activity, earlier than follicles from wild-type ewes. However, the preovulatory follicles from mutant ewes collectively secrete similar quantities of oestradiol, androstenedione and inhibin A in exactly the same pattern as wild-type ewes, which result in similar concentrations of FSH. The available evidence strongly indicates that the Booroola mutation exerts its action at the ovary rather than by altering gonadotrophin secretion. The bone morphogenetic protein (BMP) receptors and putative ligands are ubiquitously expressed within the ovary and BMPs seem to be involved in the paracrine regulation of FSH action. Thus, if the mutation is causing a reduction in BMPR1 B signalling, it may act on an inhibitor of follicle differentiation. Further research in this area will concentrate on the elucidation of the natural ligands for BMPR1 B at different stages of follicle development and examine the effect of BMPR1 B mutation on the downstream signalling cascade.

Animals↗
PubMed1990

Photodynamic action of methylene blue: repair and mutation in Escherichia coli.

The effects of the photodynamic action of methylene blue (MB-PDA) on strains of Escherichia coli were investigated to determine whether the dye could be used in photodynamic therapy (PDT). Using the method of alkaline sucrose gradient sedimentation, it was shown that in darkness MB induces a type of prelesion in DNA that transforms into single-strand breaks in alkaline conditions, provided that the dye is present during the processing of the gradient. This prelesion is completely reversible if the cells are washed immediately to remove the dye. However, after illumination with white light, the prelesions become "fixed" stable lesions, irreversible even after successive washings. The lethal damage induced by MB-PDA in E. coli can be repaired by the excision-repair system (about 30%) and by the recA-dependent repair system (about 70%). Polymerase I enzyme participates actively in the repair of the damage. MB-PDA is a weak mutagen and the induction of mutations by this treatment is restricted to high survival rates. Moreover, MB-PDA does not induce the SOS system (an inducible repair system dependent of the recA and loxA genes products), as measured by Weigle reactivation. However, it seems that this treatment can impair the repair systems in E. coli.

Bacteriophage lambda↗
PubMed2006

Mutation, selection, and ancestry in branching models: a variational approach.

We consider the evolution of populations under the joint action of mutation and differential reproduction, or selection. The population is modelled as a finite-type Markov branching process in continuous time, and the associated genealogical tree is viewed both in the forward and the backward direction of time. The stationary type distribution of the reversed process, the so-called ancestral distribution, turns out as a key for the study of mutation-selection balance. This balance can be expressed in the form of a variational principle that quantifies the respective roles of reproduction and mutation for any possible type distribution. It shows that the mean growth rate of the population results from a competition for a maximal long-term growth rate, as given by the difference between the current mean reproduction rate, and an asymptotic decay rate related to the mutation process; this tradeoff is won by the ancestral distribution. We then focus on the case when the type is determined by a sequence of letters (like nucleotides or matches/mismatches relative to a reference sequence), and we ask how much of the above competition can still be seen by observing only the letter composition (as given by the frequencies of the various letters within the sequence). If mutation and reproduction rates can be approximated in a smooth way, the fitness of letter compositions resulting from the interplay of reproduction and mutation is determined in the limit as the number of sequence sites tends to infinity. Our main application is the quasispecies model of sequence evolution with mutation coupled to reproduction but independent across sites, and a fitness function that is invariant under permutation of sites. In this model, the fitness of letter compositions is worked out explicitly. In certain cases, their competition leads to a phase transition.

Animals↗
PubMed1998

[Pathogenesis of disorder due to thyroid hormone receptor mutation].

Thyroid hormone action is mediated through its nuclear receptor (thyroid hormone receptor; TR). A mutation in the TR causes resistance to thyroid hormone (RTH) with autosomal dominant inheritance. Using T3 binding assay, gel shift assay and transfection study, the pathogenesis of this disorder was elucidated at a molecular level. Mutant TRs lose their T3 binding activity, and inhibit wild type TR action in a dominant negative manner. For this dominant negative inhibition, the preservation of DNA binding and dimerization activities are required. It is indicated that the mutant TR complex competes with wild type TR complex for the DNA binding site to exert dominant negative inhibition. Thus, the functional analyses of mutant TRs are useful to elucidate the molecular basis of this disorder.

Humans↗