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PubMed1984

[Mutagenic action of tritium on the germ cells of male mice. I. The induction of dominant lethal mutations by tritium oxide and an assessment of its relative biological effectiveness].

The frequency of dominant lethal mutations (DLM) in the germ cells of male mice exposed to either THO- or gamma-irradiation of 137Cs at the dose of 0.5-3.7 Gr was studied. The distribution of the number of dead embryos in females from the control group as well as that at a dose of irradiation less than 1 Gr, corresponds to the Poisson rule. Spermatids appeared to be more radiosensitive than spermatocytes and spermatozoa. The rate of the effect caused by irradiation of both spermatocytes and spermatids did not depend on the dose rate of gamma-irradiation ranging from 1.10(-3) to 1.7 Gr per min. The linear quadratic formula of aD+bD2 describes the relation of the DLM frequency to the doses of either beta- or gamma-irradiation. The THO RBE increases with the decrease of the dose reaching 2.5 at the D less than 0.1 Gr.

Animals↗
PubMed1994

Inhibition of human immunodeficiency virus type 1 Tat-dependent activation of translation in Xenopus oocytes by the benzodiazepine Ro24-7429 requires trans-activation response element loop sequences.

Two benzodiazepine compounds, [7-chloro-5-(2-pyrryl)-3H-1,4 benzodiazapin-2-(H)-one] (Ro5-3335) and [7-chloro-5-(1H-pyrrol-2-yl)-3H-benzo[e] [1,4] diazepin-2-yl]- methylamine (Ro24-7429), inhibit human immunodeficiency virus type 1 (HIV-1) replication via a specific effect on the function of the transactivator protein, Tat. To gain further insight into the mechanism of action of these compounds, we have tested their effects in an alternative assay for Tat activation in Xenopus oocytes. In this system, translation of trans-activation response element (TAR)-containing RNA is activated by Tat. Both compounds specifically blocked activation of translation in a dose-dependent fashion, with Ro24-7429 showing the greater potency. In the Xenopus oocyte system, as in mammalian cells, mutation of the TAR loop sequences abolishes Tat action. However, it is possible to obtain TAR-specific, Tat-dependent activation of a target RNA with a mutation in the loop provided that this target is in large excess. This result has been interpreted as indicating that a negative factor has been titrated (M. Braddock, R. Powell, A.D. Blanchard, A.J. Kingsman, and S.M. Kingsman, FASEB J. 7:214-222, 1993). Interestingly Ro24-7429 was unable to inhibit the TAR-specific but loop sequence-independent mode of translational activation. This finding suggests that a specific loop-binding cellular factor may mediate the effects of this inhibitor of Tat action. Consistent with this notion, we could not detect any effect of Ro24-7429 on the efficiency of specific Tat binding to TAR in vitro.

Animals↗
PubMed1985

[Analysis of the visible mutations induced by x-rays and ethyl methanesulfonate in the mature spermatozoa of Drosophila melanogaster].

The recessive visible mutations spectrum of chromosome II induced by X-rays and ethylmethanesulfonata (EMS) in mature Drosophila melanogaster spermatozoa has been studied. Treatment of both mutagens resulted in mutations in all 5 genes in stock mei-9LI and only 4--in D-32. The comparison of mutation frequencies of the same genes in two stocks under EMS-treatment demonstrated the statistical difference of mutation frequencies j, pr, cn of two stocks, genes b and vg did not differ. Under the influence of X-rays the differencies have been observed only for gene b. In stock D-32 the mutation frequency differes from the control for b and vg (EMS treatment) and j, pr, vg (under the action of X-rays), in mei-9LI--the mutation frequency of all 5 genes (under the X-rays) and 4 of 5 genes (EMS treatment).

Animals↗
PubMed2004

Inhibition of type A GABA receptors by L-type calcium channel blockers.

Modulation of type A GABA receptors (GABAA) by L-type Ca++ channel blockers was investigated. The dihydropyridines nifedipine and nitrendipine, and the phenylalkylamine verapamil inhibited recombinant rat alpha1beta2gamma2 receptors recorded from human embryonic kidney (HEK) 293 cells; nifedipine at low concentrations also elicited modest stimulatory effects on GABA-gated current. The IC50 for GABA current inhibition was lowest for nitrendipine (17.3 +/- 1.3 microM), so subsequent studies were focused on further exploring its mechanism and possible site of action. When co-applied with GABA, nitrendipine had minimal effects on initial current amplitude, but significantly enhanced current decay rate. Nitrendipine-mediated inhibition was subunit-selective, as its IC50 was 10-fold lower in alpha1beta2 receptors. Nitrendipine's effect in recombinant human alpha1beta2gamma2 receptors was similar (IC50=23.0 +/- 1.3 microM) to that observed in rat receptors of the same configuration, indicating the site of action is conserved in the two species. The inhibitory effects were dependent on channel gating, were independent of transmembrane voltage, and were also observed in GABAA receptors recorded from hypothalamic brain slices. The pharmacologic mechanism of inhibition by nitrendipine was non-competitive, indicating it does not act at the GABA binding site. Nitrendipine block was retained in the presence of the benzodiazepine antagonist flumazenil, indicating it does not interact at the benzodiazepine site. The actions of nitrendipine were not affected by a mutation (beta2T246F) that confers resistance to the channel blocker picrotoxin, and they were not altered in the presence of the picrotoxin site antagonist alpha-isopropyl-alpha-methyl-gamma-butyrolactone, demonstrating nitrendipine does not act at the picrotoxin site of the GABAA receptor. Possible interaction of nitrendipine with the Zn++ site was also eliminated, as mutation of beta2 H267 to A, which confers resistance to Zn++, had no effect on nitrendipine-mediated inhibition. Our data suggest some of the central effects of dihydropyridines may be due to actions at GABAA receptors. Moreover, the effects may be mediated through interaction with a novel modulatory site on the GABAA receptor.

4-Butyrolactone↗
PubMed1990

A directly repeated sequence in the beta-globin promoter regulates transcription in murine erythroleukemia cells.

We have identified a previously undetected cis-acting element in the mouse beta-major globin promoter region that is necessary for maximal transcription levels of the gene in the inducible preerythroid murine erythroleukemia (MEL) cell line. This element, termed the beta-globin direct-repeat element (beta DRE), consists of a directly repeated 10-base-pair sequence, 5'-AGGGCAG(G)AGC-3', that lies just upstream from the TATA box of the promoter. The beta DRE motif is highly conserved in all adult mammalian beta-globin promoter sequences known. Mutation of either single repeat alone caused less than a twofold decrease in transcript levels. However, simultaneous mutation of both repeated regions resulted in a ninefold decrease in accumulated transcripts when the gene was transiently transfected into MEL cells. Attachment of the beta DRE to a heterologous promoter had little effect on levels of accumulated transcripts initiated from the promoter in undifferentiated MEL cells but resulted in a threefold increase in transcript levels in induced (differentiated) MEL cells. Similarly, a comparison of the relative effects of mutations in the beta DRE in uninduced and induced MEL cells indicated that the element was more active in induced cells. The increase in beta DRE activity upon MEL cell differentiation and the more pronounced effects of mutations in both repeats of the beta DRE have implications for the mechanism of action of the element in regulating beta-globin transcription and for mutational studies of other repetitive or redundant transcription elements.

Animals↗
PubMed1986

Induction of deletion and insertion mutations by 9-aminoacridine. An in vitro model.

The ability of 9-aminoacridine to induce mutagenic lesions during DNA replication in vitro was investigated. The ampicillinase gene of pBR322 was replicated in vitro in the presence of 9-aminoacridine. Transfection of the replicated DNA into Escherichia coli gave Amps mutants. Determination of the base changes in 76 of these mutants indicated that the spectrum of mutations induced by 9-aminoacridine was consistent with its action in vivo. Both large (407-base) and small (1- and 2-base) deletions were induced at repetitive sequences. The frequency of deletion mutations depended on the identity of the base deleted and sequences surrounding the deletions. The characteristics of the frameshift mutations induced were consistent with the interactions of 9-aminoacridine with DNA. These results establish that 9-aminoacridine can induce frameshift mutations during the replication process and provide an in vitro model of frameshift induction for mechanistic studies.

Aminacrine↗
PubMed2006

C-terminal HERG (LQT2) mutations disrupt IKr channel regulation through 14-3-3epsilon.

Beta-adrenergic receptor-mediated cAMP or protein kinase A (PKA)-dependent modulation of cardiac potassium currents controls ventricular action potential duration (APD) at faster heart rates. HERG (KCNH2) gene mutations are associated with congenital long-QT syndrome (LQT2) and affect IKr activity, a key determinant in ventricular repolarization. Physical activity or emotional stress often triggers lethal arrhythmias in LQT2 patients. Beta-adrenergic stimulation of HERG channel activity is amplified and prolonged in vitro by the adaptor protein 14-3-3epsilon. In LQT2 families, we identified three novel heterozygous HERG mutations (G965X, R1014PfsX39, V1038AfsX21) in the C-terminus that led to protein truncation and loss of a PKA phosphorylation site required for binding of 14-3-3epsilon. When expressed in CHO cells, the mutants produced functional HERG channels with normal kinetic properties. We now provide evidence that HERG channel regulation by 14-3-3epsilon is of physiological significance in humans. Upon co-expression with 14-3-3epsilon, mutant channels still bound 14-3-3epsilon but did not respond with a hyperpolarizing shift in voltage dependence as seen in wild-type channels. Co-expression experiments of wild-type and mutant channels revealed dominant-negative behavior of all three HERG mutations. Simulations of the effects of sympathetic stimulation of HERG channel activity on the whole-cell action potential suggested a role in rate-dependent control of APD and an impaired ability of mutant cardiac myocytes to respond to a triggered event or an ectopic beat. In summary, the attenuated functional effects of 14-3-3epsilon on C-terminally truncated HERG channels demonstrate the physiological importance of coupling beta-adrenergic stimulation and HERG channel activity.

14-3-3 Proteins↗
PubMed2001

Synergistic growth inhibition of prostate cancer cells by 1 alpha,25 Dihydroxyvitamin D(3) and its 19-nor-hexafluoride analogs in combination with either sodium butyrate or trichostatin A.

Prostate cancer is a major cause of male cancer death. In vitro and in vivo data support a role for 1 alpha,25 Dihydroxyvitamin D(3) (1 alpha,25(OH)(2)D(3)) in regulating the growth and differentiation of the normal prostate gland yet prostate cancer cells appear significantly less sensitive to this action. Vitamin D(3) receptor (VDR) content or mutational status do not correlate clearly with the antiproliferative effects of 1 alpha,25(OH)(2)D(3) and therefore it is unclear why prostate cancer cell lines are significantly less sensitive to this action. We hypothesized that the antiproliferative responses of prostate cancer cells to 1 alpha,25(OH)(2)D(3) are suppressed by a process involving histone deacetylation. Sodium butyrate (NaB) and trichostatin A (TSA) are inhibitors of histone deacetylase (HDAC) activity. Low doses of NaB or TSA (300 microM and 15 nM respectively), which alone were relatively inactive, synergized with 1 alpha,25(OH)(2)D(3) in liquid and semi-solid agar to inhibit the growth of LNCaP, PC-3 and DU-145 prostate cancer cells. Still greater synergy was observed between vitamin D(3) hexafluoride analogs and either NaB or TSA. The mechanism appeared to involve neither the cyclin-dependent kinase inhibitor, p21((waf1/cip1)) nor cell cycle arrest, but rather induction of apoptosis. These data suggest that cells dysregulate the normal pro-apoptotic signals of 1 alpha,25(OH)(2)D(3) during prostate cancer development by a mechanism involving histone deacetylation. Combination therapy with potent vitamin D(3) analogs and clinically approved HDAC inhibitors may overcome this lesion and improve the treatment of both androgen-dependent and independent prostate cancer.

Antineoplastic Agents↗
PubMed2006

The variation of the sarcolipin gene (SLN) in atrial fibrillation, long QT syndrome and sudden arrhythmic death syndrome.

BACKGROUND: Mutations in genes responsible for the cardiac action potential and control of intracellular Ca(2+)-distribution are associated with cardiac arrhythmia and sudden death. Sarcolipin is a 31-amino acid protein that inhibits the sarcoplasmic reticulum Ca(2+) ATPase pump (SERCA2). The sarcolipin gene, SLN, is expressed in the heart and a candidate gene for cardiomyopathy as well as atrial fibrillation (AF), long QT syndrome (LQTS) or sudden arrhythmic death syndrome (SADS). We examined the genetic variation of SLN in patients with the arrhythmic disorders AF, LQTS and SADS. METHODS: We screened the coding region of SLN for mutations using single strand conformation polymorphism/heteroduplex analysis on PCR-amplified genomic DNA from 95 unrelated LQTS patients, 59 SADS cases and 147 patients with atrial fibrillation (AF) and 92 controls. Aberrant conformers were sequenced. RESULTS: No mutations or polymorphisms were found in the coding sequence. A G>C transition in the highly conserved position +1 of the 3'untranslated region (3'UTR) was found in two SADS cases. A polymorphism, a G>C transition at position -65 in the 5'untranslated region (5'UTR), was found with a G allele frequency of 0.48. A borderline significant difference in genotype distribution of the latter polymorphism was found between the AF group and controls. CONCLUSION: Mutations in the coding region of SLN are not frequently involved in LQTS, SADS or AF. Whether the described 3'- and 5'UTR variants have functional significance must await further studies.

Adolescent↗
PubMed1992

Mutations of Ha-ras p21 that define important regions for the molecular mechanism of the SDC25 C-domain, a guanine nucleotide dissociation stimulator.

The SDC25 C-domain is a very active guanine nucleotide dissociation stimulator (GDS) isolated from Saccharomyces cerevisiae which acts equally well on Ha-ras p21 and yeast RAS2. These properties make the SDC25 C-domain a suitable tool to study the basic mechanism of a GDS. The action of the SDC25 C-domain was analysed by mutation of structurally important regions of p21. Substitutions that influence the coordination of Mg2+.GDP or the interaction of the guanine ring were found to stimulate the intrinsic dissociation of GDP and suppress the action of the SDC25 C-domain. No relevant effects were observed with mutations in the phosphate binding loop L1 or by deleting the last 23 C-terminal residues of p21. Substitutions in the switch region 1 (loop L2) and 2 (loop L4) of p21 strongly impaired the action of this GDS; however, we show that this effect is not related to a decreased affinity of the SDC25 C-domain for the mutated p21. No functional competition could be found between this GDS and the catalytic domain of the human GTPase activating protein (GAP). This indicates that GDS and GAP bind to different sites of the p21.nucleotide complex, even though the same mutations in loops L2 and L4 regions affect the activity of both effectors. Since these two regions appear not to be involved directly in the interaction with GDS, we conclude that the negative effect induced by their mutation is related to their function as switches of selective conformations during the GDP to GTP exchange reaction catalysed by GDS.

Escherichia coli↗
PubMedDate not supplied

[Activated protein C resistance as a cause of thrombophilia].

The proportion of identifiable causes of familial thrombophilia has increased from 5-10% to 60-70% since the identification of activated protein C resistance (aPCR) in February 1993 by Dahlbäck et al. A mutation in the factor V gene (G-->A, 1691) leads to the so called Leiden mutation (R 506 Q) that produces a mutated factor V resistant to the catalytic action of activated protein C (aPC), yet normal in its procoagulant properties. This recently identified aPCR is in Nordic populations the most prevalent and well defined genetic defect associated with disease so far described. Its prevalence in the general population ranges from 0% to up to 15% and suggests that a positive genetic selection pressure has been involved. The aPCR phenotype can be assessed in vitro by measurement of the prolongation of the activated partial thromboplastin time in the presence of aPC, whereas the aPCR genotype is studied using polymerase chain reaction searching for the Arg to Gln mutation in the coagulation factor V gene. Some acquired conditions such as the presence of lupus anticoagulants, antiphospholipid antibodies, pregnancy, liver disease and contraceptives may lead into the aPCR phenotype. The aPCR search must be the initial step in the study of a patient with thrombophilia, either inherited or acquired aPCR together with protein C, protein S and antithrombin III explain 60 to 70% of cases of familial thrombophilia.

Antithrombin III↗
PubMed2006

Temporal and spatial delineation of mouse Otx2 functions by conditional self-knockout.

To identify the independent spatial and temporal activities of the essential developmental gene the Otx2, the germline mutation of which is lethal at embryonic day 8.5, we floxed one allele and substituted the other with an inducible CreER recombinase gene. This makes 'trans' self-knockout possible at any developmental stage. The transient action of tamoxifen pulses allows time-course mutation. We demonstrate efficient temporal knockout and demarcate spatio-temporal windows in which Otx2 controls the head, brain structures and body development.

Animals↗
PubMed1977

[Study of the mechanism of reduction of the virulence in the Shigella flexneri strains resistant to streptomycin].

Streptomycin-resistant mutations in donor strains of E. coli K12 Hfr AB 313 and AT-13 are not associated with the action of the strA-gene limiting the suppression. Shigella mutations str-r obtained by the method described in this paper also failed to limit the suppression. Changes in the virulence observed in transmission strA region from the str-r strains of E. coli and shigellae used in this work were not connected with mutations limiting the suppression (with competent mutations).

Alleles↗
PubMed2006

Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy.

Voltage-gated sodium channels (Na(V)) are critical for initiation of action potentials. Heterozygous loss-of-function mutations in Na(V)1.1 channels cause severe myoclonic epilepsy in infancy (SMEI). Homozygous null Scn1a-/- mice developed ataxia and died on postnatal day (P) 15 but could be sustained to P17.5 with manual feeding. Heterozygous Scn1a+/- mice had spontaneous seizures and sporadic deaths beginning after P21, with a notable dependence on genetic background. Loss of Na(V)1.1 did not change voltage-dependent activation or inactivation of sodium channels in hippocampal neurons. The sodium current density was, however, substantially reduced in inhibitory interneurons of Scn1a+/- and Scn1a-/- mice but not in their excitatory pyramidal neurons. An immunocytochemical survey also showed a specific upregulation of Na(V)1.3 channels in a subset of hippocampal interneurons. Our results indicate that reduced sodium currents in GABAergic inhibitory interneurons in Scn1a+/- heterozygotes may cause the hyperexcitability that leads to epilepsy in patients with SMEI.

Action Potentials↗
PubMed1987

A calcium-dependent potassium current is increased by a single-gene mutation in Paramecium.

The membrane currents of wild type Paramecium tetraurelia and the behavioral mutant teaA were analyzed under voltage clamp. The teaA mutant was shown to have a greatly increased outward current which was blocked completely by the combined use of internally delivered Cs+ and external TEA+. This, along with previous work (Satow, Y., Kung, C., 1976, J. Exp. Biol. 65:51-63) identified this as a K+ current. It was further found to be a calcium-activated K+ current since this increased outward K+ current cannot be elicited when the internal calcium is buffered with injected EGTA. The mutation pwB, which blocks the inward calcium current, also blocks this increased outward K+ current in teaA. This shows that this mutant current is activated by calcium through the normal depolarization-sensitive calcium channel. While tail current decay kinetic analysis showed that the apparent inactivation rates for this calcium-dependent K+ current are the same for mutant and wild type, the teaA current activates extremely rapidly. It is fully activated within 2 msec. This early activation of such a large outward current causes a characteristic reduction in the amplitude of the action potential of the teaA mutant. The teaA mutation had no effect on any of the other electrophysiological parameters examined. The phenotype of the teaA mutant is therefore a general decrease in responsiveness to depolarizing stimuli because of a rapidly activating calcium-dependent K+ current which prematurely repolarizes the action potential.

Animals↗
PubMed2004

Phase II study of alternating chemotherapy regimens for advanced non-small cell lung cancer.

PURPOSE: We assessed the use of alternating drugs with differing mechanisms of action as treatment for advanced non-small cell lung cancer. BACKGROUND: We hypothesized that the shape of a dose-response curve would be determined by the major mechanisms of resistance of a cancer to the drug being studied. Assessment of data from published clinical trials suggested that if our hypothesis were correct resistance of non-small cell lung cancer to most agents is due to "saturable passive" resistance mechanisms (non-competitive inhibition of drug effect due to deficiency of a factor required for drug effect) rather than to "active" resistance mechanisms (competitive inhibition of drug effect due to excess of a factor) or to "non-saturable passive resistance (due to factor alteration or mutation). Using drugs with differing mechanisms of action is a strategy that might be of value against passive resistance. METHOD: In patients with advanced non-small cell lung cancer, we used four alternating cisplatin-based regimens. In each regimen, cisplatin 80 mg/m(2) was given iv on day 1 of each course. The regimens were: cisplatin + vinorelbine 25 mg/m(2) days 1, 8 and 15, cisplatin + gemcitabine 1000 mg/m(2) days 1, 8 and 15, cisplatin + paclitaxel 200 mg/m(2) day 1 iv over 1h, and cisplatin + etoposide 100 mg/m(2) po days 1-6. Patients were assigned randomly to different regimen sequences. Patients first received 1 course of each of these 4 regimens, then received 1 further course of each of single agent vinorelbine, gemcitabine, paclitaxel and etoposide (at the same doses as in courses 1-4), without cisplatin. (Cisplatin was omitted from courses 5-8 to limit cumulative toxicity.) Change in tumor size was measured after each course. RESULTS: Thirty-six patients were entered. One patient achieved complete remission and nine achieved partial remissions, for an objective response rate of 28% (95% confidence intervals, 13-43%). Nineteen patients (53%) (95% confidence intervals, 37-69%) had stable disease. Eleven patients had growth of >10% on one regimen followed by tumor shrinkage of >10% on a later one. Median survival was 8.1 months, 1-year survival was 28% and 2-year survival was 6%. No unexpected toxicity was seen. CONCLUSIONS: The use of four alternating regimens is feasible in advanced non-small cell lung cancer. Response rates and survival times were comparable to those observed for standard chemotherapy approaches, suggesting that this strategy does not offer any major advantage. We plan to explore the hypothesis that this approach failed since tumor cells surviving first exposure to chemotherapy rapidly down-regulate their ability to undergo apoptosis.

Administration, Oral↗
PubMed1995

[Molecular endocrinology of thyroid diseases].

Over the last decade it has become possible to investigate the molecular basis of functional and neoplastic thyroid diseases, leading to the elucidation of various genetic defects at the level of the pituitary, thyroid and target organs. Mutations in either the pituitary-specific transcription factor Pit-1 or its target gene, TSH beta, lead to rare forms of hereditary congenital hypothyroidism. However, somatic mutations in thyroid epithelial cells causing an increase in hormone production and/or cellular proliferation are much more frequent. Nearly 50% of all toxic adenomas were shown to harbour activating mutations in either the TSH-receptor or certain G-proteins. In contrast, follicular and papillary thyroid malignancies are associated with mutations in the ras and ret genes respectively. Intriguingly, different mutations and rearrangements in the ret gene were shown to cause medullary thyroid cancer and MEN2 as well as to be specifically associated with papillary thyroid cancer. In contrast, mutations in thyroid-specific genes, such as thyroid peroxidase and thyroglobulin, causing congenital hypothyroidism are extremely rare. Besides the molecular abnormalities at the pituitary and thyroidal level leading to altered hormone secretion, genetic defects impairing thyroid hormone action at the target level also occur. Specifically, mutations in one of the thyroid hormone receptor genes (the proto-oncogene c-erbA beta) were shown to cause the autosomal dominant syndrome of resistance to thyroid hormone. The quest for a better understanding of the molecular defects in the pituitary-thyroid axis has led to the cloning of some of the key proteins, which can now be used for diagnostic purposes in vivo and in vitro. The use of recombinant thyroid peroxidase and TSH-receptor proteins has made possible the development of more sensitive and specific in vitro assays for autoantibodies. In addition, recombinant TSH was recently shown to be effective in stimulating radioiodine uptake in patients with residual differentiated thyroid cancer who remained on suppressive thyroid hormone therapy. Recombinant human TSH may therefore become a convenient diagnostic tool in the follow-up of patients with thyroid cancer by allowing for thyroglobulin measurements and radioiodine scanning without the need for the patient to become hypothyroid.

DNA-Binding Proteins↗