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At least 793 records · Page 44Linked to original sources
PubMed1998

The genetics of caloric restriction in Caenorhabditis elegans.

Low caloric intake (caloric restriction) can lengthen the life span of a wide range of animals and possibly even of humans. To understand better how caloric restriction lengthens life span, we used genetic methods and criteria to investigate its mechanism of action in the nematode Caenorhabditis elegans. Mutations in many genes (eat genes) result in partial starvation of the worm by disrupting the function of the pharynx, the feeding organ. We found that most eat mutations significantly lengthen life span (by up to 50%). In C. elegans, mutations in a number of other genes that can extend life span have been found. Two genetically distinct mechanisms of life span extension are known: a mechanism involving genes that regulate dauer formation (age-1, daf-2, daf-16, and daf-28) and a mechanism involving genes that affect the rate of development and behavior (clk-1, clk-2, clk-3, and gro-1). We find that the long life of eat-2 mutants does not require the activity of DAF-16 and that eat-2; daf-2 double mutants live even longer than extremely long-lived daf-2 mutants. These findings demonstrate that food restriction lengthens life span by a mechanism distinct from that of dauer-formation mutants. In contrast, we find that food restriction does not further increase the life span of long-lived clk-1 mutants, suggesting that clk-1 and caloric restriction affect similar processes.

Aging↗
PubMed2000

The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01.

A checkpoint operating in the G(2) phase of the cell cycle prevents entry into mitosis in the presence of DNA damage. UCN-01, a protein kinase inhibitor currently undergoing clinical trials for cancer treatment, abrogates G(2) checkpoint function and sensitizes p53-defective cancer cells to DNA-damaging agents. In most species, the G(2) checkpoint prevents the Cdc25 phosphatase from removing inhibitory phosphate groups from the mitosis-promoting kinase Cdc2. This is accomplished by maintaining Cdc25 in a phosphorylated form that binds 14-3-3 proteins. The checkpoint kinases, Chk1 and Cds1, are proposed to regulate the interactions between human Cdc25C and 14-3-3 proteins by phosphorylating Cdc25C on serine 216. 14-3-3 proteins, in turn, function to keep Cdc25C out of the nucleus. Here we report that UCN-01 caused loss of both serine 216 phosphorylation and 14-3-3 binding to Cdc25C in DNA-damaged cells. In addition, UCN-01 potently inhibited the ability of Chk1 to phosphorylate Cdc25C in vitro. In contrast, Cds1 was refractory to inhibition by UCN-01 in vitro, and Cds1 was still phosphorylated in irradiated cells treated with UCN-01. Thus, neither Cds1 nor kinases upstream of Cds1, such as ataxia telangiectasia-mutated, are targets of UCN-01 action in vivo. Taken together our results identify the Chk1 kinase and the Cdc25C pathway as potential targets of G(2) checkpoint abrogation by UCN-01.

Alkaloids↗
PubMed1995

The ncl-1 gene and genetic mosaics of Caenorhabditis elegans.

A ncl-1 mutation results in enlarged nucleoli, which can be detected in nearly all cells of living animals by Nomarski microscopy. Spontaneous mitotic loss of a ncl-1(+)-containing free duplication in an otherwise homozygous ncl-1 mutant animal results in mosaicism for ncl-1 expression, and the patterns of mosaicism lead us to conclude that ncl-1 acts cell autonomously. The probability of mitotic loss of the duplication sDp3 is approximately constant over many cell divisions. About 60% of the losses of sDp3 at the first embryonic cell division involve nondisjunction. Frequencies of mitotic loss of different ncl-1(+)-bearing free duplications varied over a 200-fold range. The frequencies of mitotic loss were enhanced by a chromosomal him-10 mutation. We have used ncl-1 as a cell autonomous marker in the mosaic analysis of dpy-1 and lin-37. The focus of action of dpy-1 is in hypodermis. A mutation in lin-37 combined with a mutation in another gene results in a synthetic multivulva phenotype. We show that lin-37 acts cell nonautonomously and propose that it plays a role, along with the previously studied gene lin-15, in the generation of an intercellular signal by hyp7 that represses vulval development.

Alleles↗
PubMed2004

Male breast cancer--an andrological disease: risk factors and diagnosis.

Gynaecomastia, the enlargement of the male breast, is considered as an andrological disease. To date, a review on male breast cancer (MBC) has not been published in an andrological journal. The papers underlying this review were published from authors of different institutions: Clinical Genetics, Dermatology, Gynaecology, Internal Medicine, Oncology, Pathology, Psychiatry, Radiology and Surgery. MBC accounts for approximately 1% of breast cancer patients. A total of 182 men died of breast cancer in 1999, in Germany. In the US, 1500 new cases per year occur. MBC accounts for <5% of surgically removed breast lumps. Diseases with increased oestrogen action increase the risk of MBC. Mutations of distinct genes are estimated to account for up to roughly 10% of MBC. BRCA1 and BRCA2 gene mutations are responsible for approximately 80% of the families with hereditary breast cancer. The diagnosis of MBC is not possible without histological examination. Different diagnostic procedures such as clinical diagnosis, sonography, mammography, fine-needle biopsy and core needle facilitate the decision whether a biopsy is necessary.

Breast Neoplasms, Male↗
PubMed2004

Cross-linking of glycine receptor transmembrane segments two and three alters coupling of ligand binding with channel opening.

Contact points between transmembrane segments (TMs) two and three of the glycine receptor are undefined and may play an important role in channel gating. We tested whether two amino acids in TM2 (S267) and TM3 (A288), known to be critical for alcohol and volatile anesthetic action, could cross-link by mutating both to cysteines and expressing the receptors in Xenopus laevis oocytes. In contrast with the wild-type receptor and single cysteine mutants, the S267C/A288C double mutant displayed unusual responses, including a tonic leak activity that was closed by strychnine and a run-down of the response upon repeated applications of glycine. We hypothesized that these characteristics were due to cross-linking of the two cysteines on opposing faces of these adjacent, alpha helical TMs. This would alter the movement of these two regions required for normal gating. To test this hypothesis, we used dithiothreitol to reduce the putative S267C-A288C disulfide bond. Reduction abolished the leak current and provided normal responses to glycine. Subsequent application of the cross-linking agent mercuric chloride caused the initial characteristics to return. These data demonstrate that S267 and A288 are near-neighbors and provide insight towards the location and role of the TM2-TM3 interface in ligand-gated ion channels.

Alcohols↗
PubMed1998

Effects of ethanol and anesthetics on type 1 and 5 metabotropic glutamate receptors expressed in Xenopus laevis oocytes.

Previous studies have demonstrated that ethanol and volatile anesthetics inhibit the function of some metabotropic (G protein-coupled) receptors, including the 5-hydroxytryptamine2 and muscarinic cholinergic receptors. The metabotropic glutamate receptors (mGluRs) show little sequence homology with most other metabotropic receptors and are important modulators of synaptic transmission in the mammalian central nervous system. It was of interest to determine drug actions on these receptors, and we investigated the effects of ethanol, halothane, the anesthetic compound F3 (1-chloro-1,2,2-trifluorocyclobutane), and the nonanesthetics F6 (1,2-dichlorohexafluorocyclobutane) and F8 (2,3-chlorooctafluorobutane) on the function of mGluR1 and mGluR5 expressed in Xenopus laevis oocytes. Halothane, F3, and ethanol inhibited mGluR5-induced Ca(2+)-dependent Cl- currents, yet pharmacologically relevant concentrations of these compounds had little effect on the glutamate-induced currents in the oocytes expressing mGluR1. F6 had inhibitory effects on both receptors, and F8 did not affect either mGluR1 or mGluR5 function. The protein kinase C (PKC) inhibitor GF109203X enhanced the glutamate-induced current, and the PKC activator phorbol-12-myristate-13-acetate inhibited this current in the oocytes expressing mGluR5, but these compounds had little effect on mGluR1 function. GF109203X abolished the inhibitory effects of halothane, F3, and ethanol on mGluR5s. Conversely, the phosphatase inhibitor calyculin A prolonged the action of halothane and ethanol. Furthermore, mutation of a PKC consensus site (Ser890) of mGluR5 abolished the inhibitory effects of halothane, F3, and ethanol. These results suggest that ethanol and volatile anesthetics inhibit mGluR5 because they promote PKC-mediated phosphorylation.

Anesthetics, Inhalation↗
PubMed2001

Evidence for direct protein kinase-C mediated modulation of N-methyl-D-aspartate receptor current.

Protein kinase-C (PKC) activation differentially affects currents from N-methyl-D-aspartate (NMDA) type glutamate receptors depending upon their subunit composition. Experiments using chimeras initially indicated that the cytoplasmic C-terminal tails of NR2B (responsive to PKC) and NR2C (unresponsive to PKC) subunits contain the amino acid residues responsible for the observed disparity of PKC effects. However, truncation and point mutation experiments have suggested that PKC action on NMDA receptors may be entirely indirect, working via the phosphorylation of associated proteins. Here we suggest that PKC does, in fact, affect NR2B/NR1-011 NMDA currents by direct phosphorylation of the NR2B tail at residues S1303 and S1323. Replacement of either of these residues with Ala severely reduces PKC potentiation. To verify that S1303 and S1323 are sites of direct phosphorylation by PKC, synthetic peptides from the regions surrounding these sites were used as substrates for in vitro assays with purified rat brain PKC. These results indicate that PKC can directly phosphorylate S1303 and S1323 in the NR2B C terminus, leading to enhanced currents through NMDA receptor channels. The direct action of PKC on certain NMDA receptor subtypes may be important in any physiological or pathological process where PKC and NR2B/NR1 receptors interact.

Animals↗
PubMed2005

Comparative antimicrobial characterization of LBM415 (NVP PDF-713), a new peptide deformylase inhibitor of clinical importance.

LBM415 (NVP PDF-713) is the first member of the peptide deformylase (PDF) inhibitor class being developed for clinical trials as a parenteral and oral agent for treatment of community-acquired respiratory tract disease and serious infections caused by antimicrobial-resistant gram-positive cocci. In this study susceptibility testing results from 1,306 recent clinical isolates selected to over-represent resistance trends among the species were summarized. All staphylococci (153 strains; MIC at which 90% of isolates were inhibited [MIC90], 2 microg/ml), Streptococcus pneumoniae (170 strains; MIC90, 1 microg/ml), other streptococci (150 strains; MIC90, 1 microg/ml), enterococci (104 strains; MIC90, 4 microg/ml), Moraxella catarrhalis (103 strains; MIC90, 0.5 microg/ml), and Legionella pneumophila (50 strains; MIC90, 0.12 microg/ml) were inhibited at < or = 8 microg of LBM415/ml, as were 97% of Haemophilus influenzae isolates (300 strains; MIC90, 4 to 8 microg/ml). Among other bacterial groups, 100% of gram-positive and -negative anaerobes, including 22 Bacteroides spp. strains (31 strains total; MIC90, 1 microg/ml), were inhibited by < or = 4 microg/ml, whereas Enterobacteriaceae (112 strains) and most nonfermentative bacilli (107 strains) were not inhibited at readily achievable concentrations. The compound was found to have a dominantly bacteriostatic action, and spontaneous single-step mutational rates occurred at low levels (10(-6) to <10(-8)). Drug interaction studies failed to identify any class-specific synergistic interactions, nor were antagonistic interactions observed. Variations in broth and agar MIC test conditions demonstrated that, whereas the agar-based method trended towards a 1-log2 dilution-higher MIC than the broth method and was inoculum dependent, other variations in incubation environment, medium supplements, pH, or calcium concentration had little influence on LBM415 MIC results. Use of the efflux inhibitor phe-arg-beta-naphthylamide showed an average of 1 log2 dilution decrease in H. influenzae MICs, demonstrating the contribution of efflux pumps in influencing susceptibility to PDF inhibitors. The in vitro activity of LBM415 against targeted bacterial species, including resistant subsets, and other laboratory characteristics of this novel compound demonstrate the potential of PDF inhibitors as a new class of antimicrobial agents.

Amidohydrolases↗
PubMed1974

Bacteriophage f1 infection and colicin tolerance.

Bacteriophage f1-infected cells are relatively insensitive to a variety of colicins including E1, E2, and K; an f1 gene product is responsible for this insensitivity. A colicin-tolerant mutant cannot grow f1 phage; f1 DNA does not penetrate this mutant properly. One interpretation of these results is that an f1 gene product blocks the penetration of colicins which is necessary for their action, whereas the colicin-tolerant mutation blocks the penetration of colicins as well as phage f1 DNA.

Binding Sites↗
PubMed2000

Asymmetry of configuration space induced by unequal crossover: implications for a mathematical theory of evolutionary innovation.

Evolution can be regarded as the exploration of genetic or morphological state space by populations. In traditional models of population and quantitative genetics, the state space can be formally represented as a configuration space with clearly defined concepts of neighborhood and distance, defined by the action of variational operators such as mutation and/or recombination. In this paper, we describe a process where no genetic configuration space closure (and hence, no non-arbitrary notion of distance and neighborhood) exists. The process is gene duplication by means of unequal crossover, which we regard as an example of an "innovation" process that changes the state space of the system rather than exploring a closed state space. We assert that such processes are qualitatively distinct from representations of the adaptation process, which occur on regular configuration spaces.

Biological Evolution↗
PubMed1997

In vivo and in vitro characterization of neonatal hyperparathyroidism resulting from a de novo, heterozygous mutation in the Ca2+-sensing receptor gene: normal maternal calcium homeostasis as a cause of secondary hyperparathyroidism in familial benign hypocalciuric hypercalcemia.

We characterized the in vivo, cellular and molecular pathophysiology of a case of neonatal hyperparathyroidism (NHPT) resulting from a de novo, heterozygous missense mutation in the gene for the extracellular Ca2+ (Ca2+(o))-sensing receptor (CaR). The female neonate presented with moderately severe hypercalcemia, markedly undermineralized bones, and multiple metaphyseal fractures. Subtotal parathyroidectomy was performed at 6 wk; hypercalcemia recurred rapidly but the bone disease improved gradually with reversion to an asymptomatic state resembling familial benign hypocalciuric hypercalcemia (FBHH). Dispersed parathyroid cells from the resected tissue showed a set-point (the level of Ca2+(o) half maximally inhibiting PTH secretion) substantially higher than for normal human parathyroid cells (approximately 1.8 vs. approximately 1.0 mM, respectively); a similar increase in set-point was observed in vivo. The proband's CaR gene showed a missense mutation (R185Q) at codon 185, while her normocalcemic parents were homozygous for wild type (WT) CaR sequence. Transient expression of the mutant R185Q CaR in human embryonic kidney (HEK293) cells revealed a substantially attenuated Ca2+(o)-evoked accumulation of total inositol phosphates (IP), while cotransfection of normal and mutant receptors showed an EC50 (the level of Ca2+(o) eliciting a half-maximal increase in IPs) 37% higher than for WT CaR alone (6.3+/-0.4 vs. 4.6+/-0.3 mM Ca2+(o), respectively). Thus this de novo, heterozygous CaR mutation may exert a dominant negative action on the normal CaR, producing NHPT and more severe hypercalcemia than typically seen with FBHH. Moreover, normal maternal calcium homeostasis promoted additional secondary hyperparathyroidism in the fetus, contributing to the severity of the NHPT in this case with FBHH.

Calcium↗
PubMed1995

The lurcher gene induces apoptotic death in cerebellar Purkinje cells.

In the neurologically mutant mouse strain lurcher (Lc), heterozygous animals display cell autonomous degeneration of cerebellar Purkinje cells beginning in the second postnatal week. During the course of our studies to identify the genetic lesion responsible for this disease (Norman et al., 1991), we have formulated an hypothesis suggesting that in Lc Purkinje cells homeostasis is sufficiently perturbed to lead to the activation of programmed cell death, thus resulting in neuronal loss and the consequent neurologic disease (Heintz, 1993). To address this possibility, we have examined the properties of Lc Purkinje cells as they die during the second postnatal week. Our light and electron microscopic studies demonstrate that dying Lc Purkinje cells exhibit the characteristic morphologic features of apoptosis, including nuclear condensation, axon beading and membrane blebbing. Using an in situ end-labeling method, we have also detected nicked nuclear DNA in these cells. Furthermore, we have examined the expression of the sulfated glycoprotein 2 (SGP2), whose mRNA is induced in both T-cells and prostate epithelial cells undergoing apoptotic death. We show by in situ hybridization that SGP2 is not expressed at detectable levels in normal Purkinje cells, but that its mRNA is present in Lc Purkinje cells prior to their death. Also expression of the Kv3.3b potassium channel, which marks the terminal phase of Purkinje cell differentiation, is evident in Lc Purkinje cells prior to their death. These data demonstrate that the Lc mutation induces apoptosis in cerebellar Purkinje cells following their maturation in postnatal cerebellum. Isolation of the Lc mutation and further analysis of its action in eliciting apoptosis can provide an important opportunity for understanding the etiology of neurodegenerative disease.

Animals↗
PubMed2006

Resistance to growth hormone releasing hormone and gonadotropins in Albright's hereditary osteodystrophy.

Heterozygous inactivating mutations in the Gs alpha gene cause Albright's hereditary osteo-dystrophy (AHO). Consistent with the observation that only maternally inherited mutations lead to resistance to hormone action (pseudohypoparathyroidism type Ia [PHP-Ia), recent studies have provided evidence for a predominant maternal origin of Gs alpha transcripts in endocrine organs, such as thyroid, gonad and pituitary. Accordingly, patients with PHP-Ia display variable degrees of resistance to parathyroid hormone (PTH), thyroid stimulating hormone (TSH), gonadotropins and growth hormone (GH) releasing hormone (GHRH). Although the incidence and the clinical and biochemical characteristics of PTH and TSH resistance have been widely investigated and described, the cause and significance of the reproductive dysfunction in AHO is still poorly understood. The clinical finding of alterations of GH secretion in these patients was described for the first time only 2 years ago. The present report briefly reviews the literature focusing on the actual knowledge about these last two subjects.

Female↗
PubMed2001

Congenital long QT syndromes and Brugada syndrome: the arrhythmogenic ion channel disorders.

Congenital long QT syndromes (LQTS) and Brugada syndrome are hereditary disorders of cardiac ion channels which result in life-threatening cardiac arrhythmias or sudden cardiac death in patients with anatomically normal hearts. The pathogenesis of these dramatic events has been partially elucidated with the identification of the individual ion channels involved and understanding of the effect of some disease-causing mutations on the membrane currents and action potential. The clinical spectrum of congenital LQTS is broader than previously thought and involves certain patients previously diagnosed with idiosyncratic drug-induced proarrhythmia. The initial treatment for congenital LQTS patients involves beta-blockers in most cases. Indications for implantable cardioverter-defibrillator (ICD) or pace-maker (PM) implantation in selected individuals continue to evolve.

Action Potentials↗
PubMed1997

Leptin sensitivity in nonobese glucagon-like peptide I receptor -/- mice.

Glucagon-like peptide I (GLP-I) stimulates glucose-dependent insulin secretion and inhibits food intake in the central nervous system. Because leptin reduces food intake but inhibits insulin secretion, we examined leptin action in mice with a null mutation in the GLP-I receptor. Intracerebroventricular leptin administration inhibited food intake in both wild-type and GLP-I receptor (GLP-IR) -/- mice, and daily intraperitoneal administration of leptin for 2 weeks produced comparable reductions in food intake and body weight in control and GLP-IR -/- mice. Glucose tolerance was improved in both wild-type and GLP-IR -/- mice, whether pair fed or leptin treated; however, blood sugars were significantly lower in the leptin-treated GLP-IR -/- mice following oral glucose challenge (P < 0.01). Glucose-stimulated insulin was reduced in both pair-fed and leptin-treated mice (P < 0.01-0.001); however, insulin levels were significantly lower in leptin-treated versus pair-fed GLP-IR -/- mice (P < 0.01). A single leptin injection had no effect on glucose tolerance in GLP-IR -/- mice, but decreased hepatic PEPCK mRNA in both wild-type and GLP-IR -/- mice. The improvement in blood glucose excursion, despite lower levels of glucose-stimulated insulin in lean leptin-treated GLP-IR -/- mice, suggests that leptin may have beneficial effects on control of blood glucose in the absence of obesity. Furthermore, the greater effects of leptin on glucose and insulin in leptin-treated versus pair-fed GLP-IR -/- mice raises the possibility that disruption of GLP-I signaling modifies the sensitivity to leptin in vivo.

Animals↗
PubMed1993

[Genetic analysis of hormone resistance].

Thyroid hormone resistance is a syndrome defined as impaired tissue response to thyroid hormone which would be excessive under normal circumstances. Although it has long been speculated that this defect was caused by an abnormality at the site of the nuclear receptor for thyroid hormone, isolation of complementary DNA encoding thyroid hormone receptors has only recently enabled us to directly examine this hypothesis. Thyroid hormone receptors are coded on two separate genes, THRA and THRB. Both genes give rise to multiple receptor subtypes through alternative splicing or different usage of promoters. Tight linkage between THRB locus and resistant phenotype was demonstrated in one affected family, and subsequently, more than 30 genetic abnormalities of the THRB gene were isolated. No abnormalities have been demonstrated on the THRA gene. Denaturing gradient gel electrophoresis was used to screen abnormalities on the THRB gene of affected families, and the results also strongly supported the hypothesis that thyroid hormone resistance is caused by an abnormal THRB gene. Molecular analysis of thyroid hormone resistance will facilitate understanding of the pathophysiology of this syndrome and the molecular mechanism of thyroid hormone action.

Humans↗
PubMed1998

A point mutation in Galphao and Galphai1 blocks interaction with regulator of G protein signaling proteins.

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize Gi-family alpha subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Galpha subunit Gpa1(sst) that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780-5784), site-directed mutagenesis of alphao and alphai1 was done. G184S alphao and G183S alphai1 show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G --> S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nM, respectively, for aluminum fluoride-activated wild type alphao and alphai1 to compete with fluorescein isothiocyanate-alphao binding to glutathione S-transferase-RGS4. The G --> S mutant proteins showed a greater than 30-100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in alphao and alphai1 that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Gi function.

Enzyme Activation↗
PubMed2004

Molecular genetic probing of dopamine receptors in drug addiction.

Drug addiction is a brain disease with complex genetic, psychological and social factors. The dopaminergic system of the brain plays a central role in natural reward and motivation and is the main neural substrate for the actions of abusive drugs. The analysis of mice with mutations in their dopamine receptor genes has provided new information regarding the influence of individual dopamine receptors on drug actions. The use of genetic manipulation of dopamine receptors, and related intracellular signaling molecules, in mice will help to enhance our understanding of the molecular and cellular mechanisms underlying compulsive drug-seeking and drug-taking behaviors, reasons for relapse into drug addiction and persistent neuronal changes in response to repeated drug use. These studies will provide new insights for improved therapeutic strategies for the prevention and treatment of drug addiction.

Animals↗