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At least 721 records · Page 40Linked to original sources
PubMed1997

Glucocorticoid and mineralocorticoid receptors: biology and clinical relevance.

Mineralocorticoid and glucocorticoid receptors act as homodimers via canonical pentadecamer hormone response elements to regulate transcription. Glucocorticoid, but as yet not mineralocorticoid, receptors have been shown also to modulate AP-1- and NF kappa B-induced transcription by direct protein-protein interactions. The role of 11 beta-hydroxysteroid dehydrogenase in conferring aldosterone specificity on epithelial mineralocorticoid receptors has been proven by the demonstration of sequence mutations in all cases of apparent mineralocorticoid excess examined to date. The autosomal form of aldosterone resistance (pseudohypoaldosteronism) has been shown to reflect loss-of-function mutations in epithelial sodium channel subunit sequence. (Patho)physiological roles for aldosterone and glucocorticoid membrane receptors, and for the recently described nuclear receptors for 11-ketosteroids in 11 beta-hydroxysteroid dehydro-genase-protected epithelia, remain to be established.

Aldosterone↗
PubMed2006

Penetrance of eye defects in mice heterozygous for mutation of Gli3 is enhanced by heterozygous mutation of Pax6.

BACKGROUND: Knowledge of the consequences of heterozygous mutations of developmentally important genes is important for understanding human genetic disorders. The Gli3 gene encodes a zinc finger transcription factor and homozygous loss-of-function mutations of Gli3 are lethal. Humans heterozygous for mutations in this gene suffer Greig cephalopolysyndactyly or Pallister-Hall syndromes, in which limb defects are prominent, and mice heterozygous for similar mutations have extra digits. Here we examined whether eye development, which is abnormal in mice lacking functional Gli3, is defective in Gli3+/- mice. RESULTS: We showed that Gli3 is expressed in the developing eye but that Gli3+/- mice have only very subtle eye defects. We then generated mice compound heterozygous for mutations in both Gli3 and Pax6, which encodes another developmentally important transcription factor known to be crucial for eye development. Pax6+/-; Gli3+/- eyes were compared to the eyes of wild-type, Pax6+/- or Gli3+/- siblings. They exhibited a range of abnormalities of the retina, iris, lens and cornea that was more extensive than in single Gli3+/- or Pax6+/- mutants or than would be predicted by addition of their phenotypes. CONCLUSION: These findings indicate that heterozygous mutations of Gli3 can impact on eye development. The importance of a normal Gli3 gene dosage becomes greater in the absence of a normal Pax6 gene dosage, suggesting that the two genes co-operate during eye morphogenesis.

Animals↗
PubMed1996

cdh-3, a gene encoding a member of the cadherin superfamily, functions in epithelial cell morphogenesis in Caenorhabditis elegans.

Several genes that encode members of the cadherin superfamily have been identified in Caenorhabditis elegans. Based on the roles of cadherins in vertebrates and Drosophila, it is expected that they function in the control of epithelial morphogenesis, an event which is poorly understood at the molecular level in C. elegans. Reporter genes under the control of upstream sequences from one of these genes, cdh-3, are expressed in developing epithelial cells, but also in a number of neuroectodermal cells that extend processes along some of these epithelial cells. We generated a loss-of-function mutation in cdh-3 by transposon-mediated deletion mutagenesis. This mutation affects the morphogenesis of a single cell, hyp10, which forms the tip of the nematode tail. The lack of detectable defects associated with the other cells expressing cdh-3 reporter constructs hints at the existence of other genes that can compensate for cdh-3 loss of function.

Alleles↗
PubMed2005

Regulation of tyrosinase trafficking and processing by presenilins: partial loss of function by familial Alzheimer's disease mutation.

Presenilins (PS) are required for gamma-secretase cleavage of multiple type I membrane proteins including the amyloid precursor protein and Notch and also have been implicated in regulating intracellular protein trafficking and turnover. Using genetic and pharmacological approaches, we reveal here a unique function of PS in the pigmentation of retinal pigment epithelium and epidermal melanocytes. PS deficiency leads to aberrant accumulation of tyrosinase (Tyr)-containing 50-nm post-Golgi vesicles that are normally destined to melanosomes. This trafficking is gamma-secretase-dependent, and abnormal localization of Tyr in the absence of PS is accompanied by the simultaneous accumulation of its C-terminal fragment. Furthermore, we show that the PS1M146V familial Alzheimer's disease mutation exhibits a partial loss-of-function in pigment synthesis. Our results identify Tyr and related proteins as physiological substrates of PS and link gamma-secretase activity with intracellular protein transport.

Alzheimer Disease↗
PubMed1997

Random mutagenesis of the cAMP chemoattractant receptor, cAR1, of Dictyostelium. Mutant classes that cause discrete shifts in agonist affinity and lock the receptor in a novel activational intermediate.

The cAMP chemoattractant receptor, cAR1, of Dictyostelium transduces extracellular cAMP signals via G protein-dependent and G protein-independent mechanisms. While site-directed mutagenesis studies of G protein-coupled receptors have provided a host of information regarding the domains essential for various functions, many mechanistic and structural questions remain to be resolved. We therefore carried out polymerase chain reaction-mediated random mutagenesis over a large part of the cAR1 sequence (from TMIII through the proximal part of the cytoplasmic tail). We devised a rapid screen for loss-of-function mutations based on the essential role of cAR1 in the developmental program of Dictyostelium. Although there were an average of two amino acid substitutions per receptor, approximately 90% of the mutants were able to substitute for wild-type cAR1 when expressed in receptor null cells. About 2% were loss-of-function mutants that expressed wild-type levels of receptor protein. We used biochemical screens to select about 100 of these mutants and chose eight representative mutants for extensive characterization. These fell into distinct classes. One class had a conditional defect in cAMP binding that was reversed by high salt. Another large class had decreased affinity under all conditions. Curiously, the decreases were clustered into three discrete intervals. One of the most interesting class of mutants lost all capacity for signal transduction but was phosphorylated in response to agonist binding. This latter finding suggests that there are at least two activated states of cAR1 that can be recognized by different downstream effectors.

Animals↗
PubMed1995

Transposon insertions causing constitutive Sex-lethal activity in Drosophila melanogaster affect Sxl sex-specific transcript splicing.

Sex-lethal (Sxl) gene products induce female development in Drosophila melanogaster and suppress the transcriptional hyperactivation of X-linked genes responsible for male X-chromosome dosage compensation. Control of Sxl functioning by the dose of X-chromosomes normally ensures that the female-specific functions of this developmental switch gene are only expressed in diplo-X individuals. Although the immediate effect of X-chromosome dose is on Sxl transcription, during most of the life cycle "on" vs. "off" reflects alternative Sxl RNA splicing, with the female (productive) splicing mode maintained by a positive feedback activity of SXL protein on Sxl pre-mRNA splicing. "Male-lethal" (SxlM) gain-of-function alleles subvert Sxl control by X-chromosome dose, allowing female Sxl functions to be expressed independent of the positive regulators upstream of Sxl. As a consequence, SxlM haplo-X animals (chromosomal males) die because of improper dosage compensation, and SxlM chromosomal females survive the otherwise lethal effects of mutations in upstream positive regulators. Five independent spontaneous SxlM alleles were shown previously to be transposon insertions into what was subsequently found to be the region of regulated sex-specific Sxl RNA splicing. We show that these five alleles represent three different mutant types: SxlM1, SxlM3, and SxlM4. SxlM1 is an insertion of a roo element 674 bp downstream of the translation-terminating male-specific exon. SxlM3 is an insertion of a hobo transposon (not 297 as previously reported) into the 3' splice site of the male exon, and SxlM4 is an insertion of a novel transposon into the male-specific exon itself. We show that these three gain-of-function mutants differ considerably in their ability to bypass the sex determination signal, with SxlM4 being the strongest and SxlM1 the weakest. This difference is also reflected in effects of these mutations on sex-specific RNA splicing and on the rate of appearance of SXL protein in male embryos. Transcript analysis of double-mutant male-viable SxlM derivatives in which the SxlM insertion is cis to loss-of-function mutations, combined with other results reported here, indicates that the constitutive character of these SxlM alleles is a consequence of an alteration of the structure of the pre-mRNA that allows some level of female splicing to occur even in the absence of functional SXL protein. Surprisingly, however, most of the constitutive character of SxlM alleles appears to depend on the mutant alleles' responsiveness, perhaps greater than wild-type, to the autoregulatory splicing activity of the wild-type SXL proteins they produce.

Amino Acid Sequence↗
PubMed2001

A SURF1 gene mutation presenting as isolated leukodystrophy.

Mitochondrial respiratory chain defects are increasingly recognized in patients with leukodystrophy. We report the first case of leukodystrophy with systemic cytochrome oxidase deficiency caused by a loss of function mutation in the SURF1 gene in a 2-year-old girl presenting with failure to thrive, global neurodevelopmental regression, and lactic acidosis. Although all previously reported mutations in the SURF1 gene have been found in patients with cytochrome oxidase (COX)-deficient Leigh syndrome, the phenotype associated with SURF1 protein deficiency should be extended to include leukodystrophy.

Acidosis, Lactic↗
PubMed2002

Anemia and iron overload due to compound heterozygosity for novel ceruloplasmin mutations.

Aceruloplasminemia is a recessive disorder characterized by anemia, iron overload, and neurodegeneration, caused by the absence of ceruloplasmin (Cp), a multicopper oxidase important for iron export. Few patients homozygous for loss of function mutations of the Cp gene have been reported. We describe a 62-year-old white woman with heavy liver iron overload, diabetes, anemia, and neurologic symptoms. She was compound heterozygote for 2 novel mutations that result in the absence of hepatocyte Cp: an adenine insertion at nucleotide 2917 causing a truncated protein and a C-G transversion causing a glutamine-->glutamic acid substitution at position 146. Although rare in whites, aceruloplasminemia should be considered in the differential diagnosis of unexplained anemia associated with iron overload, because these features anticipate progressive neurologic symptoms. We propose that anemia, secondary to the impaired macrophage iron release, plays a major role in hepatic iron overload through increased absorption mediated by the erythroid regulator.

Anemia↗
PubMed2006

Kallmann syndrome: mutations in the genes encoding prokineticin-2 and prokineticin receptor-2.

Kallmann syndrome combines anosmia, related to defective olfactory bulb morphogenesis, and hypogonadism due to gonadotropin-releasing hormone deficiency. Loss-of-function mutations in KAL1 and FGFR1 underlie the X chromosome-linked form and an autosomal dominant form of the disease, respectively. Mutations in these genes, however, only account for approximately 20% of all Kallmann syndrome cases. In a cohort of 192 patients we took a candidate gene strategy and identified ten and four different point mutations in the genes encoding the G protein-coupled prokineticin receptor-2 (PROKR2) and one of its ligands, prokineticin-2 (PROK2), respectively. The mutations in PROK2 were detected in the heterozygous state, whereas PROKR2 mutations were found in the heterozygous, homozygous, or compound heterozygous state. In addition, one of the patients heterozygous for a PROKR2 mutation was also carrying a missense mutation in KAL1, thus indicating a possible digenic inheritance of the disease in this individual. These findings reveal that insufficient prokineticin-signaling through PROKR2 leads to abnormal development of the olfactory system and reproductive axis in man. They also shed new light on the complex genetic transmission of Kallmann syndrome.

Chromosome Segregation↗
PubMed2005

Characterization of the reconstituted gamma-secretase complex from Sf9 cells co-expressing presenilin 1, nicastrin [correction of nacastrin], aph-1a, and pen-2.

Gamma-secretase catalyzes the proteolytic processing of a number of integral membrane proteins, including amyloid precursor protein (APP) and Notch. The native gamma-secretase is a heterogeneous population of large membrane protein complexes containing presenilin 1 (PS1) or presenilin 2 (PS2), aph-1a or aph-1b, nicastrin, and pen-2. Here we report the reconstitution of a gamma-secretase complex in Sf9 cells by co-infection with baculoviruses carrying the PS1, nicastrin, pen-2, and aph-1a genes. The reconstituted enzyme processes C99 and the Notch-like substrate N160 and displays the characteristic features of gamma-secretase in terms of sensitivity to a gamma-secretase inhibitor, upregulation of Abeta42 production by a familial Alzheimer's disease (FAD) mutation in the APP gene, and downregulation of Notch processing by PS1 FAD mutations. However, the ratio of Abeta42:Abeta40 production by the reconstituted gamma-secretase is significantly higher than that of the native enzyme from 293 cells. Unlike in mammalian cells where PS1 FAD mutations cause an increase in Abeta42 production, PS1 FAD missense mutations in the reconstitution system alter the cleavage sites in the C99 substrate without changing the Abeta42:Abeta40 ratio. In addition, PS1DeltaE9 is a loss-of-function mutation in both C99 and N160 processing. Reconstitution of gamma-secretase provides a homogeneous system for studying the individual gamma-secretase complexes and their roles in Abeta production, Notch processing and AD pathogenesis. These studies may provide important insight into the development of a new generation of selective gamma-secretase inhibitors with an improved side effect profile.

Alzheimer Disease↗
PubMed2002

Characterization of a spontaneous 9.5-kilobase-deletion mutant of murine gammaherpesvirus 68 reveals tissue-specific genetic requirements for latency.

Murine gammaherpesvirus 68 (gammaHV68 [also known as MHV-68]) establishes a latent infection in mice, providing a small-animal model with which to identify host and viral factors that regulate gammaherpesvirus latency. While gammaHV68 establishes a latent infection in multiple tissues, including splenocytes and peritoneal cells, the requirements for latent infection within these tissues are poorly defined. Here we report the characterization of a spontaneous 9.5-kb-deletion mutant of gammaHV68 that lacks the M1, M2, M3, and M4 genes and eight viral tRNA-like genes. Previously, this locus has been shown to contain the latency-associated M2, M3, and viral tRNA-like genes. Through characterization of this mutant, we found that the M1, M2, M3, M4 genes and the viral tRNA-like genes are dispensable for (i) in vitro replication and (ii) the establishment and maintenance of latency in vivo and reactivation from latency following intraperitoneal infection. In contrast, following intranasal infection with this mutant, there was a defect in splenic latency at both early and late times, a phenotype not observed in peritoneal cells. These results indicate (i) that there are different genetic requirements for the establishment of latency in different latent reservoirs and (ii) that the genetic requirements for latency depend on the route of infection. While some of these phenotypes have been observed with specific mutations in the M1 and M2 genes, other phenotypes have never been observed with the available gammaHV68 mutants. These studies highlight the importance of loss-of-function mutations in defining the genetic requirements for the establishment and maintenance of herpesvirus latency.

3T3 Cells↗
PubMed2001

Crystal structure of baculovirus P35 reveals a novel conformational change in the reactive site loop after caspase cleavage.

Baculovirus P35 is a universal suppressor of apoptosis that stoichiometrically inhibits cellular caspases in a novel cleavage-dependent mechanism. Upon caspase cleavage at Asp-87, the 10- and 25-kDa cleavage products of P35 remain tightly associated with the inhibited caspase. Mutations in the alpha-helix of the reactive site loop preceding the cleavage site abrogate caspase inhibition and antiapoptotic activity. Substitution of Pro for Val-71, which is located in the middle of this alpha-helix, produces a protein that is cleaved at the requisite Asp-87 but does not remain bound to the caspase. This loss-of-function mutation provided the opportunity to structurally analyze the conformational changes of the P35 reactive site loop after caspase cleavage. We report here the 2.7 A resolution crystal structure of V71P-mutated P35 after cleavage by human caspase-3. The structure reveals a large movement in the carboxyl-terminal side of the reactive site loop that swings down and forms a new beta-strand that augments an existing beta-sheet. Additionally, the hydrophobic amino terminus releases and extends away from the protein core. Similar movements occur when P35 forms an inhibitory complex with human caspase-8. These findings suggest that the alpha-helix mutation may alter the sequential steps or kinetics of the conformational changes required for inhibition, thereby causing P35 loss of function.

Amino Acid Sequence↗
PubMedDate not supplied

Mutations and promoter SNPs in RUNX2, a transcriptional regulator of bone formation.

Cleidocranial dysplasia (CCD) is a dominantly inherited skeletal malformation syndrome with high penetrance and variable expressivity. It is caused by loss of function mutations in the RUNX2 gene that encodes for a transcription factor essential for osteoblast differentiation and chondrocyte maturation. To identify new pathogenic mutations associated with CCD we screened 38 CCD patients for mutations in the RUNX2 coding sequence. We also report the mutation screening of the "bone-related" RUNX2 promoter in CCD patients without mutation in the RUNX2 coding region. We identify eight new and three previously described mutations in the RUNX2 gene. Additionally, a total of five sequence variants in the RUNX2 promoter were detected. Three of them occur within putative zinc finger transcription factor binding sites. DHPLC analysis of chromosomes from the control population and CCD patients showed that two promoter sequence variants were unique for CCD families. Electrophoretic mobility shift assay (EMSA) with protein extracts from ROS17/2.8 and C3H10T1/2 cell lines demonstrated that the promoter sequence variants altered DNA-protein binding specificity. Moreover, one of the variants significantly decreased the expression of a RUNX2 reporter gene in osteoblastic ROS17/2.8 cells, but not in multipotent, mesenchymal C3H10T1/2 cells. Interestingly, one of these sites bound the TRPS1 transcription factor and we demonstrated that TRPS1 is able to repress the RUNX2 promoter. The in vitro functional studies in conjunction with analysis of clinical phenotype of CCD patients suggest that these promoter sequence variants may affect transcriptional activity of the RUNX2 gene. Analysis of the promoter variants and RUNX2-interacting proteins may help to identify important cis-elements and trans-factors that regulate the RUNX2 transcriptional network and identify new susceptibility markers for more common bone disorders.

Animals↗
PubMedDate not supplied

Influence of fadAG203R and deltaflbA mutations on morphology and physiology of submerged Aspergillus nidulans cultures.

Morphologic and physiologic changes taking place in carbon-limited submerged cultures of Aspergillus nidulans deltaflbA and fadAG203R strains were studied. Loss-of-function mutation of the flbA gene resulted in an altered germination with unusually thick germination tubes, "fluffy" pellet morphology, as well as a reduced fragmentation rate of hyphae during autolysis. In the fadAG203R mutant strain, conidiophores formed in the stationary phase of growth, and the size of pellets shrank considerably. There were no significant differences in the generation of reactive oxygen species (ROS) and in the specific catalase and superoxide dismutase activities by the tested mutants and the appropriate parental strains. Therefore, the participation of ROS or antioxidative enzymes in FadA/FlbA signaling pathways seems to be unlikely in submerged cultures. On the other hand, earlier increases in the extracellular protease and ammonia production were recorded with the deltaflbA strain, whereas the protease and ammonia production of the fadAG203R mutant lagged behind those of the wild-type strains. Similar changes in the time courses of the induction of gamma-glutamyltranspeptidase and the degradation of glutathione were observed. These results suggest that FadA/FlbA signaling may be involved in the mobilization of protein and peptide reserves as energy sources during carbon starvation.

Aspergillus nidulans↗
PubMed1995

Identification of a novel response regulator required for the swarmer-to-stalked-cell transition in Caulobacter crescentus.

The onset of motility late in the Caulobacter crescentus cell cycle depends on a signal transduction pathway mediated by the histidine kinase PleC and response regulator DivK. We now show that pleD, whose function is required for the subsequent loss of motility and stalk formation by the motile swarmer cell, encodes a 454-residue protein with tandem N-terminal response regulator domains D1 and D2 and a novel C-terminal GGDEF domain. The identification of pleD301, a semidominant suppressor of the pleC Mot phenotype, as a mutation predicted to result in a D-53-->G change in the D1 domain supports a role for phosphorylation in the PleD regulator. Disruptions constructed in the pleD open reading frame demonstrated that the gene is not essential and that the pleC phenotype can also be suppressed by a recessive, loss-of-function mutation. These results suggest that PleD is part of a signal transduction pathway controlling stalked-cell differentiation early in the C. crescentus cell cycle.

Alleles↗
PubMed2000

Transgenic rescue of aganglionosis and piebaldism in lethal spotted mice.

Complete colonization of the gut by enteric neural precursors depends on activation of ednrB and Ret receptors by their respective ligands, edn3 and gdnf. Mutations that eliminate expression of either ligand or either receptor produce intestinal aganglionosis in rodents and humans. Embryos homozygous for the lethal spotted (ls) allele, a loss of function mutation in the edn3 gene, have no ganglion cells in their terminal large intestines and are spotted, due to incomplete colonization of the skin by melanocyte precursors. Expression of edn3 in enteric neural precursors of transgenic mice compensates fully for deficient endogenous edn3 in ls/ls embryos. The effects of the edn3 transgene are dose-dependent, as lower levels of expression in one line prevent aganglionosis in only a subset of animals and reduce, but fail to eliminate, piebaldism. In contrast, expression of neither constitutively active Ret nor activated ras in enteric neural progenitors alters the severity of aganglionosis or piebaldism in ls/ls mice. Given the spatial and temporal pattern of edn3-transgene expression, our results suggest that edn3/ednrB signals are not required prior to the arrival of crest cells in the gut and endrB stimulation elicits distinct cellular responses from Ret or ras activation. Dev Dyn 2000;217:120-132.

Animals↗
PubMed2002

Congenital hypomyelinating neuropathy, central dysmyelination, and Waardenburg-Hirschsprung disease: phenotypes linked by SOX10 mutation.

A unique phenotype of Waardenburg-Hirschsprung disease (WS4) accompanied by peripheral neuropathy and central dysmyelination has been recognized recently in association with SOX10 mutations. We report an infant boy with lethal congenital hypomyelinating neuropathy and WS4 who had a heterozygous SOX10 mutation (Q250X). Histopathological studies showed an absence of peripheral nerve myelin despite normal numbers of Schwann cells and profound dysmyelination in the central nervous system. These observations suggest that some SOX10 mutations such as Q250X may allow Schwann cells and oligodendrocytes to proliferate but interfere with further differentiation to form myelin. In contrast with the SOX10 loss-of-function mutations causing only WS4, mutations associated with both peripheral and central dysmyelination may affect pathology through a dominant-negative mechanism.

DNA-Binding Proteins↗
PubMed2006

GnRH receptor and GPR54 inactivation in isolated gonadotropic deficiency.

Isolated hypogonadotropic hypogonadism (IHH) is defined by a complete or partial impaired secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In the regulation of the gonadotropic axis, the gonadotropin-releasing hormone (GnRH) and its receptor have evolved as a central element in fetal life, at puberty, and for reproduction in adulthood. GnRH resistance due to GnRH receptor (GnRHR) germ-line mutations was the first genetic alteration identified in patients with IHH. GnRHR mutated receptors are associated with impaired GnRH binding, intracellular trafficking or ligand-induced signal transduction, leading to various degrees of LH and FSH deficiency. Loss-of-function mutations of the GnRH receptor account for 50% of familial cases of IHH without anosmia. In 2003, mutations of GPR54 were identified in patients with IHH, opening a new pathway in the physiological regulation of puberty and reproduction. Kisspeptins, which are the natural ligands of GPR54, are potent stimulators of the LH and FSH secretion via the control of GnRH secretion or modulation of the pituitary response to GnRH stimulation. Genotype-phenotype correlations in IHH due to GnRHR and GPR54 mutations indicate that similar mutations may lead to a variable phenotype and suggest that the pituitary might have its own pubertal maturation independent from GnRH. These two causes of IHH result in a more quantitative than qualitative defect of the gonadotropic axis activation. Molecular genetics of IHH has led to a major breakthrough in the neuroendocrine regulation of the gonadotropic axis. New insights into the understanding of the initiation of puberty and in the therapeutic management of defects of the gonadotropic axis have emerged from these studies.

Animals↗