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At least 55 records · Page 3Linked to original sources
PubMed2006

Analog of H2 relaxin exhibits antagonistic properties and impairs prostate tumor growth.

Hormone antagonists can be effective tools to delineate receptor signaling pathways and their resulting downstream physiological actions. Mutation of the receptor binding domain (RBD) of human H2 relaxin (deltaH2) impaired its biological function as measured by cAMP signaling. In a competition assay, deltaH2 exhibited antagonistic activity by blocking recombinant H2 relaxin from binding to receptors on THP-1 cells. In a flow cytometry-based binding assay, deltaH2 demonstrated weak binding to 293T cells expressing the LGR7 receptor in the presence of biotinylated H2 relaxin. When human prostate cancer cell lines (PC-3 and LNCaP) were engineered to overexpress eGFP, wild-type (WT) H2, or deltaH2, and subsequently implanted into NOD/SCID mice, tumor xenografts overexpressing deltaH2 displayed smaller volumes compared to H2 and eGFP controls. Plasma osmolality readings and microvessel density and area assessment suggest that deltaH2 modulates physiological parameters in vivo. In a second murine model, intratumoral injections of lentivectors engineered to express deltaH2/eGFP led to suppressed tumor growth compared to controls. This study provides further evidence supporting a role for H2 relaxin in prostate tumor growth. More importantly, we report how mutation of the H2 relaxin RBD confers the hormone derivative with antagonistic properties, offering a novel reagent for relaxin research.

Animals↗
PubMed2003

immX immunity region of rhizobium phage 16-3: two overlapping cistrons of repressor function.

16-3 is a temperate phage of the symbiotic nitrogen-fixing bacterium Rhizobium meliloti 41. Its prophage state and immunity against superinfection by homoimmune phages are governed by a complex set of controls: the immC and immX repressor systems and the avirT element are all located in well-separated, distinct regions which span 25 kb on the bacteriophage chromosome. The anatomy and function of the immC region are well documented; however, fewer analyses have addressed the immX and avirT regions. We focused in this paper on the immX region and dissected it into two major parts: X(U/L) and X(V). The X(U/L) part (0.6 kb) contained two overlapping cistrons, X(U) and X(L), coding for proteins pXU and pXL, respectively. Inactivation of either gene inactivated the repressor function of the immX region. Loss-of-function mutants of X(U) and X(L) complemented each other in trans in double lysogens. The X(V) part (1 kb) contained a target for X(U/L) repressor action. Mutations at three sites in X(V) led to various degree of ImmX insensitivity in a hierarchic manner. Two sites (X(V1) and X(V3)) exhibited the inverted-repeat structures characteristic of many repressor binding sites. However, X(V1) could also be folded into a transcription terminator. Of the two immunity regions of 16-3, immX seems to be unique both in its complex genetic anatomy and in its sequence. To date, no DNA or peptide sequence homologous to that of ImmX has been found in the data banks. In contrast, immC shares properties of a number of immunity systems commonly found in temperate phages.

Bacteriophages↗
PubMed2006

Downregulation of gamma interferon receptor 1 by Kaposi's sarcoma-associated herpesvirus K3 and K5.

Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway. In order to complete their life cycles, viruses must modulate the host IFN-mediated immune response. The K3 and K5 proteins of a human tumor-inducing herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV), have been shown to downregulate the surface expression of host immune modulatory receptors by increasing their endocytosis rates, which leads to suppression of cell-mediated immunity. In this report, we demonstrate that K3 and K5 both specifically target gamma interferon receptor 1 (IFN-gammaR1) and induce its ubiquitination, endocytosis, and degradation, resulting in downregulation of IFN-gammaR1 surface expression and, thereby, inhibition of IFN-gamma action. Mutational analysis indicated that K5 appeared to downregulate IFN-gammaR1 more strongly than K3 and that the amino-terminal ring finger motif and the carboxyl-terminal region of K5 were necessary for IFN-gammaR1 downregulation. These results suggest that KSHV K3 and K5 suppress both cytokine-mediated and cell-mediated immunity, which ensures efficient viral avoidance of host immune controls.

Cell Line↗
PubMed1995

One of three CCArGG box/serum response elements of the beta-actin gene is an insulin-responsive element.

The cytoskeletal actins are abundant proteins in mammalian nonmuscle cells. We have previously reported that physiological concentrations of insulin induced beta-actin transcription in rat H4 hepatoma cells. To define whether one or more of the three CCArGG box elements or other elements within the beta-actin gene promoter is an insulin response element, we transfected H4 cells with regions of the human beta-actin gene promoter fused to the chloramphenicol acetyltransferase gene. A 350-basepair DNA fragment was isolated that mediates both insulin and serum effects. This fragment contains at least two up-stream elements, a CCAAT box and a CCArGG box, and accounts for more than 70% of the basal activity of the beta-actin promoter in H4 cells. There was a small, but significant, stimulatory effect of insulin over maximal serum induction, suggesting a difference in their mechanisms of action. Mutation of the CCAAT box drastically reduced basal expression, with no effect on insulin induction. In contrast, a mutation of the CCArGG element reduced basal expression and completely abolished insulin inducibility. Electrophoretic mobility shift assays suggested that insulin regulated the activity, but not the binding, of a factor(s) that associates with the CCArGG box. These data demonstrate that in H4 cells, insulin induction of beta-actin gene expression was mediated at least in part through one of the three beta-actin CCArGG elements.

Actins↗
PubMed1994

Androgen action: molecular mechanism and medical application.

Androgen action in many organs, such as prostate and skin, is dependent on the conversion of testosterone by 5 alpha-reductase to 5 alpha-dihydrotestosterone. 5 alpha-Dihydrotestosterone then binds to the androgen receptor to regulate specific gene expression. Inhibitors of 5 alpha-reductase are useful for the selective treatment of prostatic cancer, benign prostate hyperplasia, acne, baldness and female hirsutism, without affecting spermatogenesis, sexual behavior and smooth muscle growth, that do not require the conversion of testosterone to 5 alpha-dihydrotestosterone. Certain unsaturated fatty acids, such as gamma-linolenic acid, are potent 5 alpha-reductase inhibitors, suggesting a linkage between unsaturated fatty acids and androgen action. Mutations in androgen receptor genes are responsible for many cases of androgen-insensitivity. In some prostate cancer cells, some antiandrogens may act like androgens in stimulating the proliferation of the cancer cells because these antiandrogens can bind to a mutated androgen receptor and transactivate target genes. Prostate cancers are usually androgen-dependent initially but can lose dependency and responsiveness. Tumor cells which are resistant to endocrine therapy ultimately proliferate. Androgen-independent or androgen-repressive cells can arise from androgen-sensitive prostate cancer cells by changes in specific gene expression over time in a clonal isolate. This change in androgen responsiveness was accompanied by a change in androgen receptor expression and transcriptional activity as well as expression of some oncogenes.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed2005

Gene action of new mutations in Arabidopsis thaliana.

For a newly arising mutation affecting a trait under selection, its degree of dominance relative to the preexisting allele(s) strongly influences its evolutionary impact. We have estimated dominance parameters for spontaneous mutations in a subset of lines derived from a highly inbred founder of Arabidopsis thaliana by at least 17 generations of mutation accumulation (MA). The labor-intensive nature of the crosses and the anticipated subtlety of effects limited the number of MA lines included in this study to 8. Each MA line was selfed and reciprocally crossed to plants representing the founder genotype, and progeny were assayed in the greenhouse. Significant mutational effects on reproductive fitness included a recessive fitness-enhancing effect in one line and fitness-reducing effects, one additive and the other slightly recessive. Mutations conferring earlier phenology or smaller leaves were significantly recessive. For effects increasing leaf number and reducing height at flowering, additive gene action accounted for the expression of the traits. The sole example of a significantly dominant mutational effect delayed phenology. Our findings of recessive action of a fitness-enhancing mutational effect and additive action of a deleterious effect counter a common expectation of (partial) dominance of alleles that increase fitness, but the frequency of occurrence of such mutations is unknown.

Alleles↗
PubMed2000

Missense point mutations of tau to segregate with FTDP-17 exhibit site-specific effects on microtubule structure in COS cells: a novel action of R406W mutation.

Missense and splicing point mutations have been found in the tau gene in families with frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17). Of these mutations, we examined four exonic missense point mutations (G272V, P301L, V337M and R406W) in 3-repeat or 4-repeat tau isoform on the transfection experiment. The effects of two mutations (G272V or P301L) on microtubules were subtle whereas those of two other mutations (V337M or R406W) were dramatically significant when these two mutations were constructed into 3-repeat tau but not into 4-repeat tau. The R406W mutation induced an alternation of microtubules to form dotted or fragmented forms retaining colocalization of tau with tubulin whereas the V337M mutation predominantly disrupted microtubule networks and diminished colocalization of tau and tubulin. The effect of the mutations on microtubules were thus site-dependent and isoform-dependent. Tau with R406W mutation was found to be colocalized with tubulin without filamentous structures on confocal views, suggesting that the carboxyl region of tau played a different role from tubulin-binding domain on microtubule assemble. Another abnormal property was identified in tau with R406W mutation that failed to suffer phosphorylation. Thus, diverse effects of tau mutations on microtubules may explain the various clinicopathologies of FTDP-17 and related tauopathies.

Animals↗
PubMed1999

Validation of the human T-lymphocyte cloning assay--ring test report from the EU concerted action on HPRT mutation (EUCAHM).

The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE (P = 0.0004) and lnMF (P = 0.03), but there was no significant laboratory effect on the lnCE (P = 0.38) or lnMF (P = 0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations.

Clone Cells↗
PubMed1979

The time of action of three mutations affecting Drosophila eye morphogenesis.

Histology and clonal analysis are used to look at the time of action of the mutant Drosophila genes eyeless, eyeless dominant and sine oculis. The findings suggest that eyeless dominant has its effect during the first two larval instars, whilst eyeless and sine oculis act during the third larval instar.

Animals↗
PubMed1993

Defective insulin action in fibroblasts from noninsulin-dependent diabetes mellitus patients with Gln1152 insulin receptor mutation.

Insulin action was investigated in cultured skin fibroblasts from two consanguineous patients with a heterozygous point mutation in the insulin receptor kinase (Arg1152-Gln). In spite of normal binding, Gln1152 insulin receptor exhibited 20% increased basal kinase activity, but significantly reduced insulin-dependent autophosphorylation and kinase activity compared to controls from either weight-matched noninsulin-dependent diabetic patients (n = 4) or normal subjects (n = 5). In fibroblasts from the mutant patients, basal alpha-aminoisobutyric acid and 2-deoxyglucose (2-DG) uptake, cytochalasin-B (CB) plasma membrane binding, and glycogen synthase activity were increased to levels similar to those in maximally insulin-stimulated control cells. No insulin stimulation of these metabolic effects was detected in the mutant cells. In spite of the high basal 2-DG uptake and CB binding and the lack of further insulin response, fibroblasts from the mutant patients responded to 12-O-tetradecanoylphorbol-13-acetate with a further 50% increase in 2-DG uptake and CB binding. The magnitude of the effects of insulin and 12-O-tetradecanoylphorbol-13-acetate in control cells were nearly identical. We conclude that the Gln1152 insulin receptor impairs insulin regulation of metabolic responses in patient cells. Its presence in fibroblasts from the mutant patients appears to be accompanied by an increased pool of glucose transporters.

Adult↗
PubMed2003

Mutations affecting gonadotropin secretion and action.

A number of mutations are known to disturb the development and function of the hypothalamic-pituitary-gonadal axis. They affect hypothalamic-pituitary-gonadal function at multiple levels, from the migration of gonadotropin releasing hormone neurons to the hypothalamus right through to gonadotropin action in the ovary and testis. Most of the mutations are inactivating, causing various forms of hypogonadism. Exceptions are the activating mutations of the luteinizing hormone receptor, causing male-limited gonadotropin-independent precocious puberty. The human mutations and genetically modified animal models have clarified the molecular pathogenesis of hypogonadism and such disorders can now be diagnosed using molecular biological techniques, enabling selection of specific treatments and appropriate counselling of patients and their families.

Amino Acid Sequence↗
PubMed1996

A mechanism of Bud1p GTPase action suggested by mutational analysis and immunolocalization.

BACKGROUND: Yeast cells polarize, bud, and divide in either of two genetically programmed patterns: axial or bipolar. The Saccharomyces cerevisiae gene BUD1 (also known as RSR1) encodes a Ras-related GTPase critical for selection of a bud sites in these patterns. To distinguish between possible mechanisms of Bud1p action, we have examined the function and subcellular localization of Bud1p in a variety of mutant situations. RESULTS: Bud1p has 57% identity to H-ras, except for an 81 amino-acid insertion near the carboxyl terminus. Mutation of the proposed BUD1 effector domain produces a protein which can neither support normal patterns of budding nor interact with CDC24, which encodes a likely Bud1p effector. A version of Bud1p deleted for the 81 amino-acid unique region is essentially wild-type. Immunofluorescence and cell fractionation indicate that Bud1p remains associated with the membrane throughout its GTPase cycle. Both potential effectors of Bud1p, Bem1p and Cdc24p, are also membrane associated even in the absence of Bud1p, suggesting that Bud1p is not required to dock these proteins from the cytosol but may couple these proteins and others within the plane of the plasma membrane. CONCLUSIONS: Based upon observations reported here and elsewhere, we propose a novel mechanism of Bud1p GTPase action. Like Ras, Bud1p GTPase is constitutively associated with the plasma membrane; however, concentrated activities of Bud5p GDP-GTP exchange factor and Bud2p GTPase-activating protein at the future bud site promote rapid cycling of Bud1p between GTP- and GDP-bound conformations in a spatially restricted manner. Local GTPase cycling serves to efficiently nucleate complexes between polarity establishment functions that direct cytoskeletal polarization towards the bud site.

Alleles↗
PubMed1999

The effect of deep pore mutations on the action of phenylalkylamines on the Kv1.3 potassium channel.

We investigated the action of the phenylalkylamines verapamil and N-methyl-verapamil on the Kv1.3 potassium channel using the whole-cell configuration of the patch-clamp technique. Our goal was to identify their binding as a prerequisite for using the phenylalkylamines as small, well-defined molecular probes, not only to expand the structural findings made with peptide toxins or by crystallization, but also to use them as lead compounds for the generation of more potent and therefore more specific K+ channel modulators. Competition experiments with charybdotoxin, known to interact with external residues of Kv1.3, showed no interaction with verapamil. The internal application of quarternary N-methyl-verapamil in combination with verapamil suggested competition for the same internal binding site. Verapamil affinity was decreased 6 fold by a mutation (M395V) in a region of the internal pore which forms part of the internal tetraethylammonium (TEA+) binding site, although mutations at neighbouring residues (T396 and T397) were without effect. Modification of C-type inactivation by mutations in the internal pore suggest that this region participates in the inactivation process. The action of phenylalkylamines and local anaesthetics on L-type Ca2+ channels and Na channels, respectively, and verapamil on Kv1.3 indicate very similar blocking mechanisms. This might allow the use of these compounds as molecular probes to map the internal vestibule of all three channel types.

Amino Acid Sequence↗
PubMed2000

The actions of ether, alcohol and alkane general anaesthetics on GABAA and glycine receptors and the effects of TM2 and TM3 mutations.

The actions of 13 general anaesthetics (diethyl ether, enflurane, isoflurane, methoxyflurane, sevoflurane, chloral hydrate, trifluoroethanol, tribromoethanol, tert-butanol, chloretone, brometone, trichloroethylene, and alpha-chloralose) were studied on agonist-activated Cl(-) currents at human GABA(A) alpha(2)beta(1), glycine alpha(1), and GABA(C) rho(1) receptors expressed in human embryonic kidney 293 cells. All 13 anaesthetics enhanced responses to submaximal (EC(20)) concentrations of agonist at GABA(A) and glycine receptors, except alpha-chloralose, which did not enhance responses at the glycine alpha(1) receptor. None of the anaesthetics studied potentiated GABA responses at the GABA(C) rho(1) receptor. Potentiation of submaximal agonist currents by the anaesthetics was studied at GABA(A) and glycine receptors harbouring mutations in putative transmembrane domains 2 and 3 within GABA(A) alpha(2), beta(1), or glycine alpha(1) receptor subunits: GABA(A) alpha(2)(S270I)beta(1), alpha(2)(A291W)beta(1), alpha(2)beta(1)(S265I), and alpha(2)beta(1)(M286W); glycine alpha(1)(S267I) and alpha(1)(A288W). For all anaesthetics studied except alpha-chloralose, at least one of the mutations above abolished drug potentiation of agonist responses at GABA(A) and glycine receptors. alpha-Chloralose produced efficacious direct activation of the GABA(A) alpha(2)beta(1) receptor (a 'GABA-mimetic' effect). The other 12 anaesthetics produced minimal or no direct activation of GABA(A) and glycine receptors. A non-anaesthetic isomer of alpha-chloralose, beta-chloralose, was inactive at GABA(A) and glycine receptors and did not antagonize the actions of alpha-chloralose at GABA(A) receptors. The implications of these findings for the molecular mechanisms of action of general anaesthetics at GABA(A) and glycine receptors are discussed.

Alcohols↗
PubMed2005

Mutational analysis of action of mitochondrial fusion factor mitofusin-2.

Mitofusin-2 (Mfn2) is an essential component of mitochondrial fusion machinery, but its molecular mechanism of action is not clear. We found that a Mfn2 deletion mutant lacking two transmembrane spans (Mfn(DeltaTM)) acts as a dominant-negative mutant and blocks mitochondrial fusion. Furthermore, detailed analysis of various mutants of Mfn(DeltaTM) revealed that GTPase activity and four regions highly conserved from nematodes to mammals are necessary for the dominant-negative effect. Immunoprecipitation studies of the N- and C-terminal cytosolic tails of Mfn2 showed that in addition to the coiled-coil domains previously identified, a highly conserved domain in the most N-terminal region and GTPase activity are necessary for the interaction between the N- and C-terminal tails, which is in turn required for the dominant-negative effect. In addition, we found unexpectedly that overexpression of the deletion mutant composed of one short region each in the N- and C-terminal tails of Mfn2 resulted in loss of mitochondrial membrane potential, suggesting that Mfn2 might also be connected to maintenance of mitochondrial membrane potential.

Amino Acid Sequence↗