scholarly journals A dominant activating mutation in the effector region of RAS abolishes IRA2 sensitivity.

1992 ◽  
Vol 12 (2) ◽  
pp. 631-637 ◽  
Author(s):  
K Tanaka ◽  
D R Wood ◽  
B K Lin ◽  
M Khalil ◽  
F Tamanoi ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Biochemical Properties ◽  
Negative Regulator ◽  
Ras Proteins ◽  
Vitro Assay ◽  
Ras Protein ◽  
Ras Genes ◽  
Activating Mutation

Previously described mutations in RAS genes that cause a dominant activated phenotype affect the intrinsic biochemical properties of RAS proteins, either decreasing the intrinsic GTPase or reducing the affinity for guanine nucleotides. In this report, we describe a novel activating mutation in the RAS2 gene of Saccharomyces cerevisiae that does not alter intrinsic biochemical properties of the mutant RAS2 protein. Rather, this mutation, RAS2-P41S (proline 41 to serine), which lies in the effector region of RAS, is shown to abolish the ability of the IRA2 protein to stimulate the GTPase activity of the mutant RAS protein. This mutation also modestly reduced the ability of the mutant protein to stimulate the target adenylate cyclase in an in vitro assay, although in vivo the phenotypes it induced suggest that it retains potency in stimulation of adenylate cyclase. Our results demonstrate that although the effector region of RAS appears to be important for interaction with both target effector and negative regulators of RAS, it is possible to eliminate negative regulator responsiveness and retain potency in effector stimulation.

A dominant activating mutation in the effector region of RAS abolishes IRA2 sensitivity

1992 ◽  
Vol 12 (2) ◽  
pp. 631-637
Author(s):  
K Tanaka ◽  
D R Wood ◽  
B K Lin ◽  
M Khalil ◽  
F Tamanoi ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Biochemical Properties ◽  
Negative Regulator ◽  
Ras Proteins ◽  
Vitro Assay ◽  
Ras Protein ◽  
Ras Genes ◽  
Activating Mutation

Previously described mutations in RAS genes that cause a dominant activated phenotype affect the intrinsic biochemical properties of RAS proteins, either decreasing the intrinsic GTPase or reducing the affinity for guanine nucleotides. In this report, we describe a novel activating mutation in the RAS2 gene of Saccharomyces cerevisiae that does not alter intrinsic biochemical properties of the mutant RAS2 protein. Rather, this mutation, RAS2-P41S (proline 41 to serine), which lies in the effector region of RAS, is shown to abolish the ability of the IRA2 protein to stimulate the GTPase activity of the mutant RAS protein. This mutation also modestly reduced the ability of the mutant protein to stimulate the target adenylate cyclase in an in vitro assay, although in vivo the phenotypes it induced suggest that it retains potency in stimulation of adenylate cyclase. Our results demonstrate that although the effector region of RAS appears to be important for interaction with both target effector and negative regulators of RAS, it is possible to eliminate negative regulator responsiveness and retain potency in effector stimulation.

scholarly journals Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

1997 ◽  
Vol 17 (3) ◽  
pp. 1057-1064 ◽  
Author(s):  
F Shima ◽  
Y Yamawaki-Kataoka ◽  
C Yanagihara ◽  
M Tamada ◽  
T Okada ◽  
...  
Keyword(s):  
Adenylyl Cyclase ◽  
Posttranslational Modification ◽  
Fold Increase ◽  
Terminal Segment ◽  
Amino Acid Residues ◽  
Ras Proteins ◽  
Ras Protein ◽  
Reconstituted System

Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.

scholarly journals Functional Expression of Nramp1 In Vitro in the Murine Macrophage Line RAW264.7

1999 ◽  
Vol 67 (5) ◽  
pp. 2225-2232 ◽  
Author(s):  
Gregory Govoni ◽  
François Canonne-Hergaux ◽  
Cheryl G. Pfeifer ◽  
Sandra L. Marcus ◽  
Scott D. Mills ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Functional Expression ◽  
Integral Membrane Protein ◽  
Biochemical Properties ◽  
Raw264.7 Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Vitro Assay

ABSTRACT Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes.Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-typeNramp1G169 cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1D169 allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane ofSalmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defectiveS. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.

scholarly journals Mutations in the SHR5 gene of Saccharomyces cerevisiae suppress Ras function and block membrane attachment and palmitoylation of Ras proteins.

1995 ◽  
Vol 15 (3) ◽  
pp. 1333-1342 ◽  
Author(s):  
V Jung ◽  
L Chen ◽  
S L Hofmann ◽  
M Wigler ◽  
S Powers
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Current Data ◽  
Ras Proteins ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ras Protein ◽  
Membrane Attachment ◽  
Extragenic Suppressors

We have identified a gene, SHR5, in a screen for extragenic suppressors of the hyperactive RAS2Val-19 mutation in the budding yeast Saccharomyces cerevisiae. SHR5 was cloned, sequenced, and found to encode a 23-kDa protein not significantly homologous to other proteins in the current data bases. Genetic evidence arguing that Shr5 operates at the level of Ras is presented. We tested whether SHR5, like previously isolated suppressors of hyperactivated RAS2, acts by affecting the membrane attachment and/or posttranslational modification of Ras proteins. We found that less Ras protein is attached to the membrane in shr5 mutants than in wild-type cells and that the Ras proteins are markedly underpalmitoylated, suggesting that Shr5 is involved in palmitoylation of Ras proteins. However, shr5null mutants exhibit normal palmitoyltransferase activity measured in vitro. Further, shr5null mutations attenuate Ras function in cells containing mutant Ras2 proteins that are not palmitoylated or farnesylated. We conclude that SHR5 encodes a protein that participates in the membrane localization of Ras but also interacts in vivo with completely unprocessed and cytosolic Ras proteins.

scholarly journals Role and Function of LitR, an Adenosyl B12-Bound Light-Sensitive Regulator of Bacillus megaterium QM B1551, in Regulation of Carotenoid Production

2015 ◽  
Vol 197 (14) ◽  
pp. 2301-2315 ◽  
Author(s):  
Hideaki Takano ◽  
Kou Mise ◽  
Kenta Hagiwara ◽  
Naoya Hirata ◽  
Shoko Watanabe ◽  
...  
Keyword(s):  
Bacillus Megaterium ◽  
Biochemical Properties ◽  
Binding Activity ◽  
Negative Regulator ◽  
Gram Positive ◽  
Content Type ◽  
Carotenoid Production ◽  
Dna Binding Activity

ABSTRACTThe LitR/CarH family of proteins is a light-sensitive MerR family of transcriptional regulators that contain an adenosyl B12(coenzyme B12or AdoB12)-binding domain at the C terminus. The genes encoding these proteins are found in phylogenetically diverse bacterial genera; however, the biochemical properties of these proteins from Gram-positive bacteria remain poorly understood. We performed genetic and biochemical analyses of a homolog of the LitR protein fromBacillus megateriumQM B1551, a Gram-positive endospore-forming soil bacterium. Carotenoid production was induced by illumination in this bacterium.In vivoanalysis demonstrated that LitR plays a central role in light-inducible carotenoid production and serves as a negative regulator of the light-inducible transcription ofcrtandlitRitself. Biochemical evidence showed that LitR in complex with AdoB12binds to the promoter regions oflitRand thecrtoperon in a light-sensitive manner.In vitrotranscription experiments demonstrated that AdoB12-LitR inhibited the specific transcription of thecrtpromoter generated by a σA-containing RNA polymerase holoenzyme under dark conditions. Collectively, these data indicate that the AdoB12-LitR complex serves as a photoreceptor with DNA-binding activity inB. megateriumQM B1551 and that its function as a transcriptional repressor is fundamental to the light-induced carotenoid production.IMPORTANCEMembers of the LitR/CarH family are AdoB12-based photosensors involved in light-inducible carotenoid production in nonphototrophic Gram-negative bacteria. Our study revealed thatBacillusLitR in complex with AdoB12also serves as a transcriptional regulator with a photosensory function, which indicates that the LitR/CarH family is generally involved in the light-inducible carotenoid production of nonphototrophic bacteria.

Epinephrine Induces von Willebrand Factor Release from Cultured Endothelial Cells: Involvement of Cyclic AMP-dependent Signalling in Exocytosis

1997 ◽  
Vol 77 (06) ◽  
pp. 1182-1188 ◽  
Author(s):  
Ulrich M Vischer ◽  
Claes B Wollheinn
Keyword(s):  
Endothelial Cells ◽  
Adenylate Cyclase ◽  
Von Willebrand Factor ◽  
Umbilical Vein ◽  
Free Calcium ◽  
Camp Content ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWf) is released from endothelial cell storage granules after stimulation with thrombin, histamine and several other agents that induce an increase in cytosolic free calcium ([Ca2+]i). In vivo, epinephrine and the vasopressin analog DDAVP increase vWf plasma levels, although they are thought not to induce vWf release from endothelial cells in vitro. Since these agents act via a cAMP-dependent pathway in responsive cells, we examined the role of cAMP in vWf secretion from cultured human umbilical vein endothelial cells. vWf release increased by 50% in response to forskolin, which activates adenylate cyclase. The response to forskolin was much stronger when cAMP degradation was blocked with IBMX, an inhibitor of phosphodiesterases (+200%), whereas IBMX alone had no effect. vWf release could also be induced by the cAMP analogs dibutyryl-cAMP (+40%) and 8-bromo-cAMP (+25%); although their effect was weak, they clearly potentiated the response to thrombin. Epinephrine (together with IBMX) caused a small, dose-dependent increase in vWf release, maximal at 10-6 M (+50%), and also potentiated the response to thrombin. This effect is mediated by adenylate cyclase-coupled β-adrenergic receptors, since it is inhibited by propranolol and mimicked by isoproterenol. In contrast to thrombin, neither forskolin nor epinephrine caused an increase in [Ca2+]j as measured by fura-2 fluorescence. In addition, the effects of forskolin and thrombin were additive, suggesting that they act through distinct signaling pathways. We found a close correlation between cellular cAMP content and vWf release after stimulation with epinephrine and forskolin. These results demonstrate that cAMP-dependent signaling events are involved in the control of exocytosis from endothelial cells (an effect not mediated by an increase in [Ca2+]i) and provide an explanation for epinephrine-induced vWf release.

scholarly journals TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

2021 ◽  
Author(s):  
Hongli Zhou ◽  
Minyu Zhou ◽  
Yue Hu ◽  
Yanin Limpanon ◽  
Yubin Ma ◽  
...  
Keyword(s):  
Cell Death ◽  
Enrichment Analysis ◽  
Angiostrongylus Cantonensis ◽  
Gene Set Enrichment Analysis ◽  
Caspase 8 ◽  
Cns Injury ◽  
Vitro Assay ◽  
Tnf Α

AbstractAngiostrongylus cantonensis (AC) can cause severe eosinophilic meningitis or encephalitis in non-permissive hosts accompanied by apoptosis and necroptosis of brain cells. However, the explicit underlying molecular basis of apoptosis and necroptosis upon AC infection has not yet been elucidated. To determine the specific pathways of apoptosis and necroptosis upon AC infection, gene set enrichment analysis (GSEA) and protein–protein interaction (PPI) analysis for gene expression microarray (accession number: GSE159486) of mouse brain infected by AC revealed that TNF-α likely played a central role in the apoptosis and necroptosis in the context of AC infection, which was further confirmed via an in vivo rescue assay after treating with TNF-α inhibitor. The signalling axes involved in apoptosis and necroptosis were investigated via immunoprecipitation and immunoblotting. Immunofluorescence was used to identify the specific cells that underwent apoptosis or necroptosis. The results showed that TNF-α induced apoptosis of astrocytes through the RIP1/FADD/Caspase-8 axis and induced necroptosis of neurons by the RIP3/MLKL signalling pathway. In addition, in vitro assay revealed that TNF-α secretion by microglia increased upon LSA stimulation and caused necroptosis of neurons. The present study provided the first evidence that TNF-α was secreted by microglia stimulated by AC infection, which caused cell death via parallel pathways of astrocyte apoptosis (mediated by the RIP1/FADD/caspase-8 axis) and neuron necroptosis (driven by the RIP3/MLKL complex). Our research comprehensively elucidated the mechanism of cell death after AC infection and provided new insight into targeting TNF-α signalling as a therapeutic strategy for CNS injury.

scholarly journals Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 930-939 ◽  
Author(s):  
SJ Szilvassy ◽  
PM Lansdorp ◽  
RK Humphries ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Simple Procedure ◽  
Hematopoietic Cells ◽  
Stem Cell Population ◽  
Proliferative Potential ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

scholarly journals DIGESTIBLE ENERGY IN FORAGE BYIN VIVOANDIN VITROASSAYS

1970 ◽  
Vol 50 (3) ◽  
pp. 557-562 ◽  
Author(s):  
J. E. TROELSEN
Keyword(s):  
Energy Analysis ◽  
Energy Content ◽  
In Vitro Assay ◽  
Pure Species ◽  
Vitro Assay ◽  
Digestible Energy ◽  
The Relationship ◽  
Cattle And Sheep

Forage of six pure species was harvested for hay at several maturity stages during four years. The digestible energy content of 102 different lots of hay was determined by feeding to four groups of sheep during the same period, and by in vitro digestions and energy analysis of the undigested residues. The relationship between digestible energy content assayed by the two methods was highly significant (r = 0.85) and did not differ between years and species. Exclusion from regression of the hays containing less than 2 or more than 3 digestible kcal/g revealed that the in vitro assay could reproduce the in vivo digestible energy value with a standard deviation of 0.31 in over 70% of the hays. This represented the maturity and quality range of forage commonly fed to cattle and sheep. The in vitro assay therefore appeared promising for commercial quality determinations.

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