INTERNATIONAL JOURNAL OF CANCER, Issue 2 2010
Renyuan Zhou
Abstract The heterogeneous nuclear ribonucleoprotein (hnRNP) K is an essential RNA and DNA binding protein involved in gene expression and signal transduction including DNA transcription, RNA splicing, RNA stability and translation. The role of hnRNP K in cancer is relatively understudied. However, several cellular functions strongly indicate that hnRNP K is involved in tumorigenesis. In this study, we investigated the altered protein expression and the subcellular distribution of the hnRNP K protein using tissue microarrays in pancreatic cancer. We showed an increased cytoplasmic hnRNP K in pancreatic cancer. This increase in hnRNP K protein occurs at the posttranscriptional level. We postulate that the cytoplasmic accumulation of hnRNP K will lead to silenced mRNA translation of tumor suppressor genes and thus contributes to pancreatic cancer development. We also demonstrated that knocking down of hnRNP K expression by siRNA inhibited pancreatic cancer cell growth and colony formation. hnRNP K was identified as a member of the p53/HDM2 pathway. Whether hnRNP K interacts with the mutant p53 is not known. Using two different pancreatic cancer cell lines, we can demonstrate that hnRNP K interacts with the mutant p53. The subcellular distribution and function of the mutant p53 and the interaction of hnRNP K/mutant p53 were affected by the Ras/MEK/ERK pathway, growth factors and the specific p53 mutations in pancreatic cancer cells. Since Kras is activated and p53 is mutated in most pancreatic cancers, these data unveiled an important new signaling pathway that linked by hnRNP K and mutant p53 in pancreatic cancer tumorigenesis. [source]

ISOLATION AND CHARACTERIZATION OF A CELL WALL-DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA),

JOURNAL OF PHYCOLOGY, Issue 6 2003
Cesar Fuentes
Cell wall,defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw-1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non-ionic detergent as compared with wild-type cells. Tetrad analysis of crosses involving the cw-1 mutant confirmed 2:2 segregation of the cw:cw+ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw-1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw-1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols. [source]

Mutant protein kinase C gamma that causes spinocerebellar ataxia type 14 (SCA14) is selectively degraded by autophagy

GENES TO CELLS, Issue 5 2010
Kazuhiro Yamamoto
Several causal missense mutations in the protein kinase C, (,PKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously showed that mutant ,PKC found in SCA14 is susceptible to aggregation and causes apoptosis. Aggregation of misfolded proteins is generally involved in the pathogenesis of many neurodegenerative diseases. Growing evidence indicates that macroautophagy (autophagy) is important for the degradation of misfolded proteins and the prevention of neurodegenerative diseases. In the present study, we examined whether autophagy is involved in the degradation of the mutant ,PKC that causes SCA14. Mutant ,PKC-GFP was transiently expressed in SH-SY5Y cells by using an adenoviral tetracycline-regulated system. Subsequently, temporal changes in clearance of aggregates and degradation of ,PKC-GFP were evaluated. Rapamycin, an autophagic inducer, accelerated clearance of aggregates and promoted degradation of mutant ,PKC-GFP, but it did not affect degradation of wild-type ,PKC-GFP. These effects of rapamycin were not observed in embryonic fibroblast cells from Atg5-deficient mice, which are not able to perform autophagy. Furthermore, lithium, another type of autophagic inducer, also promoted the clearance of mutant ,PKC aggregates. These results indicate that autophagy contributes to the degradation of mutant ,PKC, suggesting that autophagic inducers could provide therapeutic potential for SCA14. [source]

In Vitro and In Vivo Complementation of the Helicobacter pylori Arginase Mutant Using an Intergenic Chromosomal Site

HELICOBACTER, Issue 5 2006
Melanie L. Langford
Abstract Background:, Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. Materials and methods:, A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203,hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). Results:, A rocF mutant unable to hydrolyze or consume l -arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3, ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. Conclusions:, This complementation strategy should greatly facilitate genetic experiments in H. pylori. [source]

Bacillus subtilis Esterase (BS2) and its Double Mutant Have Different Selectivity in the Removal of Carboxyl Protecting Groups

ADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 14-15 2009
Efrosini Barbayianni
Abstract An esterase from Bacillus subtilis (BS2) and its double mutant E188W/M193C quickly hydrolyze n -butyl, n -propyl, methoxyethyl and allyl esters. The wild-type BS2 preferentially removes such esters from the ,-position of glutamate diesters, while the engineered enzyme has a reversed selectivity removing esters from the ,-position of glutamate diesters. Automated docking and molecular dynamic simulations were performed to understand the molecular reason for the different regioselectivity. [source]

Partial Recovery of Light-Independent Chlorophyll Biosynthesis in the chlL -Deletion Mutant of Synechocystis sp.

IUBMB LIFE, Issue 5 2001
PCC 680
Abstract A chlL -deletion mutant of Synechocystis sp. PCC 6803 designated as chlL - was unable to make significant amounts of chlorophyll in darkness. However, an apparent pseudorevertant has been generated spontaneously that can synthesize an increased amount of chlorophyll under light-activated heterotrophic growth conditions. Under these conditions, the chlorophyll content in this pseudorevertant was about 20% of that in the wild-type strain and about 4 times more than that in the original and in the recently recreated chlL -deletion mutant. This is paralleled by increased performance of dark-grown cells in terms of chlorophyll fluorescence induction and oxygen evolution rates in the pseudorevertant versus in the original mutant. PCR analysis confirmed that the chlL - pseudorevertant mutant still lacked the chlL gene. These results imply that the light-independent chlorophyll biosynthesis pathway was partly recovered. [source]

Resistance of Nutrient-Deprived Listeria monocytogenes 10403S and a ,sigB Mutant to Chemical Stresses in the Presence or Absence of Oxygen

JOURNAL OF FOOD SCIENCE, Issue 7 2008
B. Lungu
ABSTRACT:, Nutrient-deprived Listeria monocytogenes have increased resistance to processing control measures. Heat-stressed L. monocytogenes cells produce higher counts under anaerobic conditions and SigB reportedly contributes to the survival of environmentally stressed Gram-positive bacteria. In this study, a wild type (wt) strain, L. monocytogenes 10403S, and a ,sigB mutant, FSLA1-254, were stressed by starvation in phosphate buffered saline coupled with exposure to chemicals with/without oxygen. In the absence of chemicals, the mutant survived starvation almost as well as the wt, suggesting that the starvation survival response (SSR) in L. monocytogenes was SigB-independent. Conversely, in the presence of chemical stresses the SSR results differed depending on the chemical used. In the presence of sodium chloride (SC), both strains were able to express an SSR under aerobic conditions but not under anaerobic conditions. However, in the presence of sodium propionate (SP), the mutant yielded counts that were 2 log CFU/mL lower than the controls and their aerobic counterparts. In the presence of sodium lactate (SL), the mutant yielded counts that were approximately 3 log CFU/mL lower than the wt under anaerobic conditions. Thus, for the chemical stress produced by SC, the SSR appeared to be SigB-independent. The SSR of L. monocytogenes appeared to be SigB-dependent following exposure to SP or SL under anaerobic conditions. Following exposure to sodium diacetate or lauric acid, both strains were unable to express an SSR. No detectable CFUs were observed after 14 to 21 d under either aerobic or anaerobic incubation. Therefore, these 2 chemicals could be used in biocidal formulations against L. monocytogenes cells under aerobic or anaerobic conditions. [source]

Characterization of Phaffia rhodozyma 3A 4,8 Generated by Low-dose ,-irradiation

JOURNAL OF FOOD SCIENCE, Issue 9 2004
S.H. Lee
ABSTRACT: Astaxanthin content, superoxide dismutase activity, catalase activity, and transmission electron microscopy (TEM) of astaxanthin-hyperproducing mutant 3A 4,8, previously isolated through repeated rounds of ,-irradiation below 10 kGy and visual screening, was examined and compared with wild strain 67,385 and parent strain 2A2N to characterize its mutant. Astaxanthin content of Phaffia rhodozyma was determined using high-performance liquid chromatography. After 10 d culture, 3A 4,8 produced 2.5 mg/g yeast, 78% higher astaxanthin content than parent strain. Mutant exhibited lower superoxide dismutase and higher catalase activities than parent strain. TEM study showed mutant had smaller-sized mitochondria than parent strain. These results indicate ,-irradiation is an effective means of mutagenesis for production of carotenoidhyperproducing mutants. [source]

Comprehensive Analysis of Expressed Sequence Tags from the Pulp of the Red Mutant ,Cara Cara' Navel Orange (Citrus sinensis Osbeck)

JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 10 2010
Jun-Li Ye
Expressed sequence tag (EST) analysis of the pulp of the red-fleshed mutant ,Cara Cara' navel orange provided a starting point for gene discovery and transcriptome survey during citrus fruit maturation. Interpretation of the EST datasets revealed that the mutant pulp transcriptome held a high section of stress responses related genes, such as the type III metallothionein-like gene (6.0%), heat shock protein (2.8%), Cu/Zn superoxide dismutase (0.8%), late embryogenesis abundant protein 5 (0.8%), etc. 133 transcripts were detected to be differentially expressed between the red mutant and its orange-color wild genotype ,Washington' via digital expression analysis. Among them, genes involved in metabolism, defense/stress and signal transduction were statistical overrepresented. Fifteen transcription factors, composed of NAM, ATAF, and CUC transcription factor (NAC); myeloblastosis (MYB); myelocytomatosis (MYC); basic helix-loop-helix (bHLH); basic leucine zipper (bZIP) domain members, were also included. The data reflected the distinct expression profile and the unique regulatory module associated with these two genotypes. Eight differently expressed genes analyzed in digital were validated by quantitative real-time polymerase chain reaction. For structural polymorphism, both simple sequence repeats and single nucleotide polymorphisms (SNP) loci were surveyed; dinucleotide presentation revealed a bias toward AG/GA/TC/CT repeats (52.5%), against GC/CG repeats (0%). SNPs analysis found that transitions (73%) outnumbered transversions (27%). Seventeen potential cultivar-specific and 387 heterozygous SNP loci were detected from ,Cara Cara' and ,Washington' EST pool. [source]

NSF binds calcium to regulate its interaction with AMPA receptor subunit GluR2

JOURNAL OF NEUROCHEMISTRY, Issue 6 2007
Jonathan G. Hanley
Abstract N -ethylmaleimide-sensitive fusion protein (NSF) is essential for numerous Ca2+ -triggered vesicle trafficking events. It functions as a molecular chaperone to regulate trafficking protein complexes such as the soluble NSF attachment protein (SNAP) receptor complex and the ,-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-protein interacting with C-kinase (PICK1) complex. AMPAR trafficking is fundamental to processes of synaptic plasticity, which may underlie learning and memory. Changes in synaptic strength brought about by AMPAR trafficking are triggered by a post-synaptic influx of Ca2+, which may have numerous molecular targets including PICK1. NSF binds AMPAR subunit glutamate receptor subunit 2 (GluR2) and functions to maintain receptors at the synapse. In this study, it was showed that NSF is a Ca2+ -binding protein and that GluR2,NSF interactions are inhibited by the presence of 15 ,mol/L Ca2+. NSF Ca2+ -binding is reciprocally inhibited by the presence of GluR2 C-terminus. Mutant of NSF that binds Ca2+ with reduced affinity and binds GluR2 with reduced sensitivity to Ca2+ was identied. In addition, the interaction of ,SNAP with PICK1 is sensitive to Ca2+. This study demonstrates that the GluR2-NSF-,SNAP-PICK1 complex is regulated directly by Ca2+, allowing for the transduction of Ca2+ signals into concerted alterations in protein,protein interactions to bring about changes in AMPAR trafficking during synaptic plasticity. [source]

Effect of a Photo-synthetic Inhibitor on Tryptamine Pathway-mediated Sekiguchi Lesion Formation in Lesion Mimic Mutant of Rice Infected with Magnaporthe grisea

JOURNAL OF PHYTOPATHOLOGY, Issue 9 2008
A. Imaoka
Abstract A lesion-mimic mutant of rice (cv. Sekiguchi-asahi) showed enhanced resistance to Magnaporthe grisea infection, thereby inducing Sekiguchi lesion (sl) formation and tryptamine accumulation under light. Both Sekiguchi lesion formation and tryptamine accumulation in leaves infected with M. grisea were inhibited by pretreatment with the photosynthetic inhibitor, 3-(3, 4-Dichlorophenyl)-1,1-dimethylurea (DCMU), which suppressed the gene expression of tryptophan decarboxylase (TDC), monoamine oxidase activity, H2O2 generation and DNA fragmentation. Catalase activity was inhibited by M. grisea infection under light, but magnitude of the inhibition was reduced in leaves pretreated with DCMU. Furthermore, tryptophan accumulated in M. grisea- infected leaves under light but not in DCMU-pretreated ones. Interestingly, such DCMU inhibition was reduced in the presence of tryptophan. Our studies suggest that chloroplasts function as the inhibitor of anti-oxidant system such as catalase activity and the supplier of a precursor of tryptamine and tryptophan in the sl mutant infected with M. grisea. [source]

Soil-Borne Wheat Mosaic Virus (SBWMV) 37 kDa Protein Rescues Cell-to-Cell and Long-Distance Movement of an Immobile Tobacco Mosaic Virus Mutant in Nicotiana benthamiana, a Non-Host of SBWMV

JOURNAL OF PHYTOPATHOLOGY, Issue 1 2005
C. Zhang
Abstract To verify the role and examine the functional range of the 37 kDa putative movement protein (MP) of soil-borne wheat mosaic virus (SBWMV), the 37 kDa gene was inserted into an infectious tobacco mosaic virus (TMV)-based expression vector (p30B), to generate p30BMP. The 30 kDa cell-to-cell MP gene of TMV was then inactivated (in p30BMP to give p30B,MP) by a frameshift mutation which removed 80 amino acids from its C-terminus. Systemic infection of Nicotiana benthamiana plants occurred following inoculation with in vitro transcripts of p30BMP or p30B,MP. Progeny viral RNAs from inoculated and systemically infected leaves were analysed by reverse transcriptase polymerase chain reaction and ApaI digestion, and by sequencing. The 30 kDa TMV MP or its truncated form were detected, respectively, in Western blots of cell wall protein extracts from p30BMP-transcript or p30B,MP-transcript inoculated or systemically infected N. benthamiana leaves. High levels of SBWMV 37 kDa MP were detected in all cases. The results suggest that the 37 kDa protein of SBWMV, a monocotyledonous-infecting furovirus, can complement both cell-to-cell and long-distance movement functions in a defective heterologous virus (TMV) in N. benthamiana, a non-host of SBWMV. [source]

Overexpression of the partially activated ,IIb,3D723H integrin salt bridge mutant downregulates RhoA activity and induces microtubule-dependent proplatelet,like extensions in Chinese hamster ovary cells

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2009
E. SCHAFFNER-RECKINGER
Summary.,Background: We have recently reported a novel mutation in the ,3 subunit of the platelet fibrinogen receptor (,IIb,3D723H) identified in a patient with dominantly inherited macrothrombocytopenia, and we have shown that this mutation promotes a new phenotype in Chinese hamster ovary (CHO) cells, characterized by fibrinogen-dependent, microtubule-driven proplatelet-like cell extensions. Results: Here we demonstrate that the partially activated ,IIb,3D723H or ,IIb,3D723A salt bridge mutants, but not fully activated ,IIb,3 mutants, cause this phenotype. Time-lapse videomicroscopy clearly differentiated these stable microtubule-driven and nocodazole-sensitive extensions from common dynamic actin-driven pseudopodia. In addition, overexpression of a mitochondrial marker confirmed their functional role in organelle transport. Comparative immunofluorescence analysis of the subcellular localization of ,IIb,3, the focal adhesion proteins talin or vinculin and actin revealed a similar membrane labeling of CHO cell extensions and CD34+ -derived megakaryocyte proplatelets. Mutant ,IIb,3D723H signaling was independent of Src, protein kinase C or phosphoinositide 3-kinase, but correlated with decreased RhoA activity as compared with wild-type ,IIb,3 signaling, reminiscent of integrin signaling during neurite outgrowth. Accordingly, overexpression of constitutively active RhoA in CHO ,IIb,3D723H cells prevented protrusion formation on fibrinogen. Most interestingly, RhoA/ROCK inhibition was necessary, but not sufficient, and integrin activity was additionally required to induce CHO cell extension formation. Conclusions: CHO ,IIb,3D723H cell protrusions and megakaryocyte proplatelets, like neuronal cell neurites, result from a common integrin-dependent signaling pathway, promoting strongly decreased RhoA activity and leading to microtubule-driven formation of cytoplasmic extensions. [source]

Protein,Protein Interaction of a Pharaonis Halorhodopsin Mutant Forming a Complex with Pharaonis Halobacterial Transducer Protein II Detected by Fourier-Transform Infrared Spectroscopy,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008
Yuji Furutani
Pharaonis halorhodopsin (pHR) functions as a light-driven inward chloride ion pump in Natoronomonas pharaonis, while pharaonis phoborhodopsin (ppR; also called pharaonis sensory rhodopsin II, pSRII), is a light sensor for negative phototaxis. ppR forms a 2:2 complex with its cognate transducer protein (pHtrII) through intramembranous hydrogen bonds: Tyr199ppR,Asn74pHtrII and Thr189ppR,Glu43 pHtrII, Ser62pHtrII. It was reported that a pHR mutant (P240T/F250Y), which possesses the hydrogen-bonding sites, impairs its pumping activity upon complexation with pHtrII. In this study, effect of the complexation with pHtrII on the structural changes upon formation of the K, L1 and L2 intermediates of pHR was investigated by use of Fourier-transform infrared spectroscopy. The vibrational changes of Tyr250pHR and Asn74pHtrII were detected for the L1 and L2 intermediates, supporting that Tyr250pHR forms a hydrogen bond with Asn74pHtrII as similarly to Tyr199ppR. The conformational changes of the retinal chromophore were never affected by complexation with pHtrII, but amide-I vibrations were clearly different in the absence and presence of pHtrII. The molecular environment around Asp156pHR in helix D is also slightly affected. These additional structural changes are probably related to blocking of translocation of a chloride ion from the extracellular to the cytoplasmic side during the photocycle. [source]

Dynamics Change of Phoborhodopsin and Transducer by Activation: Study Using D75N Mutant of the Receptor by Site-directed Solid-state 13C NMR,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008
Izuru Kawamura
Pharaonis phoborhodopsin (ppR or sensory rhodopsin II) is a negative phototaxis receptor of Natronomonas pharaonis, and forms a complex, which transmits the photosignal into cytoplasm, with its cognate transducer (pHtrII). We examined a possible local dynamics change of ppR and its D75N mutant complexed with pHtrII, using solid-state 13C NMR of [3- 13C]Ala- and [1- 13C]Val-labeled preparations. We distinguished Ala C,13C signals of relatively static stem (Ala221) in the C-terminus of the receptors from those of flexible tip (Ala228, 234, 236 and 238), utilizing a mutant with truncated C-terminus. The local fluctuation frequency at the C-terminal tip was appreciably decreased when ppR was bound to pHtrII, while it was increased when D75N, that mimics the signaling state because of disrupted salt bridge between C and G helices prerequisite for the signal transfer, was bound to pHtrII. This signal change may be considered with the larger dissociation constant of the complex between pHtrII and M-state of ppR. At the same time, it turned out that fluctuation frequency of cytoplasmic portion of pHtrII is lowered when ppR is replaced by D75N in the complex with pHtrII. This means that the C-terminal tip partly participates in binding with the linker region of pHtrII in the dark, but this portion might be released at the signaling state leading to mutual association of the two transducers in the cytoplasmic regions within the ppR/pHtrII complex. [source]

Low-temperature Spectroscopy of Met100Ala Mutant of Photoactive Yellow Protein,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008
Yasushi Imamoto
The trans -to- cis photoisomerization of the p -coumaroyl chromophore of photoactive yellow protein (PYP) triggers the photocycle. Met100, which is located in the vicinity of the chromophore, is a key residue for the cis -to- trans back-isomerization of the chromophore, which is a rate-determining reaction of the PYP photocycle. Here we characterized the photocycle of the Met100Ala mutant of PYP (M100A) by low temperature UV,visible spectroscopy. Irradiation of M100A at 80 K yielded a 380 nm species (M100ABL), while the corresponding intermediate of wild type (WT; PYPBL) is formed above 90 K. The amounts of redshifted intermediates produced from M100A (M100AB, and M100AL) were substantially less than those from WT. While the near-UV intermediate (PYPM) is not formed from WT in glycerol samples at low temperature, M100AM was clearly observed above 190 K. These alterations of the photocycle of M100A were explained by the shift in the equilibrium between the intermediates. The carbonyl oxygen of the thioester linkage of the cis -chromophore in the photocycle intermediates is close to the phenyl ring of Phe96 (<3.5 Å), which would be displaced by the mutation of Met100. These findings imply that the interaction between chromophore and amino acid residues near Met100 is altered during the early stage of the PYP photocycle. [source]

Interaction of the Halobacterial Transducer to a Halorhodopsin Mutant Engineered so as to Bind the Transducer: Cl, Circulation Within the Extracellular Channel,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2007
Chisa Hasegawa
An alkali-halophilic archaeum, Natronomonas pharaonis, contains two rhodopsins that are halorhodopsin (phR), a light-driven inward Cl, pump and phoborhodopsin (ppR), the receptor of negative phototaxis functioning by forming a signaling complex with a transducer, pHtrII (Sudo Y. et al., J. Mol. Biol. 357 [2006] 1274). Previously, we reported that the phR double mutant, P240T/F250YphR, can bind with pHtrII. This mutant itself can transport Cl,, while the net transport was stopped upon formation of the complex. The flash-photolysis data were analyzed by a scheme in which phR,P1,P2,P3,P4,phR. The P3 of the wild-type and the double mutant contained two components, X- and O-intermediates. After the complex formation, however, the P3 of the double mutant lacked the X-intermediate. These observations imply that the X-intermediate (probably the N-intermediate) is the state having Cl, in the cytoplasmic binding site and that the complex undergoes an extracellular Cl, circulation because of the inhibition of formation of the X-intermediate. [source]

Molecular and Physiological Characterisation of an Insertion Mutant in the ARR21 Putative Response Regulator Gene from Arabidopsis thaliana

PLANT BIOLOGY, Issue 3 2003
J. Horák
Abstract: In our search for insertion mutants of Arabidopsis response regulator (ARR) genes, we identified a candidate for an ARR21 dSpm transposon insertion line in the SLAT collection by searching the SINS sequence database. Molecular characterisation of this line revealed that the transposon is integrated as a single copy 1727 bases downstream of the ATG signal, within the third intron of the ARR21 gene. The transposon insertion segregated in a Mendelian fashion from heterozygous plants that were allowed to self-pollinate. RT-PCR-based expression analysis showed that ARR21 transcript predominantly accumulates in siliques. In contrast, the full-length ARR21 transcript was not detectable in the ARR21 transposon insertion line, indicating that it harbours an insertion of dSpm transposon in the ARR21 gene. The ARR21 insertion mutant (arr21-1) was subjected to several physiological tests for a possible insertion-linked phenotype. However, our results revealed that the insertion in the ARR21 gene did not cause any alterations in viability and fertility, flowering time, sensitivity to ethylene, cytokinin or red light. We discuss these results in the light of recent findings about the function of the other members of the response regulator gene family of Arabidopsis. [source]

Physiological changes in white lupin associated with variation in root-zone CO2 concentration and cluster-root P mobilization

PLANT CELL & ENVIRONMENT, Issue 10 2005
M. D. CRAMER
ABSTRACT White lupin (Lupinus albus L.) mobilizes insoluble soil phosphorus through exudation of organic acids from ,cluster' roots. Organic acid synthesis requires anaplerotic carbon derived from dark CO2 fixation involving PEP-carboxylase. We tested the hypothesis that variation in root-zone CO2 concentration would influence organic acid synthesis and thus P mobilization. Root-zone CO2 concentrations and soil FePO4 concentrations supplied to sand-grown white lupin (cv. Kiev Mutant) were varied. More biomass accumulated in plants supplied with 360 µL L,1 CO2 to the root-zone, compared with those aerated with either 100 or 6000 µL L,1 CO2. Increased FePO4 in the sand resulted in greater leaf P concentrations, but root-zone [CO2] did not influence leaf P concentration. Suppression of cluster-root development in plants supplied with 100 µL L,1 root-zone CO2 was correlated with increased leaf [P]. However, at both 360 and 6000 µL L,1 CO2, cluster-root development was suppressed only at the highest leaf P concentration. Phloem sap [P] was significantly increased by greater [FePO4] in the sand, but was reduced with increased root-zone [CO2], and this may have triggered increased cluster-root initiation. Succinate was the major organic acid (carboxylate) in the phloem sap (minor components included malate, citrate, fumarate) and was increased at greater [FePO4], suggesting that this shoot-derived carboxylate might provide an important source of organic acids for root metabolism. Since cluster root development was inhibited by increasing concentrations of FePO4 in the sand, it is possible that succinate was utilized for the functioning of the root-nodules. [source]

A Preliminary Study of Solid Embryonic Cerebellar Graft Survival in Adult B6CBA Lurcher Mutant and Wild Type Mice

THE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 12 2009
Jan Cendelín
Abstract Lurcher mutant mice represent a model of olivocerebellar degeneration. They suffer from complete loss of Purkinje cells and a reduction of granule cells and inferior olive neurons. Their wild type littermates serve as healthy controls. The aim of the work was to compare solid embryonic cerebellar graft survival within a period of 9 weeks after their transplantation in adult Lurcher mutant and wild type mice of the B6CBA strain. The solid grafts were injected through a hole in the occipital bone. Host mice were sacrificed 3, 6, or 9 weeks after the transplantation and their cerebella and brain-stems were examined histologically to assess graft presence and structure. We did not find significant differences in graft survival rates between Lurcher mutant and wild type mice. The frequency of graft presence did not differ between mice examined 3, 6, and 9 weeks after the transplantation, neither in Lurchers nor in wild type mice. The grafts were of various sizes. In some cases, only small residua of the grafts were found. Nerve fiber sprouting and cell migration from the graft to the host tissue were observed more often in wild type mice than in Lurchers when examined 6 weeks after surgery. In the period 3,9 weeks after transplantation, massive dying out of the grafts was not observed despite regressive processes in some of the grafts. The degenerative changes in the Lurcher mutant cerebellum do not have strong impact on the fate of the solid cerebellar graft. Anat Rec, 292:1986,1992, 2009. © 2009 Wiley-Liss, Inc. [source]

Hush Puppy: A New Mouse Mutant With Pinna, Ossicle, and Inner Ear Defects,

THE LARYNGOSCOPE, Issue 1 2005
FRCSEd, Henry Pau MD
Abstract Objectives/Hypothesis: Deafness can be associated with abnormalities of the pinna, ossicles, and cochlea. The authors studied a newly generated mouse mutant with pinna defects and asked whether these defects are associated with peripheral auditory or facial skeletal abnormalities, or both. Furthermore, the authors investigated where the mutation responsible for these defects was located in the mouse genome. Methods: The hearing of hush puppy mutants was assessed by Preyer reflex and electrophysiological measurement. The morphological features of their middle and inner ears were investigated by microdissection, paint-filling of the labyrinth, and scanning electron microscopy. Skeletal staining of skulls was performed to assess the craniofacial dimensions. Genome scanning was performed using microsatellite markers to localize the mutation to a chromosomal region. Results: Some hush puppy mutants showed early onset of hearing impairment. They had small, bat-like pinnae and normal malleus but abnormal incus and stapes. Some mutants had asymmetrical defects and showed reduced penetrance of the ear abnormalities. Paint-filling of newborns' inner ears revealed no morphological abnormality, although half of the mice studied were expected to carry the mutation. Reduced numbers of outer hair cells were demonstrated in mutants' cochlea on scanning electron microscopy. Skeletal staining showed that the mutants have significantly shorter snouts and mandibles. Genome scan revealed that the mutation lies on chromosome 8 between markers D8Mit58 and D8Mit289. Conclusion: The study results indicate developmental problems of the first and second branchial arches and otocyst as a result of a single gene mutation. Similar defects are found in humans, and hush puppy provides a mouse model for investigation of such defects. [source]

Gene Transduction of an Active Mutant of Akt Exerts Cytoprotection and Reduces Graft Injury After Liver Transplantation

AMERICAN JOURNAL OF TRANSPLANTATION, Issue 4 2007
M. Morales-Ruiz
Akt is expected to be an effective target for the treatment of ischemia-reperfusion injury (I/R) due to its anti-apoptotic properties and its ability to activate the endothelial nitric oxide synthase (eNOS) enzyme. Therefore, this study was aimed to determine the efficacy of an active mutant of Akt (myr-Akt) to decrease I/R injury in a model of orthotopic liver transplantation in pigs. In addition, we analyzed the contribution of nitric oxide in the Akt-mediated effects by using an eNOS mutant (S1179DeNOS) that mimics the phosphorylation promoted by Akt in the eNOS sequence. Donors were treated with adenoviruses codifying for myr-Akt, S1179DeNOS or ,-galactosidase 24 h before liver harvesting. Then, liver grafts were orthotopically transplanted into their corresponding recipients. Levels of transaminases and lactate dehydrogenase (LDH) increased in all recipients after 24 h of transplant. However, transaminases and LDH levels were significantly lower in the myr-Akt group compared with vehicle. The percentage of apoptotic cells and the amount of activated-caspase 3 protein were also markedly reduced in myr-Akt-treated grafts after 4 days of liver transplant compared with vehicle and S1179DeNOS groups. In conclusion, myr-Akt gene therapy effectively exerts cytoprotection against hepatic I/R injury regardless of the Akt-dependent eNOS activation. [source]

Mutant p53 and cyclin A1 protein expression in primary laryngeal squamous cell carcinomas do not correlate to second primary tumours of the head and neck,

ANZ JOURNAL OF SURGERY, Issue 1-2 2009
Ross D. Farhadieh
Abstract Background:, Field cancerization is a feature of head and neck squamous cell carcinoma. No biological marker in the index tumour has been correlated to the development of second primary tumours (SPT). Cyclin A1 is a cell cycle regulator and a downstream target of p53. This study assessed predictive correlation of cyclin A1 and mut-p53 with clinicopathological parameters and occurrence of (SPT) 7in the head and neck. Methods:, Using immunohistochemistry 106 patients treated for primary laryngeal squamous cell carcinoma were investigated for expression of cyclin A1 and mut-p53. Results:, Expression of cyclin A1 and mut-p53 were noted in 83 of 106 (78.3%) and 25 of 106 (23.6%) patients. There was a weak but significant correlation between mut-p53 and cyclin A1 (r = 0.301, P = 0.002) expression. During the follow-up period (median 41.0 months (range 1,205 months)), 21 of 106 (19.8%) patients developed an SPT. There was no statistically significant correlation between the markers investigated and disease recurrence, SPT diagnosis or clinicopathological parameters. Conclusion:, Second primary tumours are an intriguing problem in treatment of HNSCC and a predictive marker identifying those greatest at risk would be a leap forward. [source]

Mutant p53 melanoma cell lines respond differently to CP-31398-induced apoptosis

BRITISH JOURNAL OF DERMATOLOGY, Issue 5 2005
C.K. Ho
Summary Background, p53, a commonly mutated gene in human cancers, participates in cell cycle arrest, DNA repair and apoptosis. A small pharmacological compound, CP-31398, was found to have the ability to promote proper p53 protein folding, activate p53 transcription of downstream targets, and slow tumour growth in mice. Additionally, CP-31398 was found to be able to convert mutant p53 to wild-type conformation in several cell lines. Objectives, To examine if CP-31398 can revert all mutant p53 proteins to wild-type function. Methods, We studied a series of apoptotic responses to CP-31398 in three melanoma cell lines varying in p53 mutation status. Results, Upon a moderate dose of CP-31398 treatment (15 µg mL,1), only the wild-type p53 MMRU and the single p53 point mutation MeWo cells exhibited apoptosis. Another melanoma cell line, Sk-mel-110, containing multiple p53 mutations, did not exhibit apoptosis. Although CP-31398 enhanced overall p53 protein level, its ability to promote proper folding of p53 protein was limited to CP-31398-sensitive MMRU and MeWo cells. These sensitive cells showed an increased Bax and PUMA transcription, altered mitochondrial membrane potential, followed by the release of cytochrome c, and cleaved caspase-9 and caspase-3. We also demonstrated that Apaf-1 was not involved in CP-31398-mediated apoptosis. Conclusions, Our results suggest that the ability of CP-31398 to revert mutant p53 proteins to wild-type conformation may be correlated to p53 mutational status. More studies are necessary, to further investigate the effect of CP-31398 on mutant p53 and its potential applications as an anticancer agent. [source]

The Role of the Conserved Threonine in P450BM3 Oxygen Activation: Substrate-Determined Hydroxylation Activity of the Thr268Ala Mutant

CHEMBIOCHEM, Issue 2 2008
Max J. Cryle Dr.
Abstract The hydroxylation activity of the Thr268Ala mutant of P450BM3 has been shown to occur to varying degrees with small alterations in the structure of a fatty-acid substrate. Ten substrates were investigated, including straight chain, branched chain and cis -cyclopropyl substituted fatty acids with a straight-chain length that varied between 12 and 16 carbon atoms. The efficacy of the hydroxylation activity appeared to be governed by the chain length of the substrate. Substrates possessing 14 to 15 carbons afforded the highest levels of activity, which were comparable with the wild-type enzyme. Outside of this window, straight-chain fatty acids showed reduced activity over the other substrate types. These results provide a cautionary tale concerning the loss of ferryl activity in such cytochrome P450 threonine to alanine mutants, as the nature of the substrate can determine the extent to which hydroxylation chemistry is abolished. [source]

Structural and Biophysical Characterization of XIAP BIR3 G306E Mutant: Insights in Protein Dynamics and Application for Fragment-Based Drug Design

CHEMICAL BIOLOGY & DRUG DESIGN, Issue 3 2009
Cathy D. Moore
Previous reports describe modulators of X-linked inhibitor of apoptosis (XIAP),caspase interaction designed from the AVPI N-terminal peptide sequence of second mitochondria-derived activator of caspase. A fragment-based drug design strategy was initiated to identify therapeutic non-peptidomimetic antagonists of X-linked inhibitor of apoptosis protein,protein interactions. Fragments that bind to the AVPI binding site of BIR3 (bacculoviral inhibitory repeat) were identified, and to further localize the fragment binding within the AVPI binding site, a point mutation was designed which alters the dynamics of flexible loops and blocks PI region of the binding cleft, thus enabling definition of weakly bound small molecules in the AV portion of the binding cleft. Nuclear magnetic resonance analysis confirmed the G306E mutation stabilizes the AV pocket. Biophysical characterization of the mutant confirms conformation change within the PI sub-pocket as evidenced by a significant diminishment in binding affinity of AVPI mimetics, yet the binding affinity of the smaller AV mimetics is maintained or slightly improved in the mutant compared with wild-type. Additional data from non-covalent mass spectrometry analysis shows enhanced binding of AV mimetics to the G306E mutant over the wild-type. The presented data outline a protein engineering strategy that allowed mapping of AV-replacements with better sensitivity and precision. [source]

Tetrazole Thioacetanilides: Potent Non-Nucleoside Inhibitors of WT HIV Reverse Transcriptase and Its K103N Mutant.

CHEMINFORM, Issue 33 2006
Ester Muraglia
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source]

A quantitative study of the optic nerve in diabetic mutant, Otsuka Long-Evans Tokushima Fatty (OLETF) rats

CONGENITAL ANOMALIES, Issue 4 2001
Kazuhiko Sawada
ABSTRACT, Optic nerves of the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, an animal model of non-insulin dependent diabetes mellitus, were examined using quantitative stereological procedures. At 67 weeks of age, OLETF rats showed a mild hyperglycemia: their blood glucose level was 196 ± 93 mg/dl, significantly higher than that of non-diabetic control Long-Evans Tokushima Otsuka (LETO) rats (110 ± 24 mg/dl). However, there were no differences in the cross sectional area of optic nerves (the mean minimum diameter), the total number and mean diameter of both myelinated and non-myelinated fibers, or the thickness of the myelin sheath between OLETF and LETO rats. The results suggested that a mild hyperglycemia in OLETF rats could not cause any morphological changes in the optic nerve. [source]

The deaf mouse mutant whirler suggests a role for whirlin in actin filament dynamics and stereocilia development

CYTOSKELETON, Issue 7 2007
Mette M. Mogensen
Abstract Stereocilia, finger-like projections forming the hair bundle on the apical surface of sensory hair cells in the cochlea, are responsible for mechanosensation and ultimately the perception of sound. The actin cytoskeleton of the stereocilia contains hundreds of tightly cross-linked parallel actin filaments in a paracrystalline array and it is vital for their function. Although several genes have been identified and associated with stereocilia development, the molecular mechanisms responsible for stereocilia growth, maintenance and organisation of the hair bundle have not been fully resolved. Here we provide further characterisation of the stereocilia of the whirler mouse mutant. We found that a lack of whirlin protein in whirler mutants results in short stereocilia with larger diameters without a corresponding increase in the number of actin filaments in inner hair cells. However, a decrease in the actin filament packing density was evident in the whirler mutant. The electron-density at the tip of each stereocilium was markedly patchy and irregular in the whirler mutants compared with a uniform band in controls. The outer hair cell stereocilia of the whirler homozygote also showed an increase in diameter and variable heights within bundles. The number of outer hair cell stereocilia was significantly reduced and the centre-to-centre spacing between the stereocilia was greater than in the wildtype. Our findings suggest that whirlin plays an important role in actin filament packing and dynamics during postnatal stereocilium elongation. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

High speed sliding of axonemal microtubules produced by outer arm dynein

CYTOSKELETON, Issue 2 2005
Raviraja N. Seetharam
Abstract To study dynein arm activity at high temporal resolution, axonemal sliding was measured field by field for wild type and dynein arm mutants of Tetrahymena thermophila. For wt SB255 cells, when the rate of data acquisition was 60 fps, about 5× greater than previously published observations, sliding was observed to be discontinuous with very high velocity sliding (average 196 ,m/sec) for a few msec (1 or 2 fields) followed by a pause of several fields. The sliding velocities measured were an order of magnitude greater than rates previously measured by video analysis. However, when the data were analyzed at 12 fps for the same axonemes, consistent with previous observations, sliding was linear as the axonemes extended several times their original length with an average velocity of ,10 ,m/sec. The pauses or stops occurred at approximately 200 and 300% of the initial length, suggesting that dynein arms on one axonemal doublet were initially active to the limit of extension, and then the arms on the next doublet became activated. In contrast, in a mutant where OADs are missing, sliding observed at 60 fps was continuous and slow (5 ,m/sec), as opposed to the discontinuous high-velocity sliding of SB255 and of the mutant at the permissive temperature where OADs are present. High-velocity step-wise sliding was also present in axonemes from an inner arm dynein mutant (KO6). These results indicate that the high-speed discontinuous pattern of sliding is produced by the mechanochemical activity of outer arm dynein. The rate of sliding is consistent with a low duty ratio of the outer arm dynein and with the operation of each arm along a doublet once per beat. Cell Motil. Cytoskeleton 60:96,103, 2005. © 2004 Wiley-Liss, Inc. [source]