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Deficient Mutant (deficient + mutant)

Distribution by Scientific Domains
Life Sciences70%
Medical Sciences20%

Selected Abstracts

Activation of p21-activated kinase 1 is required for lysophosphatidic acid-induced focal adhesion kinase phosphorylation and cell motility in human melanoma A2058 cells

FEBS JOURNAL, Issue 8 2004
In Duk Jung
Lysophosphatidic acid (LPA), one of the naturally occurring phospholipids, stimulates cell motility through the activation of Rho family members, but the signaling mechanisms remain to be elucidated. In the present study, we investigated the roles of p21-activated kinase 1 (PAK1) on LPA-induced focal adhesion kinase (FAK) phosphorylation and cell motility. Treatment of human melanoma cells A2058 with LPA increased phosphorylation and activation of PAK1, which was blocked by treatment with pertussis toxin and by inhibition of phosphoinositide 3-kinase (PI3K) with an inhibitor LY294002 or by overexpression of catalytically inactive mutant of PI3K,, indicating that LPA-induced PAK1 activation was mediated via a Gi protein and the PI3K, signaling pathway. In addition, we demonstrated that Rac1/Cdc42 signals acted as upstream effector molecules of LPA-induced PAK activation. However, Rho-associated kinase, MAP kinase kinase 1/2 or phospholipase C might not be involved in LPA-induced PAK1 activation or cell motility stimulation. Furthermore, PAK1 was necessary for FAK phosphorylation by LPA, which might cause cell migration, as transfection of the kinase deficient mutant of PAK1 or PAK auto-inhibitory domain significantly abrogated LPA-induced FAK phosphorylation. Taken together, these findings strongly indicated that PAK1 activation was necessary for LPA-induced cell motility and FAK phosphorylation that might be mediated by sequential activation of Gi protein, PI3K, and Rac1/Cdc42. [source]

Carbofuran degradation mediated by three related plasmid systems

FEMS MICROBIOLOGY ECOLOGY, Issue 3 2000
A.V. Ogram
Abstract Two carbofuran-metabolizing Sphingomonas strains, TA and CD, were isolated from soils with differing histories of exposure to carbofuran. These strains were compared with a previously described strain, Sphingomonas sp. CFO6, with regard to growth rate, formation of metabolites, and plasmid content and structure. Extensive regions of similarity were observed between the three different plasmid systems as evidenced by cross hybridization. In addition, all three systems harbor IS1412, an insertion sequence (IS) element involved in heat-induced loss of carbofuran phenotype in CFO6, and heat-induced carbofuran deficient mutants of all three strains correlated with loss of IS1412. A carbofuran deficient mutant of TA generated by induction of IS elements was complemented by reintroduction of the wild-type plasmid, confirming the presence of genes required for carbofuran metabolism on this plasmid. Carbofuran metabolism in these three strains is clearly linked via plasmids of different numbers and sizes that share extensive common regions, and carbofuran-degrading genes may be associated with active IS elements. [source]

Nitric oxide scavenging and detoxification by the Mycobacterium tuberculosis haemoglobin, HbN in Escherichia coli

MOLECULAR MICROBIOLOGY, Issue 5 2002
Ranjana Pathania
Summary Nitric oxide (NO), generated in large amounts within the macrophages, controls and restricts the growth of internalized human pathogen, Mycobacterium tuberculosis H37Rv. The molecular mechanism by which tubercle bacilli survive within macrophages is currently of intense interest. In this work, we have demonstrated that dimeric haemoglobin, HbN, from M. tuberculosis exhibits distinct nitric oxide dioxygenase (NOD) activity and protects growth and cellular respiration of heterologous hosts, Escherichia coli and Mycobacterium smegmatis, from the toxic effect of exogenous NO and the NO-releasing compounds. A flavohaemoglobin (HMP)-deficient mutant of E. coli, unable to metabolize NO, acquired an oxygen-dependent NO consumption activity in the presence of HbN. On the basis of cellular haem content, the specific NOD activity of HbN was nearly 35-fold higher than the single-domain Vitreoscilla haemoglobin (VHb) but was sevenfold lower than the two-domain flavohaemoglobin. HbN-dependent NO consumption was sustained with repeated addition of NO, demonstrating that HbN is catalytically reduced within E. coli. Aerobic growth and respiration of a flavohaemoglobin (HMP) mutant of E. coli was inhibited in the presence of exogenous NO but remained insensitive to NO inhibition when these cells produced HbN, VHb or flavohaemoglobin. M. smegmatis, carrying a native HbN very similar to M. tuberculosis HbN, exhibited a 7.5-fold increase in NO uptake when exposed to gaseous NO, suggesting NO-induced NOD activity in these cells. In addition, expression of plasmid-encoded HbN of M. tuberculosis in M. smegmatis resulted in 100-fold higher NO consumption activity than the isogenic control cells. These results provide strong experimental evidence in support of NO scavenging and detoxification function for the M. tuberculosis HbN. The catalytic NO scavenging by HbN may be highly advantageous for the survival of tubercle bacilli during infection and pathogenesis. [source]

Identification of mutants in phosphorus metabolism

ANNALS OF APPLIED BIOLOGY, Issue 1 2001
JULIE C LLOYD
Summary Phosphorus availability is often limiting for plant growth. However, little is known of the pathways and mechanisms that regulate phosphorus (P) uptake and distribution in plants. We have developed a screen based on the induction of secreted root acid phosphatase activity by low-P stress to identify mutants of Arabidopsis thaliana with defects in P metabolism. Acid phosphatase activity was detected visually in the roots of A. thaliana seedlings grown in vitro on low-P medium, using the chromogenic substrate, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP). In low-P stress conditions the roots of wild-type plants stained blue, as the induced root acid phosphatase cleaved BCIP to release the coloured product. Potential mutants were identified as having white, or pale blue, roots under these conditions. Out of approximately 79 000 T-DNA mutagenised seedlings screened, two mutants with reduced acid phosphatase staining were further characterised. Both exhibited reduced growth and differences in their P contents when compared to wild-type A. thaliana. The mutant with the most severe phenotype, pho3, accumulated high levels of anthocyanins and starch in a distinctive visual pattern within the leaves. The phenotypes of these mutants are distinct from two previously identified phosphorus mutants (phol and pho2) and from an acid phosphatase deficient mutant (pupl) of A. thaliana. This suggested that the screening method was robust and might lead to the identification of further mutants with the potential for increasing our understanding of P nutrition. [source]

Metabolism in 1,3-propanediol fed-batch fermentation by a D -lactate deficient mutant of Klebsiella pneumoniae

BIOTECHNOLOGY & BIOENGINEERING, Issue 5 2009
Yun-Zhen Xu
Abstract Klebsiella pneumoniae HR526, a new isolated 1,3-propanediol (1,3-PD) producer, exhibited great productivity. However, the accumulation of lactate in the late-exponential phase remained an obstacle of 1,3-PD industrial scale production. Hereby, mutants lacking D -lactate pathway were constructed by knocking out the ldhA gene encoding fermentative D -lactate dehydrogenase (LDH) of HR526. The mutant K. pneumoniae LDH526 with the lowest LDH activity was studied in aerobic fed-batch fermentation. In experiments using pure glycerol as feedstock, the 1,3-PD concentrations, conversion, and productivity increased from 95.39,g,L,1, 0.48 and 1.98,g,L,1,h,1 to 102. 06,g,L,1, 0.52,mol,mol,1 and 2.13,g,L,1,h,1, respectively. The diol (1,3-PD and 2,3-butanediol) conversion increased from 0.55,mol,mol,1 to a maximum of 0.65,mol,mol,1. Lactate would not accumulate until 1,3-PD exceeded 84,g,L,1, and the final lactate concentration decreased dramatically from more than 40,g,L,1 to <3,g,L,1. Enzymic measurements showed LDH activity decreased by 89,98% during fed-batch fermentation, and other related enzyme activities were not affected. NADH/NAD+ enhanced more than 50% in the late-exponential phase as the D -lactate pathway was cut off, which might be the main reason for the change of final metabolites concentrations. The ability to utilize crude glycerol from biodiesel process and great genetic stability demonstrated that K. pnemoniae LDH526 was valuable for 1,3-PD industrial production. Biotechnol. Bioeng. 2009; 104: 965,972. © 2009 Wiley Periodicals, Inc. [source]

The Critical Role of IL-12p40 in Initiating, Enhancing, and Perpetuating Pathogenic Events in Murine Experimental Autoimmune Neuritis

BRAIN PATHOLOGY, Issue 4 2002
Lei Bao
Interleukin 12 (IL-12) is a proinflammatory cytokine with important immunoregulatory activities and is critical in determining the differentiation and generation of Th1 cells. For the present study, we investigated the role of endogenous IL-12 in the pathogenesis of experimental autoimmune neuritis (EAN), which is a CD4+ T-cell mediated autoimmune inflammatory disease of the peripheral nervous system. EAN is used as an animal model for Guillain-Barré syndrome of humans. Here, EAN was established in IL-12 p40 deficient mutant (IL-12 -/- ) C57BL/6 mice by immunization with P0 peptide 180,199, a purified component of peripheral nerve myelin, and Freund's complete adjuvant. In these IL-12 -/- mice the onset of clinical disease was delayed, and the incidence and severity of EAN were significantly reduced compared to that in wild-type mice. The former group's clinical manifestations were associated with less P0-peptide 180,199 induced secretion of interferon-, (IFN-,) by splenocytes in vitro and low production of anti-P0-peptide 180,199 IgG2b antibodies in serum. Fewer IFN-, and TNF-, producing cells, but more cells secreting IL-4, were found in sciatic nerve sections from IL-12 -/- mice, consistent with impaired Th1 functions and response. However, the IL-12 deficiency appeared not to affect P0 peptide 180,199-specific T-cell proliferation. These results indicate that IL-12 has a major role in the initiation, enhancement and perpetuation of pathogenic events in EAN by promoting a Th1 cell-mediated immune response and suppressing the Th2 response. This information augments consideration of IL-12 as a therapeutic target in Guillain-Barré syndrome and other T-cell-mediated autoimmune diseases. [source]

Carbofuran degradation mediated by three related plasmid systems

FEMS MICROBIOLOGY ECOLOGY, Issue 3 2000
A.V. Ogram
Abstract Two carbofuran-metabolizing Sphingomonas strains, TA and CD, were isolated from soils with differing histories of exposure to carbofuran. These strains were compared with a previously described strain, Sphingomonas sp. CFO6, with regard to growth rate, formation of metabolites, and plasmid content and structure. Extensive regions of similarity were observed between the three different plasmid systems as evidenced by cross hybridization. In addition, all three systems harbor IS1412, an insertion sequence (IS) element involved in heat-induced loss of carbofuran phenotype in CFO6, and heat-induced carbofuran deficient mutants of all three strains correlated with loss of IS1412. A carbofuran deficient mutant of TA generated by induction of IS elements was complemented by reintroduction of the wild-type plasmid, confirming the presence of genes required for carbofuran metabolism on this plasmid. Carbofuran metabolism in these three strains is clearly linked via plasmids of different numbers and sizes that share extensive common regions, and carbofuran-degrading genes may be associated with active IS elements. [source]

Methionine gamma-lyase: The unique reaction mechanism, physiological roles, and therapeutic applications against infectious diseases and cancers

IUBMB LIFE, Issue 11 2009
Dan Sato
Abstract Sulfur-containing amino acids (SAAs) are essential components in many biological processes and ubiquitously distributed to all organisms. Both biosynthetic and catabolic pathways of SAAs are heterogeneous among organisms and between developmental stages, and regulated by the environmental changes. Limited lineage of organisms ranging from archaea to plants, but not human, possess a unique enzyme methionine gamma-lyase (MGL, EC 4.4.1.11) to directly degrade SAA to ,-keto acids, ammonia, and volatile thiols. The reaction mechanisms and the physiological roles of this enzyme are partially demonstrated by the enzymological analyzes, structure determination, isotopic labeling of the intermediate metabolites, and functional analyzes of deficient mutants. MGL has been exploited as a drug target for the infectious diseases caused by parasitic protozoa and anaerobic periodontal bacteria. In addition, MGL has been utilized to develop therapeutic interventions of various cancers, by introducing recombinant proteins to deplete methionine essential for the growth of cancer cells. In this review, we discuss the current understanding of enzymological properties, putative physiological roles, and therapeutic applications of MGL. © 2009 IUBMB IUBMB Life, 61(11): 1019,1028, 2009 [source]

Role of prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein-specific signal peptidase II (LspA) in localization and physiological function of lipoprotein MsmE in Streptococcus mutans

MOLECULAR ORAL MICROBIOLOGY, Issue 6 2008
T. Arimoto
Introduction:, To clarify the role that prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein-specific signal peptidase II (LspA) play in the physiological function of MsmE, we constructed lgt -deficient and lspA -deficient mutants of Streptococcus mutans 109c and examined the potential role of Lgt and LspA in membrane anchoring and growth in a melibiose medium of S. mutans. Methods:, The lgt -, lspA -, and msmE -deficient mutants of S. mutans 109c were constructed by double-crossover recombination of their respective genes. Localization of MsmE was demonstrated by Western blot analysis with an MsmE antiserum. The growth of S. mutans cells was examined in a Trypton medium containing melibiose or glucose. Results:, In the S. mutans lgt mutant, localization of the surface lipoprotein MsmE changed with the culture supernatant. The growth of the S. mutans lgt and lspA mutants was remarkably reduced in the melibiose medium; however, growth was recovered in the strains complemented with the lgt or the lspA gene. Therefore, lipid-modification by Lgt and subsequent signal peptide cleavage by LspA were crucial for membrane anchoring and the physiological function of MsmE in S. mutans. Conclusion:, These results demonstrate that MsmE is required for melibiose metabolism in S. mutans and that modification by Lgt and LspA are important processes for the physiological function of MsmE. [source]

Involvement of trichothecenes in fusarioses of wheat, barley and maize evaluated by gene disruption of the trichodiene synthase (Tri5) gene in three field isolates of different chemotype and virulence

MOLECULAR PLANT PATHOLOGY, Issue 6 2006
FRANK J. MAIER
SUMMARY Fusarium graminearum is the main causative agent of Fusarium head blight on small grain cereals and of ear rot on maize. The disease leads to dramatic yield losses and to an accumulation of mycotoxins. The most dominant F. graminearum mycotoxins are the trichothecenes, with deoxynivalenol and nivalenol being the most prevalent derivatives. To investigate the involvement of trichothecenes in the virulence of the pathogen, the gene coding for the initial enzyme of the trichothecene pathway was disrupted in three field isolates, differing in chemotype and in virulence. From each isolate three individual disruption mutants were tested for their virulence on wheat, barley and maize. Despite the different initial virulence of the three wild-type progenitor strains on wheat, all disruption mutants caused disease symptoms on the inoculated spikelet, but the symptoms did not spread into other spikelets. On barley, the trichothecene deficient mutants showed no significant difference compared to the wild-type strains: all were equally aggressive. On maize, mutants derived from the NIV-producing strain caused less disease than their wild-type progenitor strain, while mutants derived from DON-producing strains caused the same level of disease as their progenitor strains. These data demonstrate that trichothecenes influence the virulence of F. graminearum in a highly complex manner, which is strongly host as well as moderately chemotype specific. [source]