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121.
Ronald Lundquist Purita Manlapaz-Fernandez Baldomero M. Olivera 《Journal of molecular biology》1974,83(4):541-543
Increased levels of dCTP increase the frequency of initiation of discontinuous DNA intermediates in a cellophane disc in vitro system. Increased levels of other ribo- and deoxyribonucleoside triphosphates show no effect. 相似文献
122.
Conotoxins 总被引:6,自引:0,他引:6
B M Olivera J Rivier J K Scott D R Hillyard L J Cruz 《The Journal of biological chemistry》1991,266(33):22067-22070
123.
Conotoxin GI: disulfide bridges, synthesis, and preparation of iodinated derivatives 总被引:3,自引:0,他引:3
W R Gray F A Luque R Galyean E Atherton R C Sheppard B L Stone A Reyes J Alford M McIntosh B M Olivera 《Biochemistry》1984,23(12):2796-2802
The 13 amino acid toxic peptide from the marine snail Conus geographus, conotoxin GI, blocks the acetylcholine receptor at the neuromuscular junction. In this report, we describe a method for analyzing disulfide bonding in nanomole amounts of small cystine-rich peptides. The procedure involves partial reduction and a double-label alkylation of cysteine residues. Using this method, we show that the natural conotoxin GI has a (2-7, 3-13) disulfide configuration. The structure of conotoxin GI has been confirmed by chemical synthesis. The preparation and purification of molecularly homogeneous, iodinated derivatives of this toxin are also described. All derivatives, including the [diiodohistidine,diiodotyrosine]conotoxin GI, retained at least half of the biological activity of unmodified toxin. Since the tetraiodinated toxin, which is greater than 25% by weight iodine, retains considerable toxicity, unmodified histidine and tyrosine residues in conotoxin GI are not crucial for biological activity. 相似文献
124.
We have located the nadD locus between lip and leuS at 14 min on the Salmonella typhimurium chromosome, and we have shown it to be the structural gene for nicotinic acid mononucleotide adenylyltransferase. This is the first indispensable gene of pyridine nucleotide metabolism that has been identified. Mutants altered at this locus, isolated by their 6-aminonicotinamide resistance phenotype, accumulate abnormally large pools of nicotinic acid mononucleotide in vivo; many exhibit a temperature-sensitive lethal phenotype. Enzyme assays reveal markedly lower transferase activity in mutant extracts than in nadD+ extracts. The partial dominance of nadD mutants when placed in a nadD+/nadD diploid suggests that nicotinic acid mononucleotide adenylyltransferase is a multimeric enzyme. 相似文献
125.
126.
Sandoval A Arias-Barrau E Arcos M Naharro G Olivera ER Luengo JM 《Environmental microbiology》2007,9(3):737-751
Functional analyses of the different proteins involved in the synthesis and accumulation of polyhydroxyalkanoates (PHAs) in P. putida U were performed using a mutant in which the pha locus had been deleted (PpUDeltapha). These studies showed that: (i) Pha enzymes cannot be replaced by other proteins in this bacterium, (ii) the transformation of PpDeltapha with a plasmid containing the locus pha fully restores the synthesis of bioplastics, (iii) the transformation of PpDeltapha with a plasmid harbouring the gene encoding the polymerase PhaC1 (pMCphaC1) permits the synthesis of polyesters (even in absence of phaC2ZDFI); however, in this strain (PpUDeltapha-pMCphaC1) the number of PHAs granules was higher than in the wild type, (iv) the expression of phaF in PpUDeltapha-pMCphaC1 restores the original phenotype, showing that PhaF is involved in the coalescence of the PHAs granules. Furthermore, the deletion of the phaDFI genes in P. putida U considerably decreases (> 70%) the biosynthesis of PHAs consisting of hydroxyalkanoates with aliphatic constituents, and completely prevents the synthesis of those ones containing aromatic monomers. Additional experiments revealed that the deletion of phaD in P. putida U strongly reduces the synthesis of PHA, this effect being restored by PhaF. Moreover, the overexpression of phaF in P. putida U, or in its DeltafadBA mutant, led to the collection of PHA over-producer strains. 相似文献
127.
Jun Zou Shao-Chun Chang Jasna Marjanovic Philip W. Majerus 《The Journal of biological chemistry》2009,284(4):2064-2071
Myotubularin-related protein 6 (MTMR6) is a catalytically active member of
the myotubularin (MTM) family, which is composed of 14 proteins. Catalytically
active myotubularins possess 3-phosphatase activity dephosphorylating
phosphatidylinositol-3-phoshate and phosphatidylinositol-3,5-bisphosphate, and
some members have been shown to form homomers or heteromeric complexes with
catalytically inactive myotubularins. We demonstrate that human MTMR6 forms a
heteromer with an enzymatically inactive member myotubularin-related protein 9
(MTMR9), both in vitro and in cells. MTMR9 increased the binding of
MTMR6 to phospholipids without changing the lipid binding profile. MTMR9
increased the 3-phosphatase activity of MTMR6 up to 6-fold. We determined that
MTMR6 is activated up to 28-fold in the presence of phosphatidylserine
liposomes. Together, MTMR6 activity in the presence of MTMR9 and assayed in
phosphatidylserine liposomes increased 84-fold. Moreover, the formation of
this heteromer in cells resulted in increased protein levels of both MTMR6 and
MTMR9, probably due to the inhibition of degradation of both proteins.
Furthermore, co-expression of MTMR6 and MTMR9 decreased etoposide-induced
apoptosis, whereas decreasing both MTMR6 and MTMR9 by RNA interference led to
increased cell death in response to etoposide treatment when compared with
that seen with RNA interference of MTMR6 alone. Thus, MTMR9 greatly enhances
the functions of MTMR6.Myotubularin proteins are a family of 14 proteins with the canonical dual
specificity protein tyrosine phosphatase active site CX5R
motif
(1–3).
Eight members of the myotubularin family possess catalytic activity,
dephosphorylating phosphatidylinositol 3-phosphate
(PtdIns-3-P)4 and
phosphatidylinositol 3,5-bisphosphate (PtdIns-3,5-P2) at the D-3
position, and six members are not catalytically active because they lack the
conserved cysteine residue in the protein tyrosine phosphatase motif that is
required for activity. Interest in this group of proteins originated from the
genetic evidence linking myotubularin, the founding member of this family, to
myotubular myopathy, an X-linked disorder characterized by severe hypotonia
and generalized muscle weakness
(4). Subsequently, mutations in
MTMR2 and in its inactive binding partner MTMR13 were linked to a subset of
Charcot-Marie-Tooth disease type 4B, a demyelinating neurodegenerative
disorder (5,
6).Despite near identical substrate specificity, biochemical and genetic
evidence supports the hypothesis that myotubularin proteins are not redundant
and have unique functions within cells
(2,
7–9).
The mechanisms by which loss of function of myotubularin proteins produce
diseases are not known. Current evidence supports the hypothesis that each
myotubularin protein regulates a specific pool of PtdIns-3-P and/or
PtdIns-3,5-P2, which in turn regulates a variety of cellular
functions. Differences in tissue expression and subcellular localization play
a role in the specificity of different myotubularins
(10–15).The functions of myotubularin proteins are altered by the formation of
heteromers between catalytically active and inactive members of the family.
The initial biochemical purification of MTM1 demonstrated the presence of MTM1
homodimers and MTM1-3-phosphatase adapter protein (3PAP) heteromers
(16), which was later
described as MTMR12 (15,
17). MTMR2 was found to form
heteromers with MTMR5 (13) and
MTMR13 (18), and MTMR7 formed
heteromers with MTMR9 (19). In
each case, a catalytically active myotubularin protein interacted with an
inactive protein. Heteromerization generated two important effects: increased
catalytic activity of the active component
(13,
15,
19,
20) and targeting of the
heteromer to specific subcellular locations
(15). Mutations in the
inactive member MTMR13 result in a similar phenotype in patients as the
mutations in its catalytically active binding partner MTMR2, indicating an
indispensable role for the catalytically inactive subunit
(21).Myotubularin proteins can be grouped into subfamilies based on homology.
Closely related MTMR6, MTMR7, and MTMR8 comprise such a subfamily. We have
previously characterized the interaction between mouse MTMR7 and MTMR9
proteins (19). In this report,
we characterize the interaction between human MTMR6 and MTMR9. MTMR6 and MTMR9
have been shown to form a heteromeric complex in mouse and Caenorhabditis
elegans (19,
22). MTMR6 has been shown to
inhibit the activity of a calcium-activated potassium channel (type KCa3.1)
(23,
24). Two screening experiments
implicate MTMR6 as a regulator of apoptosis. By RNA microarray analysis,
increased MTMR6 expression was observed in B cell chronic lymphoid leukemia
cells with increased resistance to irradiation-induced apoptosis
(25), whereas in an RNA
interference screen in HeLa cells, decreased MTMR6 expression promoted
apoptosis (26).Here we show that MTMR6 interacts with MTMR9 in vitro and in human
cells. This interaction increases the phospholipid binding and enzymatic
activity of MTMR6 in vitro. Co-expression of either subunit in cells
dramatically increased the protein levels of the individual binding partners,
suggesting that heteromer formation increases the stability of the proteins.
Finally, MTMR9 was found to potentiate the effects of MTMR6 on apoptosis. 相似文献
128.
Rasić-Marković A Krstić D Vujović Z Jakovljevic V Stanojlović O Hrncić D Djurić D Loncar-Stevanović H 《Molecular and cellular biochemistry》2008,308(1-2):111-116
Alcohol intake is associated with numerous degenerative disorders, and the detrimental effects of alcohol may be due to its
influence on plasma membrane and cellular transport systems. The aim of the present study was to compare in vitro and in vivo
effects of ethanol on rabbit erythrocyte ATPase activities and correlate them with ethanol-induced oxidative stress. Age-matched
male rabbits were given 5% ethanol in 2% sucrose solution, for 6 weeks ad libitum; control animals were given tap water. Daily
intake of ethanol was 5 g/kg body weight; this experimental regimen resulted in an average serum ethanol concentration of
16.77 ± 2.00 mM/l. After this period, blood was collected, serum ethanol concentration was determined and erythrocyte membranes
were prepared according to the method of Post et al. Activities of Na+/K+- and Mg2+-ATPases were determined. Thiobarbituric acid-reactive substance (TBARS) assay was used to detect levels of lipid peroxidation,
a major indicator of oxidative stress. In vitro ethanol inhibits both Na+/K+-ATPase and Mg2+-ATPase, but Na+/K+-ATPase is more sensitive to the ethanol-induced inhibition. Increasing concentration of ethanol increased TBARS production,
but significant difference was attained only at 5 and 12.5 mM of ethanol. Chronic ethanol consumption induced significant
increase in Na+/K+- and Mg2+-ATPase activity, and TBARS production. Our results suggest that increased ATPase activity induced by chronic ethanol consumption
is due to oxidative, induced modification of membrane phospholipids and proteins, which are responsible for inhibition of
ATPase activity. Increased production of TBARS induced by in vitro exposure to ethanol is not the only factor that influences
ATPases activity. Further research is needed to elucidate this relationship. 相似文献
129.
Induction of Specific MicroRNAs Inhibits Cutaneous Wound Healing 总被引:1,自引:0,他引:1
I Pastar AA Khan O Stojadinovic EA Lebrun MC Medina H Brem RS Kirsner JJ Jimenez C Leslie M Tomic-Canic 《The Journal of biological chemistry》2012,287(35):29324-29335
Chronic nonhealing wounds, such as venous ulcers (VUs), are a widespread and serious medical problem with high morbidity and mortality. The molecular pathology of VUs remains poorly understood, impeding the development of effective treatment strategies. Using mRNA expression profiling of VUs biopsies and computational analysis, we identified a candidate set of microRNAs with lowered target gene expression. Among these candidates, miR-16, -20a, -21, -106a -130a, and -203 were confirmed to be aberrantly overexpressed in a cohort study of 10 VU patients by quantitative PCR and in situ hybridizations. These microRNAs were predicted to target multiple genes important for wound healing, including early growth response factor 3, vinculin, and leptin receptor (LepR). Overexpression of the top up-regulated miRNAs, miR-21 and miR-130a, in primary human keratinocytes down-regulated expression of the endogenous LepR and early growth response factor 3. The luciferase reporter assay verified LepR as a direct target for miR-21 and miR-130a. Both miR-21 and miR-130a delayed epithelialization in an acute human skin wound model. Furthermore, in vivo overexpression of miR-21 inhibited epithelialization and granulation tissue formation in a rat wound model. Our results identify a novel mechanism in which overexpression of specific set of microRNAs inhibits wound healing, resulting in new potential molecular markers and targets for therapeutic intervention. 相似文献
130.
Peressutti SR Olivera NL Babay PA Costagliola M Alvarez HM 《Journal of applied microbiology》2008,105(2):476-484
Aims: To isolate and identify linear alkylbenzene sulfonate (LAS)-degrading bacteria from Río de la Plata and adjacent waters, and to assay their degradation capability as a consortium and as single organisms.
Methods and Results: A consortium consisting of four bacterial strains: Aeromonas caviae (two strains), Pseudomonas alcaliphila and Vibrio sp. was identified by 16S rRNA analysis. Isolates grown as a consortium produced higher biomass from LAS and CO2 release (mineralization) than individual cultures, and degraded 86% of LAS (20 mg l−1 ), whereas pure strains degraded between 21% and 60%. Bacterial desulfonation from LAS was evidenced in the consortium and A. caviae strains. A complete disappearance of LAS (10 mg l−1 ) was accomplished, and LAS levels of 50 and 100 mg l−1 led to a pronounced decrease in the biodegradation extent and inhibition of culture growth.
Conclusions: A bacterial consortium capable of complete LAS degradation was isolated from the Río de la Plata and adjacent waters. This consortium was more efficient for LAS degradation than individual cultures, and was sensitive to high LAS concentrations.
Significance and Impact of the Study: The autochthonous consortium with high effectiveness on LAS biodegradation is a useful tool for LAS depletion from these polluted ecosystems. 相似文献
Methods and Results: A consortium consisting of four bacterial strains: Aeromonas caviae (two strains), Pseudomonas alcaliphila and Vibrio sp. was identified by 16S rRNA analysis. Isolates grown as a consortium produced higher biomass from LAS and CO
Conclusions: A bacterial consortium capable of complete LAS degradation was isolated from the Río de la Plata and adjacent waters. This consortium was more efficient for LAS degradation than individual cultures, and was sensitive to high LAS concentrations.
Significance and Impact of the Study: The autochthonous consortium with high effectiveness on LAS biodegradation is a useful tool for LAS depletion from these polluted ecosystems. 相似文献