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1.
Leukemia ; 27(12): 2301-10, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23508117

ABSTRACT

Fms-like tyrosine kinase-3 is a commonly mutated gene in acute myeloid leukemia, with about one-third of patients carrying an internal-tandem duplication of the juxtamembrane domain in the receptor (FLT3-ITD). FLT3-ITD exhibits altered signaling quality, including aberrant activation of STAT5. To identify genes affecting FLT3-ITD-mediated STAT5 signaling, we performed an esiRNA-based RNAi screen utilizing a STAT5-driven reporter assay. Knockdowns that caused reduced FLT3-ITD-mediated STAT5 signaling were enriched for genes encoding proteins involved in protein secretion and intracellular protein transport, indicating that modulation of protein transport processes could potentially be used to reduce constitutive STAT5 signaling in FLT3-ITD-positive cells. The relevance of KDELR1, a component involved in the Golgi-ER retrograde transport, was further analyzed. In FLT3-ITD-expressing leukemic MV4-11 cells, downregulation of KDELR1 resulted in reduced STAT5 activation, proliferation and colony-forming capacity. Stable shRNA-mediated depletion of KDELR1 in FLT3-ITD-expressing 32D cells likewise resulted in reduced STAT5 signaling and cell proliferation. Importantly, these cells also showed a reduced capacity to generate a leukemia-like disease in syngeneic C3H/HeJ mice. Together our data suggest intracellular protein transport as a potential target for FLT3-ITD driven leukemias, with KDELR1 emerging as a positive modulator of oncogenic FLT3-ITD activity.


Subject(s)
Genome , Proteins/physiology , RNA Interference , Signal Transduction/physiology , fms-Like Tyrosine Kinase 3/metabolism , Animals , Base Sequence , DNA Primers , HEK293 Cells , Humans , Male , Mice , Mice, Inbred C3H , Real-Time Polymerase Chain Reaction , STAT5 Transcription Factor/metabolism
2.
Biofouling ; 28(3): 351-62, 2012.
Article in English | MEDLINE | ID: mdl-22509741

ABSTRACT

Control or removal of undesired biofilms has frequently been found to be quite difficult. In addition to biocidal or antibiotic chemicals or materials designed to prevent biofouling, biological control agents appear to be promising. Reports of bacterial predators eradicating biofilms or eliminating pathogens motivate a more systematic screening of biofilm-eliminating bacterial predators. Unfortunately, the analysis of the eradication process is demanding. In the present study, chip-calorimetry was applied to monitor the elimination of Pseudomonas sp. biofilms by Bdellovibrio bacteriovorus. The method uses metabolic heat as a real-time parameter for biofilm activity. The method is non-invasive, fast and convenient due to real-time data acquisition. In addition, heat-production data can reveal information about the energetics of the predator-prey interaction. The calorimetric results were validated by confocal laser scanning microscopy. The approach described may be useful for the screening of biofilm susceptibility to different predators.


Subject(s)
Bdellovibrio/physiology , Biofilms/growth & development , Calorimetry/methods , Pseudomonas/growth & development , Antibiosis , Bdellovibrio/growth & development , Bdellovibrio/metabolism , Calorimetry/instrumentation , Colony Count, Microbial , Microscopy, Confocal , Pseudomonas/metabolism
3.
J Microbiol Methods ; 82(1): 42-8, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20385177

ABSTRACT

The environmental fate and, in particular, biodegradation rates of hydrophobic organic compounds (HOC) are of high interest due to the ubiquity, persistence, and potential health effects of these compounds. HOC tend to interact with bioreactor materials and sampling devices and are frequently volatile, so that conventionally derived degradation parameters are often biased. We report on the development and validation of a novel calorimetric approach that serves to gain real time information on the kinetics and the physiology of HOC bioconversion in aqueous systems while overcoming weaknesses of conventional biodegradation experiments. Soil bacteria Mycobacterium frederiksbergense LB501T, Rhodococcus erythropolis K2-3 and Pseudomonas putida G7 were exposed to pulsed titrations of dissolved anthracene, 4-(2,4-dichlorophenoxy)butyric acid or naphthalene, respectively, and the thermal responses were monitored. The combinations of strains and pollutants were selected as examples for complete and partial biodegradation and complete degradation with storage product formation, respectively. Heat production signals were interpreted thermodynamically and in terms of Michaelis-Menten kinetics. The half-saturation constant k(D) and the degradation rate r(D)(Max) were derived. Comparison with conventional methods shows the suitability to extract kinetic degradation parameters of organic trace pollutants from simple ITC experiments, while thermodynamic interpretation provided further information about the metabolic fate of HOC compounds.


Subject(s)
Bacteria/chemistry , Bacteria/metabolism , Calorimetry/methods , Soil Microbiology , Water Pollutants, Chemical/metabolism , 2,4-Dichlorophenoxyacetic Acid/analogs & derivatives , 2,4-Dichlorophenoxyacetic Acid/chemistry , 2,4-Dichlorophenoxyacetic Acid/metabolism , Anthracenes/chemistry , Anthracenes/metabolism , Bacteria/isolation & purification , Biodegradation, Environmental , Kinetics , Naphthalenes/chemistry , Naphthalenes/metabolism , Water Pollutants, Chemical/chemistry
4.
Antimicrob Agents Chemother ; 54(1): 312-9, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19822705

ABSTRACT

Chip calorimetry is introduced as a new monitoring tool that provides real-time information about the physiological state of biofilms. Its potential for use for the study of the effects of antibiotics and other biocides was tested. Established Pseudomonas putida biofilms were exposed to substances known to cause toxicity by different mechanisms and to provoke different responses of defense and resistance. The effects of these compounds on heat production rates were monitored and compared with the effects of these compounds on the numbers of CFU and intracellular ATP contents. The real-time monitoring potential of chip calorimetry was successfully demonstrated by using as examples the fast-acting poisons formaldehyde and 2,4-dinitrophenol (DNP). A dosage of antibiotics initially increased the heat production rate. This was discussed as being the effect of energy-dependent resistance mechanisms (e.g., export and/or transformation of the antibiotic). The subsequent reduction in the heat production rate was attributed to the loss of activity and the death of the biofilm bacteria. The shapes of the death curves were in agreement with the assumed variation in the levels of exposure of cells within the multilayer biofilms. The new monitoring tool provides fast, quantitative, and mechanistic insights into the acute and chronic effects of a compound on biofilm activity while requiring only minute quantities of the biocide.


Subject(s)
Anti-Bacterial Agents/pharmacology , Biofilms/drug effects , Calorimetry/methods , Microbial Sensitivity Tests/methods , 2,4-Dinitrophenol/pharmacology , Adenosine Triphosphate/metabolism , Calorimetry/instrumentation , Ciprofloxacin/pharmacology , Formaldehyde/pharmacology , Kanamycin/pharmacology , Microbial Sensitivity Tests/instrumentation , Microcomputers , Pseudomonas putida/drug effects , Pseudomonas putida/growth & development , Tetracycline/pharmacology
5.
J Microbiol Methods ; 74(2-3): 74-81, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18502524

ABSTRACT

The partial dissipation of Gibbs energy as heat reflects the metabolic dynamic of biofilms in real time and may also allow quantitative conclusions about the chemical composition of the biofilm via Hess' law. Presently, the potential information content of heat is hardly exploited due to the low flexibility, the low throughput and the high price of conventional calorimeters. In order to overcome the limitations of conventional calorimetry a miniaturized calorimeter for biofilm investigations has been evaluated. Using four thermopiles a heat production with spatial and temporal resolutions of 2.5 cm(-1) and 2 s(-1) could be determined. The limit of detection of the heat flow measurement was 20 nW, which corresponds to the cell density of an early stage biofilm (approx. 3x10(5) cells cm(-2)). By separating biofilm cultivation from the actual heat measurement, a high flexibility and a much higher throughput was achieved if compared with conventional calorimeters. The approach suggested allows cultivation of biofilms in places of interest such as technological settings as well as in nature followed by highly efficient measurements in the laboratory. Functionality of the miniaturized calorimeter was supported by parallel measurements with confocal laser scanning microscopy and a fiber optic based oxygen sensor using the oxycaloric equivalent (-460 kJ mol-O2(-1)).


Subject(s)
Biofilms , Calorimetry/methods , Pseudomonas putida/physiology , Biosensing Techniques , Microscopy, Confocal , Oxygen/analysis , Sensitivity and Specificity , Time Factors
6.
Oncogene ; 25(6): 929-39, 2006 Feb 09.
Article in English | MEDLINE | ID: mdl-16247465

ABSTRACT

AML1 (RUNX1) encodes a DNA-binding subunit of the CBF transcription factor family and is required for the establishment of definitive hematopoiesis. AML1 is one of the most frequently mutated genes associated with human acute leukemia, suggesting that genetic alterations of the gene contribute to leukemogenesis. Here, we report the analysis of mice carrying conditional AML1 knockout alleles that were inactivated using the Cre/loxP system. AML1 was deleted in adult mice by inducing Cre activity to replicate AML1 deletions found in human MDS, familial platelet disorder and rare de novo human AML. At a latency of 2 months after induction, the thymus was reduced in size and frequently populated by immature double negative thymocytes, indicating defective T-lymphocyte maturation, resulting in lymphatic diseases with 50% penetrance, including atypical hyperplasia and thymic lymphoma. Metastatic lymphomas to the liver and the meninges were observed. Mice also developed splenomegaly with an expansion of the myeloid compartment. Increased Howell-Jolly body counts indicated splenic hypofunction. Thrombocytopenia occurred due to immaturity of mini-megakaryocytes in the bone marrow. Together with mild lymphocytopenia in the peripheral blood and increased fractions of immature cells in the bone marrow, AML1 deficient mice display features of a myelodysplastic syndrome, suggesting a preleukemic state.


Subject(s)
Core Binding Factor Alpha 2 Subunit/genetics , Gene Deletion , Lymphoma/genetics , Splenomegaly/genetics , Animals , Bone Marrow Cells/pathology , Core Binding Factor Alpha 2 Subunit/metabolism , Exons , Genetic Engineering/methods , Lymphoma/pathology , Mice , Mice, Transgenic , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Poly I-C/pharmacology , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , Thrombocytopenia/genetics , Thrombocytopenia/pathology , Thymus Gland/pathology
7.
Apoptosis ; 10(5): 1163-74, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16151649

ABSTRACT

Erucylphosphocholine (ErPC) exerts strong anticancer activity in vivo and in vitroand induces apoptosis even in chemoresistant glioma cell lines. We investigated the contribution of Apaf-1 and caspase-3 to the apoptotic response to ErPC using RNA interference (RNAi) in human glioblastoma cells. We could demonstrate that human glioma cell lines are susceptible to RNAi. Apaf-1 and caspase-3 are amenable to specific small interfering RNA (siRNA)-induced degradation resulting in a reduction of protein levels to 8-33% (Apaf-1) and to 30-50% (caspase-3). Transfection of siRNA directed to Apaf-1 and caspase-3 specifically reduced caspase-3 processing induced by ErPC treatment and yielded a reduction in cells that undergo ErPC-induced apoptosis to 17-33% (Apaf-1) and to 38-50% (caspase-3). The caspase-3 siRNA experiments were corroborated in caspase-3-deficient and -reconstituted MCF-7 breast cancer cells. Survival assays and morphological observations revealed that caspase-3 reconstitution significantly sensitized MCF-7 cells to ErPC. Exploring the caspase cascade responsible for ErPC-induced apoptosis MCF-7 cells provided evidence that caspase-3 is required for the activation of caspases-2, -6 and -8 and also participates in a feedback amplification loop. Our results provide evidence that Apaf-1 and caspase-3 are major determinants of ErPC-induced apoptosis and the possible use of ErPC in a clinical setting is discussed.


Subject(s)
Apoptosis/drug effects , Caspases/biosynthesis , Down-Regulation/physiology , Glioblastoma/physiopathology , Intracellular Signaling Peptides and Proteins/metabolism , Phosphorylcholine/analogs & derivatives , Proteins/metabolism , RNA Interference , Apoptosis/physiology , Apoptotic Protease-Activating Factor 1 , BH3 Interacting Domain Death Agonist Protein/metabolism , Breast Neoplasms/physiopathology , Caspase 3 , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Phosphorylcholine/pharmacology , Proto-Oncogene Proteins c-bcl-2/physiology
8.
Comp Biochem Physiol B Biochem Mol Biol ; 135(4): 707-19, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12892763

ABSTRACT

Energy metabolism in early life stages of the shrimp Farfantepenaeus paulensis subjected to temperature reduction (26 and 20 degrees C) was determined using the activities of citrate synthase (CS) and pyruvate kinase (PK). At both temperatures, weight-specific activity of CS decreased throughout the ontogenetic development from protozoea II (PZ II) to postlarva XII-XIV (PL XII-XIV). PK activity reached a pronounced peak in PL V-VI, followed by a further decrease in PL XII-XIV. Temperature reduction produced variation in oxygen consumption rates (QO(2)), ammonia-N excretion and in enzyme activities. Ammonia-N excretion was higher at 20 degrees C in mysis III (M III), PL V-VI and PL XII-XIV, resulting in substantially lower O:N ratios in these stages. QO(2) was increased in protozoea II (PZ II) and mysis I (M I) at 26 degrees C, while no difference in QO(2) was detected in the subsequent stages at either temperature. This fact coincided with higher CS and PK activities in M III, PL V-VI and PL XII-XIV at 20 degrees C compared with 26 degrees C. Regressions between individual enzyme activities and dry weight exhibited slope values of 0.85-0.92 for CS and 1.1-1.2 for PK and temperature reduction was reflected by higher slope values at 20 than at 26 degrees C for both enzymes. Weight-specific CS activity was positively correlated with QO(2) at 20 and 26 degrees C, and may thus be used as an indicator of aerobic metabolic rate throughout the early stages of F. paulensis. The variation in enzyme activities is discussed in relation to possible metabolic adaptations during specific ontogenetic events of the F. paulensis life cycle. Here, the catalytic efficiency of energy-metabolism enzymes was reflected in ontogenetic shifts in behaviour such as larval settlement and the adoption of a benthic existence in early postlarvae. In most cases, enhanced enzyme activities appeared to counteract negative effects of reduced temperature.


Subject(s)
Citrate (si)-Synthase/metabolism , Penaeidae/embryology , Penaeidae/enzymology , Pyruvate Kinase/metabolism , Ammonia/metabolism , Animals , Energy Metabolism/physiology , Larva/physiology , Nitrogen/metabolism , Oxygen/metabolism , Regression Analysis , Temperature
9.
Genesis ; 32(1): 19-26, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11835670

ABSTRACT

By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.


Subject(s)
Cloning, Molecular/methods , Codon , Integrases/genetics , Viral Proteins/genetics , Animals , Base Sequence , Genetic Code , Mice , Mice, Transgenic , Molecular Sequence Data , Stem Cells
10.
Nat Biotechnol ; 19(11): 1047-52, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11689850

ABSTRACT

Directed molecular evolution was applied to generate Cre recombinase variants that recognize a new DNA target sequence. Cre was adapted in a three-stage strategy to evolve recombinases to specifically recombine the new site. This complex multicycle task was made feasible by an improved directed-evolution procedure that relies on placing the recombination substrate next to the recombinase coding region. Consequently, those DNA molecules carrying the coding region for a successful recombinase are physically marked by the action of that recombinase on the linked substrate and are easily retrieved from a large background of unsuccessful candidates by PCR amplification. We term this procedure substrate-linked protein evolution (SLiPE). The method should facilitate the development of new recombinases and other DNA-modifying enzymes for applications in genetic engineering, functional genomics, and gene therapy.


Subject(s)
Directed Molecular Evolution/methods , Integrases/genetics , Integrases/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , 3T3 Cells , Animals , Base Sequence , Binding Sites , CHO Cells , Cricetinae , DNA Mutational Analysis , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Humans , Integrases/chemistry , Mice , Models, Molecular , Mutagenesis, Site-Directed , Protein Engineering/methods , Protein Structure, Secondary , Recombination, Genetic , Substrate Specificity , Viral Proteins/chemistry
11.
Biotechniques ; 31(4): 906-8, 910, 912, 914, 916, 918, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11680722

ABSTRACT

We have developed a novel way to use the Cre/loxP system for in vitro manipulation of DNA and a technique to clone DNA into circular episomes. The method is fast, reliable, and allowsflexible cloning of DNA fragments into episomes containing a loxP site. We show that a loxP site can serve as a universal target site to clone a DNA fragment digested with any restriction enzyme(s). This technique abolishes the need for compatible restriction sites in cloning vectors and targets by generating custom-designed 5' 3', or blunt ends in the desired orientation and reading frame in the vector Therefore, this method eliminates the limitations encountered when DNA fragments are cloned into vectors with a confined number of cloning sites. The 34-bp loxP sequence assures uniqueness, even when large episomes are manipulated. We present three examples, including the manipulation of a bacterial artificial chromosome. Because DNA manipulation takes place at a loxP site, we refer to this technique as loxP-directed cloning.


Subject(s)
Cloning, Molecular/methods , Integrases , Viral Proteins , Amino Acid Sequence , Base Sequence , Binding Sites , Biotechnology , Chromosomes, Artificial, Bacterial/genetics , DNA Primers/genetics , DNA Restriction Enzymes , Genetic Vectors , HeLa Cells , Humans , Molecular Sequence Data , Plasmids/genetics , Recombinant Fusion Proteins/genetics
12.
Genes Dev ; 14(15): 1971-82, 2000 Aug 01.
Article in English | MEDLINE | ID: mdl-10921910

ABSTRACT

The initial steps of double-stranded break (DSB) repair by homologous recombination mediated by the 5'-3' exonuclease/annealing protein pairs, RecE/RecT and Redalpha/Redbeta, were analyzed. Recombination was RecA-independent and required the expression of both components of an orthologous pair, even when the need for exonuclease activity was removed by use of preresected substrates. The required orthologous function correlated with a specific protein-protein interaction, and recombination was favored by overexpression of the annealing protein with respect to the exonuclease. The need for both components of an orthologous pair was observed regardless of whether recombination proceeded via a single-strand annealing or a putative strand invasion mechanism. The DSB repair reactions studied here are reminiscent of the RecBCD/RecA reaction and suggest a general mechanism that is likely to be relevant to other systems, including RAD52 mediated recombination.


Subject(s)
Bacterial Proteins/metabolism , DNA Repair/physiology , DNA-Binding Proteins , Escherichia coli Proteins , Exodeoxyribonucleases/metabolism , Recombination, Genetic , Bacterial Proteins/genetics , Base Sequence , DNA/genetics , DNA/metabolism , Exodeoxyribonucleases/genetics , Molecular Sequence Data , Rec A Recombinases/genetics , Rec A Recombinases/metabolism
14.
Article in English | MEDLINE | ID: mdl-10840643

ABSTRACT

The two species of isopods, Idotea baltica (Pallas) and Idotea emarginata (Fabricius), co-occur frequently near Helgoland, North Sea, occupying different ecological niches. Respiration rates and kinetic properties of citrate synthase (CS) were compared in these species in order to identify possible mechanisms of temperature adaptation. Specimens were acclimated to 5 and 15 degrees C prior to further investigations. Respiration rates were measured under normoxic conditions at 5, 10 and 15 degrees C. CS was partly purified chromatographically and influences of temperature, pH, substrate saturation and ATP-concentration on enzyme activity were examined. In both species, rising temperatures led to linearly increasing oxygen consumption, with estimated Q10 values between 3.2 and 4.2. Only I. baltica showed an effect of short term acclimation: warm adapted animals had always higher respiration rates than cold adapted ones. In I. emarginata, the acclimation temperature had no effect on oxygen consumption. Furthermore, its CS slightly indicates higher affinity to oxaloacetic acid when specimens were adapted to 15 degrees C compared to those maintained at 5 degrees C. Any effect of the experimental temperature on CS in I. baltica was negligible. The results are discussed in view of the different habitats occupied by the species compared.


Subject(s)
Citrate (si)-Synthase/metabolism , Crustacea/physiology , Animals , Energy Metabolism , Enzyme Activation , Kinetics , Oxygen Consumption , Species Specificity , Temperature
15.
EMBO Rep ; 1(2): 133-9, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11265752

ABSTRACT

Transgenic mice have been used to explore the role of chromosomal translocations in the genesis of tumors. But none of these efforts has actually involved induction of a translocation in vivo. Here we report the use of Cre recombinase to replicate in vivo the t(8;21) translocation found in human acute myeloid leukemia (AML). As in the human tumors, the murine translocation fuses the genes AML1 and ETO. We used homologous recombination to place loxP sites at loci that were syntenic with the break points for the human translocation. Cre activity was provided in mice by a transgene under the control of the Nestin promoter, or in cultured B cells by infecting with a retroviral vector encoding Cre. In both instances, Cre activity mediated interchromosomal translocations that fused the AML1 and ETO genes. Thus, reciprocal chromosomal translocations that closely resemble rearrangements found in human cancers can be achieved in mice.


Subject(s)
DNA-Binding Proteins/genetics , Integrases/metabolism , Nerve Tissue Proteins , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Proteins , Transcription Factors/genetics , Translocation, Genetic/genetics , Viral Proteins , Animals , B-Lymphocytes/physiology , Base Sequence , Cells, Cultured , Core Binding Factor Alpha 2 Subunit , Disease Models, Animal , Genes, Reporter/genetics , Genetic Engineering , Humans , Integrases/genetics , Intermediate Filament Proteins/genetics , Leukemia, Myeloid, Acute/genetics , Mice , Mice, Transgenic , Molecular Sequence Data , Nestin , Oncogene Proteins, Fusion/metabolism , Polymerase Chain Reaction , RUNX1 Translocation Partner 1 Protein , Recombinant Fusion Proteins/metabolism , Retroviridae/genetics , Retroviridae/metabolism , Transcription Factors/metabolism , Transgenes/genetics
16.
Article in English | MEDLINE | ID: mdl-11154947

ABSTRACT

The specific activity and the kinetic properties of partly purified pyruvate kinase (PK) (EC 2.7.1.40) from the Northern Krill, Meganyctiphanes norvegica, were investigated in relation to varying food resources. In order to evaluate the effect of starvation on the total energy metabolism, the respiration rates of fed and unfed krill were determined. The FPLC-elution profile of PK displayed two distinct peaks - PK I and II. The first isoform represented 80% of the total PK activity in the organism, and 20% was contributed by the second isoform. PK I was inhibited by ATP but was not influenced by fructose-1,6-bisphosphate (FBP). In contrast, PK II showed ATP inhibition and up to 2.5-fold increased activity by addition of 17 micromol.l(-1) FBP. The Michaelis-Menten constants of both isoforms were 2-10-fold higher for ADP than for phosphoenolpyruvate (PEP). Alanine showed no regulatory effect on PK I and II. In specimens starved for 7 days oxygen consumption decreased by 20%. Neither the feeding experiments nor the animals captured in the field during low and high productive seasons indicate that PK properties of M. norvegica are modified in relation to food supply. Accordingly, alternative mechanisms are involved in the depression of the metabolic rate in terms of oxygen consumption.


Subject(s)
Crustacea/physiology , Oxygen Consumption , Pyruvate Kinase/metabolism , Animal Nutritional Physiological Phenomena , Animals , Chromatography, Gel , Chromatography, Ion Exchange , Crustacea/enzymology , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Kinetics , Pyruvate Kinase/isolation & purification , Seasons
17.
Comp Biochem Physiol B Biochem Mol Biol ; 120(4): 743-51, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9854820

ABSTRACT

Two forms of the chitinolytic enzyme N-acetyl-beta-D-glucosaminidase (NAGase, EC 3.2.1.52) have been isolated from the Antarctic krill, Euphausia superba, in order to study their potential role in temperature adaptation processes. A chromatographic protocol was developed that allowed complete separation of the two enzyme forms, named NAGase B and NAGase C. The latter was purified to homogeneity with 600-fold enrichment and a yield of 17%. The molecular mass was 150 kDa. NAGase B showed characteristics of a glycoprotein due to affinity towards concanavalin A sepharose, while NAGase C did not. Highly specific polyclonal antibodies to NAGase C [anti-(E. superba-NAGase C)-IgG] showed only negligible cross-reactivity with NAGase B isoforms. A comparison with the Northern krill, Meganyctiphanes norvegica, revealed a corresponding chromatographic pattern with two main activity peaks, for differentiation named NAGase II and NAGase III. Application of the antibody on M. norvegica revealed a high specificity toward NAGase III and a low cross-reactivity with NAGase II. First indication is given that the two forms are no isoenzymes in a strict sense but instead may have different functions in the metabolism of krill.


Subject(s)
Acetylglucosaminidase/isolation & purification , Crustacea/enzymology , Isoenzymes/isolation & purification , Acetylglucosaminidase/chemistry , Acetylglucosaminidase/immunology , Animals , Antibody Formation , Chromatography , Cross Reactions , Electrophoresis, Polyacrylamide Gel , Isoenzymes/chemistry , Isoenzymes/immunology , Molecular Weight , Rabbits , Species Specificity
18.
J Mol Biol ; 284(2): 363-84, 1998 Nov 27.
Article in English | MEDLINE | ID: mdl-9813124

ABSTRACT

The integrase class site specific recombinases FLP from Saccharomyces cerevisiae, and Cre from bacteriophage P1, have been extensively used to direct DNA rearrangements in heterologous organisms. Although their reaction mechanisms have been relatively well characterised, little comparative analysis of the two enzymes has been published. We present a comparative kinetic analysis of FLP and Cre, which identifies important differences. Gel mobility shift assays show that Cre has a higher affinity for its target, loxP (7. 4x10(10) M-1), than FLP for its target, FRT (8.92x10(8) M-1). We show that both recombinases bind the two halves of their target sites cooperatively, and that Cre shows approximately threefold higher cooperativity than FLP. Using a mathematical model describing the sequential binding of recombinase monomers to DNA, we have determined values for the association and dissociation rate constants for FLP and Cre.FLP and Cre also showed different characteristics in in vitro recombination assays. In particular, approximately tenfold more active FLP was required than Cre to optimally recombine a given quantity of excision substrate. FLP was able to reach maximum excision levels approaching 100%, whilst Cre-mediated excision did not exceed 75%. To investigate possible reasons for these differences a mathematical model describing the excision recombination reaction was established. Using measured DNA binding parameters for FLP and Cre in the model, and comparing simulated and experimental recombination data, the values of the remaining unknown parameters were determined. This analysis indicates that the synaptic complex is more stable for Cre than for FLP.


Subject(s)
DNA Nucleotidyltransferases/metabolism , Integrases/metabolism , Recombination, Genetic , Allosteric Regulation , Bacteriophage P1/enzymology , Cell-Free System , Fungal Proteins/metabolism , Kinetics , Models, Theoretical , Protein Binding , Saccharomyces cerevisiae/enzymology , Viral Proteins/metabolism
19.
Nat Genet ; 20(2): 123-8, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9771703

ABSTRACT

A straightforward way to engineer DNA in E. coli using homologous recombination is described. The homologous recombination reaction uses RecE and RecT and is transferable between E. coli strains. Several target molecules were manipulated, including high copy plasmids, a large episome and the E. coli chromosome. Sequential steps of homologous or site-specific recombination were used to demonstrate a new logic for engineering DNA, unlimited by the disposition of restriction endonuclease cleavage sites or the size of the target DNA.


Subject(s)
DNA-Binding Proteins , Escherichia coli Proteins , Escherichia coli/genetics , Genetic Engineering/methods , Recombination, Genetic , Bacterial Proteins/metabolism , DNA, Bacterial/metabolism , Exodeoxyribonucleases/metabolism , Plasmids/metabolism , Point Mutation , Polymerase Chain Reaction
20.
Cancer Lett ; 129(2): 157-63, 1998 Jul 17.
Article in English | MEDLINE | ID: mdl-9719457

ABSTRACT

The newly designed pyridine derivative B9309-068 and a series of structurally different compounds were tested for their ability to modulate rhodamine 123 (RHO) efflux from CD56+ hematopoietic cells in the presence of either 10% fetal calf serum or undiluted human AB serum. Furthermore, efflux modulation was investigated on CD34+ blast populations obtained from four patients with relapsed state AML. Target cells were specified throughout by labeling with peridinine chlorophyll protein (PerCP)-conjugated monoclonal antibodies, allowing clear differentiation from RHO emission spectrum by flow cytometry. In the presence of low serum each compound efficiently modulated RHO efflux without significant differences in the range of final concentrations (1.0-3.0 microM). At 0.1 microM, however, RHO efflux was differentially modulated following the series GF120918 approximately B9309-068 > PSC 833 > DNIG approximately DVER. With CD56+ cells in the presence of undiluted human AB serum at a final modulator concentration of 0.1 microM, all chemosensitizers tested were found to be inefficient. At final concentrations of 0.3 microM or higher, distinct RHO efflux modulation was found with the following efficacies: B9309-068 approximately GF120918 > PSC 833 >> DVER approximately DNIG. The efficacies seen in undiluted human AB serum at 3.0 microM were comparable to those seen on CD56+ cells at final modulator concentrations of 0.1 microM in low serum. Our results identify the pyridine derivative B9309-068 as a promising compound for modulating P-glycoprotein-mediated drug resistance under conditions resembling the clinical setting. Nonetheless, modulation potencies of a series of structurally very different chemosensitizers was revealed to be substantially diminished at high serum concentrations in vitro.


Subject(s)
Blood Proteins/pharmacology , Leukocytes, Mononuclear/metabolism , Morpholines/pharmacology , Pyrimidines/pharmacology , Rhodamines/pharmacokinetics , Tetrahydroisoquinolines , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP-Binding Cassette Transporters/genetics , Acridines/pharmacology , Antigens, CD34/immunology , CD56 Antigen/immunology , Calcium Channel Blockers/pharmacology , Cyclosporins/pharmacology , Dihydropyridines/pharmacology , Dose-Response Relationship, Drug , Flow Cytometry , Fluorescent Dyes/pharmacokinetics , Gene Expression , Humans , Isoquinolines/pharmacology , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/immunology , Multidrug Resistance-Associated Proteins , Rhodamine 123 , Verapamil/pharmacology
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