A new logic for DNA engineering using recombination in Escherichia coli.

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.

Chip-calorimetry provides real time insights into the inactivation of biofilms by predatory bacteria.

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.

Chip calorimetry for fast and reliable evaluation of bactericidal and bacteriostatic treatments of biofilms.

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.

Miniaturized calorimetry - a new method for real-time biofilm activity analysis.

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)).

AML1 deletion in adult mice causes splenomegaly and lymphomas.

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.

Alteration of Cre recombinase site specificity by substrate-linked protein evolution.

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.

Improved properties of FLP recombinase evolved by cycling mutagenesis.

The site-specific recombinases FLP and Cre are useful for genomic engineering in many living systems. Manipulation of their enzymatic properties offers a means to improve their applicability in different host organisms. We chose to manipulate the thermolability of FLP recombinase. A lacZ-based recombination assay in Escherichia coli was used for selection in a protein evolution strategy that relied on error-prone PCR and DNA shuffling. Improved FLP recombinases were identified through cycles of increasing stringency imposed by both raising temperature and reducing protein expression, combined with repetitive cycles of screening at the same stringency to enrich for clones with improved fitness. An eighth generation clone (termed FLPe) showed improved properties in E. coli, in vitro, in human 293- and mouse ES-cells.

Comparative kinetic analysis of FLP and cre recombinases: mathematical models for DNA binding and recombination.

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.

LoxP-directed cloning: use of Cre recombinase as a universal restriction enzyme.

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.

Rhodamine 123 efflux modulation in the presence of low or high serum from CD56+ hematopoietic cells or CD34+ leukemic blasts by B9309-068, a newly designed pyridine derivative.

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.

Effects of temperature on the respiration rates and the kinetics of citrate synthase in two species of Idotea (Isopoda, Crustacea).

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.

Isoforms of an N-acetyl-beta-D-glucosaminidase from the Antarctic krill, Euphausia superba: purification and antibody production.

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.

Citrate synthase and pyruvate kinase activities during early life stages of the shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae): effects of development and temperature.

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.

Histopathology of tubal pregnancy.

The histopathological examination of several sections of tubal ectopic pregnancies shows that the growth of the trophoblast usually takes place in an extraluminal subperitoneal site and dilation of the tube is caused by coagulated blood and not by the trophoblast. For conservative surgery of the tube it is necessary to keep in mind the histopathological findings of tubal pregnancies.

CIN and preclinical cervical carcinoma. A study of morbidity trends over a 10-year period.

Between 1971 and 1980 1312 cone biopsies were performed at the Department of Gynecology and Obstetrics, Kiel. Comparing the two intervals from 1971 to 1975 and 1976 to 1980 the number of cases with dysplasia and Carcinoma in situ significantly rose in the last 5 years in patients 16-30 years of age. Amongst other reasons for this increase, on the basis of our investigation, the regular gynecological examination in combination with the prescription of the ovulation inhibitors seems to be responsible. The average age of patients (36-44 years) undergoing cone biopsy for the first time decreased in the period between 1971 and 1980 (approx. 8 years). The excision of carcinoma in situ in women under 30 years of age mostly showed margins free of pathological epithelium. With increasing age the cases of not free margins increased to 70%. For the hemostasis of the conization crater up to 1977, the conventional suture technique was applied, since 1977 cone coagulation probe according to Semm was used. In this group (1977-1980) treated with coagulation after cone biopsy, 45% of the cases with carcinoma in situ not free margins had histologically normal epithelium of the extirpated uterus. In patients with the desire for a child or in pregnant women a secondary coagulation of the wound crater is achieved to preserve the uterus.

White sponge nevus of the vulva.

The white sponge nevus is a hereditary leukokerotosis localized preferably in the oral mucosa, but may simultaneously manifest itself in other regions, e.g. perianally. We report the case of a 18-year-old patient with a primarily and exclusively extraorally located white sponge nevus of the vulva. The differential diagnosis and clinical as well as therapeutical problems of this extremely rare disease are discussed.

Lymphoreticular cells in invasive carcinoma of the uterine cervix: an immunohistological study.

In the present study the immunohistology of the cellular stromal reaction of invasive squamous cell carcinoma of the cervix is investigated. Tumor tissue from 10 patients with invasive squamous cell carcinoma of the uterine cervix (stages Ib-IIb, according to FIGO) was immunostained by the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method. The monoclonal antibodies OKT3, OKT4, OKT8, TO15, Ki-M1, and Ki-M6 were applied. The cells in the stroma and the tumor foci were evaluated separately. In all cases, the overwhelming majority of lymphoreticular cells were found in the stroma and the tumor-cell complexes contained relatively low numbers of these cells. While B-lymphocytes were present only in low numbers or were virtually absent from the lymphoreticular infiltrates, cells of the mononuclear-phagocyte system were found to be another prominent constituent of the tumor's cellular stromal reaction.

Macrophage- and lymphocyte-subtypes in the endometrium during different phases of the ovarian cycle.

The presence of immunocompetetive cells in the endometrium during the proliferative and secretory phase of the ovarian cycle is demonstrated on the light and electron microscopic level using monoclonal antibodies (MoAb). Subtypes of monocytes, macrophages and T-lymphocytes appear during the different phases in variable extent and different localization. Some subpopulations of the monocyte/macrophage system and T-helper lymphocytes increase in number on day 21/22. Our observations indicate that cells with bone marrow origin take part in functional events of the endometrium during the ovarian cycle.

A genome-wide RNAi screen identifies proteins modulating aberrant FLT3-ITD signaling.

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.