Hybrid Modelling of Transarterial Chemoembolisation Therapies (TACE) for Hepatocellular Carcinoma (HCC).

We extend an agent-based multiscale model of vascular tumour growth and angiogenesis to describe transarterial chemoembolisation (TACE) therapies. The model accounts for tumour and normal cells that are both nested in a vascular system that changes its structure according to tumour-related growth factors. Oxygen promotes nutrients to the tissue and determines cell proliferation or death rates. Within the extended model TACE is included as a two-step process: First, the purely mechanical influence of the embolisation therapy is modelled by a local occlusion of the tumour vasculature. There we distinguish between partial and complete responders, where parts of the vascular system are occluded for the first and the whole tumour vasculature is destroyed for the latter. In the second part of the model, drug eluding beads (DEBs) carrying the chemotherapeutic drug doxorubicin are located at destroyed vascular locations, releasing the drug over a certain time-window. Simulation results are parameterised to qualitatively reproduce clinical observations. Patients that undergo a TACE-treatment are categorised in partial and complete responders one day after the treatment. Another 90 days later reoccurance or complete response are detected by volume perfusion computer tomography (VPCT). Our simulations reveal that directly after a TACE- treatment an unstable tumour state can be observed, where regrowth and total tumour death have the same likeliness. It is argued that this short time-window is favorable for another therapeutical intervention with a less radical therapy. This procedure can shift the outcome to more effectiveness. Simulation results with an oxygen therapy within the unstable time-window demonstrate a potentially positive manipulated outcome. Finally, we conclude that our TACE model can motivate new therapeutical strategies and help clinicians analyse the intertwined relations and cross-links in tumours.

Correlative microscopy approach for biology using X-ray holography, X-ray scanning diffraction and STED microscopy.

We present a correlative microscopy approach for biology based on holographic X-ray imaging, X-ray scanning diffraction, and stimulated emission depletion (STED) microscopy. All modalities are combined into the same synchrotron endstation. In this way, labeled and unlabeled structures in cells are visualized in a complementary manner. We map out the fluorescently labeled actin cytoskeleton in heart tissue cells and superimpose the data with phase maps from X-ray holography. Furthermore, an array of local far-field diffraction patterns is recorded in the regime of small-angle X-ray scattering (scanning SAXS), which can be interpreted in terms of biomolecular shape and spatial correlations of all contributing scattering constituents. We find that principal directions of anisotropic diffraction patterns coincide to a certain degree with the actin fiber directions and that actin stands out in the phase maps from holographic recordings. In situ STED recordings are proposed to formulate models for diffraction data based on co-localization constraints.

Evidence of placental haemorrhage and preterm delivery.

OBJECTIVE: To evaluate evidence of placental haemorrhage (PH) obtained through maternal interviews, patient charts and placental pathology examinations as potential indicators of a 'bleeding pathway' to preterm delivery (PTD). DESIGN: Prospective cohort. SETTING: Fifty-two clinics in five communities in Michigan, USA (1998-2004). POPULATION: A subset (n = 996) of cohort participants with complete placental pathology data. METHODS: First-trimester bleeding and placental abruption were ascertained by mid-trimester interviews and chart review, respectively. Disc-impacting blood clot was defined as a gross placental examination finding of a blood clot impacting adjacent tissue. Microscopic haemorrhage was defined as 'high' (top quintile) scores on an aggregate measure of placental pathology findings suggestive of atypical maternal vessel haemorrhage. These four PH indicators were compared with one another and with risk of PTD assessed by logistic regression analyses. MAIN OUTCOME MEASURES: Preterm delivery and PTD subtypes (i.e. <35 weeks, 35-36 weeks; spontaneous, medically indicated) compared with term deliveries. RESULTS: Placental abruption cases had 2.3-fold to 5.5-fold increased odds of the other three PH indicators. Disc-impacting blood clots and microscopic haemorrhage were associated with one another (odds ratio [OR] = 4.6), but not with first-trimester bleeding. In a multivariable model that included all four PH indicators and confounders, risk of PTD < 35 weeks was elevated with first-trimester bleeding (OR = 1.9 [1.0, 3.4]), placental abruption (OR = 5.2 [1.7, 16.2]), disc-impacting blood clots (OR = 2.3 [1.0, 5.0]) and microscopic haemorrhage (OR = 2.4 [1.4, 4.2]). CONCLUSIONS: Multiple clinical and subclinical PH indicators are associated with PTD, particularly early PTD.

Polymorphisms in thrombophilia and renin-angiotensin system pathways, preterm delivery, and evidence of placental hemorrhage.

OBJECTIVE: The purpose of this study was to analyze functional polymorphisms in candidate genes (methylenetetrahydrofolate reductase [MTHFR]677C>T, MTHFR1298A>C, factor 5 1691G>A [FVL], and angiotensinogen (AGT)-6G>A) in relation to a hypothesized placental hemorrhage pathway to preterm delivery (PTD). STUDY DESIGN: We assessed maternal genotypes, pregnancy outcomes, and placental pathologic evidence among 560 white and 399 black women who were recruited at mid trimester into a prospective cohort study (1998-2004). Odds of dominant genotypes were calculated for PTDs with (n = 56) or without (n = 177) evidence of placental hemorrhage (referent = term) with the use of race-stratified polytomous logistic regression models. RESULTS: Among white women, FVL GA/AA and AGT(-6) GA/AA were both associated with hemorrhage-related PTDs (odds ratio [OR], 4.8; 95% confidence interval [CI], 1.6-14.2 and OR, 3.8; 95% CI, 1.3-10.5, respectively), but not other PTDs (ORs, 1.2 and 0.9, respectively). FVL GA/AA was associated with placental abruption (OR, 5.8; 95% CI, 1.1-30) among white women. All results were null for MTHFR genotypes. CONCLUSION: FVL and AGT variant genotypes were associated specifically with hemorrhage-related PTDs.

Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains.

In biotechnology, the heterologous biosynthesis of isoprenoid compounds in Escherichia coli is a field of great interest and growth. In order to achieve higher isoprenoid yields in heterologous E. coli strains, it is necessary to quantify the pathway intermediates and adjust gene expression. In this study, we developed a precise and sensitive nonradioactive method for the simultaneous quantification of the isoprenoid precursors farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in recombinant and wild-type E. coli cells. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography-mass spectrometry. The integration of a geranylgeranyl diphosphate synthase gene into the E. coli chromosome leads to the accumulation of GGPP, generating quantities as high as those achieved with a multicopy expression vector.

Global transcription and metabolic flux analysis of Escherichia coli in glucose-limited fed-batch cultivations.

A time series of whole-genome transcription profiling of Escherichia coli K-12 W3110 was performed during a carbon-limited fed-batch process. The application of a constant feed rate led to the identification of a dynamic sequence of diverse carbon limitation responses (e.g., the hunger response) and at the same time provided a global view of how cellular and extracellular resources are used: the synthesis of high-affinity transporters guarantees maximal glucose influx, thereby preserving the phosphoenolpyruvate pool, and energy-dependent chemotaxis is reduced in order to provide a more economic "work mode." sigma(S)-mediated stress and starvation responses were both found to be of only minor relevance. Thus, the experimental setup provided access to the hunger response and enabled the differentiation of the hunger response from the general starvation response. Our previous topological model of the global regulation of the E. coli central carbon metabolism through the crp, cra, and relA/spoT modulons is supported by correlating transcript levels and metabolic fluxes and can now be extended. The substrate is extensively oxidized in the tricarboxylic acid (TCA) cycle to enhance energy generation. However, the general rate of oxidative decarboxylation within the pentose phosphate pathway and the TCA cycle is restricted to a minimum. Fine regulation of the carbon flux through these pathways supplies sufficient precursors for biosyntheses. The pools of at least three precursors are probably regulated through activation of the (phosphoenolpyruvate-)glyoxylate shunt. The present work shows that detailed understanding of the genetic regulation of bacterial metabolism provides useful insights for manipulating the carbon flux in technical production processes.

Smoking, alcohol and caffeine in relation to ovarian age during the reproductive years.

BACKGROUND: We sought to determine whether smoking, alcohol and caffeine are related to four indicators of ovarian age: antral follicle count (AFC), follicle stimulating hormone (FSH), inhibin B and estradiol. METHODS: Analyses drew on ultrasound scans and sera from 188 women, aged 22-49. We used least squares regression to estimate differences in AFC and hormone levels for women who smoke cigarettes or who drink alcohol or caffeine. RESULTS: Current smoking is related to elevated FSH (beta for ln(FSH) = 0.21, 95% CI 0.04, 0.39), but not to AFC, inhibin B or estradiol. Neither alcohol nor caffeine is related to any ovarian age indicator. Exploratory analyses suggest that the association of current smoking with FSH varies with age: comparing current with never smokers, at ages 30, 35, 40 and 45, estimated differences in mean FSH are 0.3, 1.3, 3.2 and 6.9 mIU/ml. CONCLUSIONS: The association of current smoking with FSH may reflect accelerated oocyte atresia, impaired follicle quality or dysregulation of the hypothalamic-pituitary-ovarian axis. Identification of the causal mechanism has implications for prevention or treatment of conception delay, infertility and morbidity associated with early menopause.

A kinetic study of lipase-catalyzed reversible kinetic resolution involving verification at miniplant-scale.

Lipase-catalyzed kinetic resolution of racemates is a popular method for synthesis of chiral synthons. Most of these resolutions are reversible equilibrium limited reactions. For the first time, an extensive kinetic model is proposed for kinetic resolution reactions, which takes into account the full reversibility of the reaction, substrate inhibition by an acyl donor and an acyl acceptor as well as alternative substrate inhibition by each enantiomer. For this purpose, the reversible enantioselective transesterification of (R/S)-1-methoxy-2-propanol with ethyl acetate catalyzed by Candida antarctica lipase B (CAL-B) is investigated. The detailed model presented here is valid for a wide range of substrate and product concentrations. Following model discrimination and the application of Haldane equations to reduce the degree of freedom in parameter estimation, the 11 free parameters are successfully identified. All parameters are fitted to the complete data set simultaneously. Six types of independent initial rate studies provide a solid data basis for the model. The effect of changes in substrate and product concentration on reaction kinetics is discussed. The developed model is used for simulations to study the behavior of reaction kinetics in a fixed bed reactor. The typical plot of enantiomeric excess versus conversion of substrate and product is evaluated at various initial substrate mixtures. The model is validated by comparison with experimental results obtained with a fixed bed reactor, which is part of a fully automated state-of-the-art miniplant.

Kinetic modeling of lipase catalyzed hydrolysis of (R/S)-1-methoxy-2-propyl-acetate as a model reaction for production of chiral secondary alcohols.

The Candida antarctica lipase B catalyzed kinetic resolution of (R/S)-1-methoxy-2-propyl-acetate was studied as a model system for the biocatalytic production of chiral secondary alcohols. For this purpose, a kinetic model is proposed involving both enantiomers of this reaction using model discrimination and parameter identification. Starting from a ping-pong bi-bi mechanism, a simplified model with sensitive parameters was derived for the R- and S-enantiomer, respectively. It was validated at pH 7.0, using time-course measurements at varying temperatures (30-60 degrees C) and initial substrate conditions (0.05-1.5 M). This model was then used for mechanistic interpretation of the kinetic resolution on a biochemical level. The effect of temperature on kinetic parameters and enantiomeric ratio was investigated and compared to findings from the field of molecular modeling to obtain a better understanding of the reaction system for process design. Values of 21.2 and 9.7 kJmol-1 were determined for the enthalpic (DeltaR-S DeltaH ++ degrees) and the entropic (-T x DeltaR-S DeltaS ++ degrees) contribution of the difference in transition state energy of both enantiomers at 30 degrees C. High enantiomeric ratio's (E of 47-110) especially at lower temperatures, in addition to enzyme activity at a wide pH range, indicate this biotransformation is a promising example for the industrial production of chiral secondary alcohols.

Predictors of antral follicle count during the reproductive years.

BACKGROUND: We sought to identify indicators of antral follicle count which would be serviceable to clinicians seeking to estimate the number of ovarian follicles without relying on sonographic counts. METHODS: We examined the relations of chronological age and four potential indicators of ovarian age-ovarian volume, FSH, dimeric inhibin B and estradiol-to antral follicle count in 176 recently pregnant women. We identified the regression models which best predict low antral follicle count (< or =10 follicles). RESULTS: Chronological age, ovarian volume, FSH and inhibin B were each significantly associated with antral follicle count. Fifty-three (30.1%) women had < or =10 antral follicles. In the total sample, at the cutpoint corresponding to 80% sensitivity, the positive predictive value for a regression model with all four variables was 60%. All regression models performed less well in women <35 years (13.9% with low count) than in women > or =35 years (52.0% with low count). In older women, the positive predictive value for the model with all four variables was 79%, compared with 60% for a model with chronological age alone. CONCLUSIONS: Our models provide a basis for advising women aged > or =35 years who are either trying to conceive or wish to learn whether they may postpone childbearing.

Trisomic pregnancy and the oocyte pool.

BACKGROUND: We tested the hypothesis that trisomy risk is increased for women with fewer oocytes (older ovarian age) than other women of the same chronological age. METHODS: Our study compared three indicators of ovarian age-number of antral follicles, level of dimeric inhibin B, level of FSH-among women who had trisomic pregnancy losses (n = 54) with those among women who had other losses (24 with other chromosomally abnormal loses, 21 with chromosomally normal losses) or who had chromosomally normal births (n = 65). RESULTS: Ovarian age indicators did not differ between women with trisomic spontaneous abortions and the three comparison groups. Compared with live birth controls, adjusting for chronological age, we estimate that, on average, among trisomy cases the geometric means of 1 + follicle count, inhibin B and FSH are about 7.5% higher, 16.6% higher and 5.5% lower, respectively, with all 95% confidence intervals including zero. The sample size was sufficient to detect moderate differences (0.52 standard errors of regression) between trisomy cases and live birth controls. CONCLUSIONS: Although our data do not support our hypothesis, they leave open the possibility that changes in follicular development unrelated to the size of the oocyte pool influence abnormal chromosome segregation.

Identification of factors impeding the production of a single-chain antibody fragment in Escherichia coliby comparing in vivo and in vitro expression.

The atrazine-specific single-chain variable antibody fragments (scFv) K411B was produced by expression in either the cytoplasm or the periplasm of Escherichia coli BL21(DE3). For periplasmic production, the pelB leader was N-terminally fused to scFv, whereas the unfused variant resulted in cytoplasmic expression. The extent of protein accumulation differed significantly. Expression of scFv with leader was 2.3 times higher than that of the protein without leader. This was further investigated by generating the respective translation profiles using coupled in vitro transcription/translation assays, the results of which were in agreement. This comparative approach was also applied to functionality: Periplasmic expression and in vitro expression resulted in only 10% correctly folded scFv, indicating that the oxidizing environment of the periplasm did not increase proper folding. Thus, the data obtained in vitro confirmed the findings observed in vivo and suggested that the discrepancy in expression levels was due to different translation efficiencies. However, the in vivo production of scFv with enhanced green fluorescent protein (EGFP) fused C-terminally (scFv-EGFP) was only successful in the cytoplasm, although in vitro the expression with and without the leader rendered the same production profile as for scFv. This indicated that neither the translation efficiency nor the solubility but other factors impeded periplasmic expression of the fusion protein.

Expression analysis of neuroglobin mRNA in rodent tissues.

Neuroglobin is a respiratory protein which was reported to be preferentially expressed in the vertebrate brain. Here we present the first detailed analysis of the expression of neuroglobin in mouse and rat tissues. Neuroglobin mRNA was detected in all brain areas studied. Most, but not all, nerve cells were labeled, suggesting differential expression of Ngb. Neuroglobin mRNA was detected in the peripheral nervous system, explaining previous northern hybridization signals in organs other than the brain. Substantial neuroglobin expression was also found in metabolically active endocrine tissues such as the adrenal and pituitary glands. The granule localization of neuroglobin transcripts in various neuronal extensions let us speculate that peripheral translation of neuroglobin protein occurs. This could have important functional consequences for synaptic plasticity, an active metabolic process that needs large amounts of oxygen. The hybridization signals suggest that the local concentration of neuroglobin is sufficient for its putative primary function as an oxygen-supplying protein.

[Motor function during patient-controlled analgesia via a lumbar epidural catheter after major abdominal surgery. Ropivacaine-sufentanil vs. bupivacaine-sufentanil]. / Motorische Blockade während einer patientenkontrollierten lumbalen Epiduralanalgesie nach abdominellen Eingriffen - Ein Vergleich zwischen Ropivacain-Sufentanil und Bupivacain-Sufentanil.

INTRODUCTION: The aim of the present study was to investigate postoperative motoric impairment during patient-controlled analgesia after major abdominal surgery with ropivacaine-sufentanil and bupivacaine-sufentanil via a lumbar epidural catheter. METHODS: After approval of the local ethics committee, 40 patients scheduled for major lower abdominal surgery were randomly allocated to receive bupivacaine 0.25 % or ropivacaine 0.2 %, both with sufentanil 2 microgram/ml in a double blind manner. General anaesthesia (midazolam, etomidate, fentanyl, vecuronium, and desflurane in N2O/O2) and postoperative management of the patients were standardised. Postoperatively, the motoric function and ability for active early mobilisation was examined clinically (application of the Bromage scale, ability to leave the bed and ability to walk). Reduction of muscular force of the legs was measured postoperatively using a scale and compared with preoperative baseline values. To ensure a similar level of analgesia, a 10-cm visual analogue scale was applied at rest and while coughing. RESULTS: The two groups did not differ with respect to the demographic data and postoperative levels of analgesia. Less reduction of motoric function at rest was observed in the ropivacaine group (p = 0,044). However, this did not lead to an increased ability to get up from bed (p = 0,57) or to walk around (p = 0,17). A high number of patients did not meet the requirements for early ambulation. Almost half of the patients of both groups were unable to leave their beds in the morning of the first postoperative day. On the second postoperative day about 25 - 30 % of the patients could not walk even when support was applied. Furthermore, median reduction (10th/90th percentile) of muscular strength was reduced to 50 % (37 %/76 %) in the ropivacaine group and to 48 % (31 %/61 %) in the bupivacaine group compared with preoperative values. DISCUSSION: While quality of analgesia was similar, mobility of the legs at rest is better preserved with ropivacaine 0.2 % than with bupivacaine 0.25 %. However, despite the fact that high dose sufentanil was added to both local anaesthetics, there was marked motoric impairment in both groups probably due to the lumbar site of the epidural catheter. This was associated with an unacceptable high incidence of patients unsuitable for early postoperative mobilisation.

Risk of cranial ultrasound abnormalities in very-low-birth-weight infants conceived with assisted reproductive techniques.

OBJECTIVE: To evaluate risks of cranial ultrasound abnormalities among very-low-birth-weight (VLBW) infants conceived with fertility therapy (ovulation induction only or with assisted reproductive techniques [ART]) and of multiple gestation pregnancies. STUDY DESIGN: The incidences of cranial ultrasound abnormalities in 1473 VLBW infants conceived with and without fertility therapy and born of multiple versus singleton pregnancies were compared, using logistic regression models. RESULTS: Infants conceived with ART were less likely to have intraventricular hemorrhage (IVH). Twins and triplets had risks of cranial ultrasound abnormalities similar to those of singletons. Twins and triplets conceived with ART were at lower risk of IVH. CONCLUSION: VLBW infants conceived with ART do not appear to be at increased risk of cranial ultrasound abnormalities. Likewise, twins and triplets were not at increased risk of these abnormalities.

Kinetic modeling and simulation of in vitro transcription by phage T7 RNA polymerase.

This study provides a mathematical model of T7 RNA polymerase (T7 RNAP) kinetics under in vitro conditions targeted at application of this model to simulation of dynamic transcription performance. A functional dependence of transcript synthesis rate is derived based on: (a) essential reactant concentrations, including T7 RNAP and its promoter, substrate nucleotides, and the inhibitory byproduct inorganic pyrophosphate; (b) a distinction among vector characteristics such as recognition sequences regulating transcription initiation and termination, respectively; and (c) specific properties of the nucleotide sequence including both transcript length and nucleotide composition. Inactivation kinetics showed a half-life of T7 RNAP activity of 50 min under the conditions applied in vitro using the isolated enzyme. Model parameters and their precision are estimated using dynamic simulation and nonlinear regression analysis. The particular novelty of this model is its capability to incorporate linear genomic sequence information for simulation of nonlinear in vitro transcription kinetics.

Nitric oxide synthase neurons in the rodent spinal cord: distribution, relation to Substance P fibers, and effects of dorsal rhizotomy.

The indirect immunofluorescent method was employed to investigate the distribution of neuronal nitric oxide synthase-like immunoreactivity(nNOS-LI) in the spinal cord of the golden hamster and to compare it to data obtained from rats. Immunoreactive neurons were found throughout the cervico-sacral extent in the dorsal horn (mainly in laminae I-III) and in the preganglionic autonomic regions, i.e., the sympathetic intermediolateral nucleus (IML), lateral funicle (LF), intercalated region (IC), the area surrounding the central canal (CA), and the sacral preganglionic parasympathetic cell group. While the distribution of immunoreactive cells was generally similar in both species, some differences were observed. For example in the hamster LF, a higher percentage of stained neurons was seen than in the IML, while the situation was rather inverse in the rat. In order to study the coincidence of nNOS-LI in the population of preganglionic sympathetic neurons (PSN) that innervate the superior cervical ganglion (SCG), these were identified by retrograde axonal transport of fluoro-gold (FG) following unilateral injection into the SCG. PSN were localized ipsilateral to the injection site mainly in the IML and LF of spinal segments C7-Th4. The portion of double-labeled neurons of the IML were lower in hamster (17% in C7, 34% in C8) of FG-labeled cells) than in rat (47% in C8, 77% in Th2), while in the LF of segments C8-Th2 in both species the majority of FG-neurons contained nNOS. While only very few double-labeled neurons were detected in the IC in hamster and rat, a striking difference was observed in the CA, where no double-labeled neurons were found in hamster, but up to 50% in rat. Double immunofluorescence detection of nNOS and substance P (SP) showed that in both the autonomic regions and the dorsal horn, SP-LI fibers and puncta were present in close spatial relationship to nNOS-LI cell bodies. These results were basically identical in the hamster and rat. Unilateral transection of the dorsal roots of segments C6-Th2 in rats resulted in a clear reduction of SP-LI structures in the dorsal horn 5 days after rhizotomy, but not in the autonomic regions. Compared to the unlesioned side, the numbers of nNOS-LI neurons in the superficial laminae of the dorsal horn were reduced to 32-46% in the lesioned segments, and to 53% and 87%, respectively, in the two segments cranial to the rhizotomized segments but remained unchanged caudally to the lesion. Numbers of nNOS-LI cell bodies in the autonomic regions were not altered following dorsal root transection. The present study provides data on the widespread distribution of nNOS in the spinal cord of golden hamster and describes the partial coincidence of the enzyme in PSN. The effects of dorsal rhizotomy on nNOS-LI neurons in the dorsal horn reveal that primary-afferent fibers provide a stimulatory influence on neurons of the dorsal horn to generate the gaseous neuroactive substance, nitric oxide.

Signal transduction dynamics of the protein kinase-A/phosphofructokinase-2 system in Saccharomyces cerevisiae.

This work focuses on the phosphofructokinase-2-system dynamics in Saccharomyces cerevisiae, in vivo. The investigations were dedicated to the development and implementation of appropriate theoretical and experimental methods toward evaluation of a quantitative strategy for the characterization of systemic mechanisms involved in the cAMP/protein kinase-A/phosphofructokinase-2 signal transduction cascade in yeast. Upon glucose pulse experiments, applied to glucose-limited continuous cultures of S. cerevisiae, the system response was determined with respect to alterations of intracellular metabolite concentrations or in vivo enzyme activities. Phosphofructokinase-2, in vivo, was found to be saturated with respect to both its substrates, F6P and ATP. This restriction results in an uncoupling of the enzyme activity and the signal transduction cascade from glycolytic flux, concluding that activation of phosphofructokinase-2 is exclusively a result of phosphorylation by protein kinase-A, which in turn is activated by increasing intracellular cAMP concentration after an extracellular glucose pulse. Signal processing from cAMP versus phosphofructokinase-2 also displays peculiar features implicated in a hysteresis behavior: when increasing cAMP concentration achieves a certain critical value, protein kinase-A switches into an active state. Posterior to this activation, the signal transform maintains autonomy and functional independence of further alterations of the intracellular cAMP concentration. Our observations, finally, allow the establishment of a representative model for the description of the signal transduction process via protein kinase-A in yeast.

High-cell-density fermentation for production of L-N-carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter.

A high-cell-density fed-batch fermentation for the production of heterologous proteins in Escherichia coli was developed using the positively regulated Escherichia coli rhaBAD promoter. The expression system was improved by reducing of the amount of expensive L-rhamnose necessary for induction of the rhamnose promoter and by increasing the vector stability. Consumption of the inducer L-rhamnose was inhibited by inactivation of L-rhamnulose kinase encoding gene rhaB of Escherichia coli W3110, responsible for the first irreversible step in rhamnose catabolism. Plasmid instability caused by multimerization of the expression vector in the recombination-proficient W3110 was prevented by insertion of the multimer resolution site cer from the ColE1 plasmid into the vector. Fermentation experiments with the optimized system resulted in the production of 100 g x L(-1) cell dry weight and 3.8 g x L(-1) of recombinant L-N-carbamoylase, an enzyme, which is needed for the production of enantiomeric pure amino acids in a two-step reaction from hydantoins.

Physiological responses to mixing in large scale bioreactors.

Escherichia coli fed-batch cultivations at 22 m3 scale were compared to corresponding laboratory scale processes and cultivations using a scale-down reactor furnished with a high-glucose concentration zone to mimic the conditions in a feed zone of the large bioreactor. Formate accumulated in the large reactor, indicating the existence of oxygen limitation zones. It is suggested that the reduced biomass yield at large scale partly is due to repeated production/re-assimilation of acetate from overflow metabolism and mixed acid fermentation products due to local moving zones with oxygen limitation. The conditions that generated mixed-acid fermentation in the scale-down reactor also induced a number of stress responses, monitored by analysis of mRNA of selected stress induced genes. The stress responses were relaxed when the cells returned to the substrate limited and oxygen sufficient compartment of the reactor. Corresponding analysis in the large reactor showed that the concentration of mRNA of four stress induced genes was lowest at the sampling port most distant from the feed zone. It is assumed that repeated induction/relaxation of stress responses in a large bioreactor may contribute to altered physiological properties of the cells grown in large-scale bioreactor. Flow cytometric analysis revealed reduced damage with respect to cytoplasmic membrane potential and integrity in cells grown in the dynamic environments of the large scale reactor and the scale-down reactor.