[Diverticulitis and diverticulosis]. / So managen Sie die Divertikelkrankheit.

Over the last 100 years, the prevalence and incidence of diverticulosis and diverticular disease have increased dramatically in western industrialized countries. The main reasons for this are considered to be changes in eating habits, and the increasing age of the population. Conservative treatment of diverticulitis is an initial period of fasting and antibiotic treatment. For recurrence prevention, a fiber-rich diet is recommended. Studies providing evidence in support of the general recommendation of recurrence prophylaxis with poorly absorbed antibiotics, mesalazine or probiotics are to date not adequate. Elective prophylactic sigmoid resection is to be recommended following an episode of diverticulitis with complications, and after an episode of uncomplicated diverticulitis in long-term immunosuppressed patients who have already had an attack. Elective sigmoid resection after a healed second attack of uncomplicated diverticulitis is controversial.

In situ microscopy for online monitoring of cell concentration in Pichia pastoris cultivations.

In situ Microscopy (ISM) is an optical non-invasive technique to monitor cells in bioprocesses in real-time. Pichia pastoris is one of the most promising protein expression systems. This yeast combines fast growth on simple media and important eukaryotic features such as glycosylation. In this work, the ISM technology was applied to Pichia pastoris cultivations for online monitoring of the cell concentration during cultivation. Different ISM settings were tested. The acquired images were analyzed with two image processing algorithms. In seven cultivations the cell concentration was monitored by the applied algorithms and offline samples were taken to determine optical density (OD) and dry cell mass (DCM). Cell concentrations up to 74g/L dry cell mass could be analyzed via the ISM. Depending on the algorithm and the ISM settings, an accuracy between 0.3 % and 12 % was achieved. The overall results show that for a robust measurement a combination of the two described algorithms is required.

The effect of pH on glucoamylase production, glycosylation and chemostat evolution of Aspergillus niger.

The effect of ambient pH on production and glycosylation of glucoamylase (GAM) and on the generation of a morphological mutant produced by Aspergillus niger strain B1 (a transformant containing an additional 20 copies of the homologous GAM glaA gene) was studied. We have shown that a change in the pH from 4 to 5.4 during continuous cultivation of the A. niger B1 strain instigates or accelerates the spontaneous generation of a morphological mutant (LB). This mutant strain produced approx. 50% less extracellular protein and GAM during both chemostat and batch cultivation compared to another strain with parental-type morphology (PS). The intracellular levels of GAM were also lower in the LB strain. In addition, cultivation of the original parent B1 strain in a batch-pulse bioreactor at pH 5.5 resulted in a 9-fold drop in GAM production and a 5-fold drop in extracellular protein compared to that obtained at pH 4. Glycosylation analysis of the glucoamylases purified from shake-flask cultivation showed that both principal forms of GAM secreted by the LB strain possessed enhanced galactosylation (2-fold), compared to those of the PS. Four diagnostic methods (immunostaining, mild methanolysis, mild acid hydrolysis and beta-galactofuranosidase digestion) provided evidence that the majority of this galactose was of the furanoic conformation. The GAMs produced during batch-pulse cultivation at pH 5.5 similarly showed an approx. 2-fold increase in galactofuranosylation compared to pH 4. Interestingly, in both cases the increased galactofuranosylation appears primarily restricted to the O-linked glycan component. Ambient pH therefore regulates both GAM production and influences its glycosylation.

RecA-dependent viral burst in bacterial colonies during the entry into stationary phase.

We have explored the nature of the sudden viral amplification observed during the ageing of P22-infected lysogenic colonies of Salmonella typhimurium [Ramírez, E, and Villaverde, A. (1997) Gene 202, 147-149]. By a comparative analysis of the wild-type P22 and a P22 integration mutant, it has been shown that the conditions promoting prophage induction occur in only a small portion of the bacterial population and briefly during the transition between the exponential growth and the stationary phase. The viral burst is RecA-dependent and cannot be reproduced in continuous culture by a mere decrease of the growth rate. This suggests that the limited viral propagation in colonies is probably linked to heterogeneous physiological conditions within colonial populations, distinct from those of the homogeneous liquid cultures.

Heat-inactivation of plasmid-encoded CI857 repressor induces gene expression from Ind- lambda prophage in recombinant Escherichia coli.

We have observed significant cell lysis upon temperature up-shift of recombinant Escherichia coli cultures harboring CI857-repressed lambda-based expression vectors. This event, that becomes evident about 30-40 min after the heat shock, takes place when using the lambda promoter system in Ind- lysogenic strains, but not in others commonly employed for recombinant gene expression. These results strongly suggest that the thermosensitive CI857 repressor, encoded by the expression vector, competes with CI Ind- molecules for binding to the prophage operator region, allowing for expression of lytic genes from the integrated Ind- viral genome upon temperature up-shift. Transcription of viral lytic genes does not include unspecific expression of a reporter sulA::lacZ gene fusion carried in the prophage genome. These results prompt, however, to carefully evaluate the limitations of expression systems based on pL/pR-CI857 in bacterial strains modified through lambda Ind- gene transfer vehicles.

Characterization of inclusion bodies in recombinant Escherichia coli producing high levels of porcine somatotropin.

The protein composition of inclusion bodies (IBs) formed in recombinant Escherichia coli producing high levels of porcine somatotropin (pST) was analyzed by one- and two-dimensional protein gel electrophoresis. Recombinant pST is exclusively recovered from the insoluble cell fraction. Results indicate that, in addition to the main species of pST, subspecies with different isoelectric points and degradative fragments are contained within IBs. The presence of outer membrane proteins in IB fractions results from coprecipitation of cell debris during IB preparation and not from specific in vivo or in vitro interaction of these proteins with IBs. Cells producing pST contain up to three IBs located in the cytoplasm. The implication of high level gene expression on the uniformity of the desired product is discussed.

Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli.

The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose.

On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.

Simple fed-batch technique for high cell density cultivation of Escherichia coli.

A simple fed-batch process for high cell density cultivation of Escherichia coli TG1 was developed. A pre-determined feeding strategy was chosen to maintain carbon-limited growth using a defined medium. Feeding was carried out to increase the cell mass concentration exponentially in the bioreactor controlling biomass accumulation at growth rates which do not cause the formation of acetic acid (mu < mu crit). Cell concentrations of 128 and 148 g per 1 dry cell weight (g l-1 DCW) were obtained using glucose or glycerol as carbon source, respectively.

Renaturation of heterodimeric platelet-derived growth factor from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography.

A procedure for renaturation of heterodimeric platelet-derived growth factor (PDGF-AB) from inclusion bodies of recombinant Escherichia coli using size-exclusion chromatography is described. Either prepurified or crude PDGF-AB inclusion bodies solubilized with guanidinium hydrochloride were subjected to buffer exchange from denaturing to renaturing conditions during chromatography. Renaturation of PDGF-AB involves folding of the solubilized and unfolded molecules into dimerization competent monomers during size-exclusion chromatography and subsequent dimerization of folded monomers into the biologically active heterodimeric growth factor. Optimized conditions result in an overall yield of 75% active PDGF-AB with respect to size-exclusion chromatography and subsequent dimerization. The described approach allows renaturation at high protein concentrations and circumvents aggregation which is observed when refolding is carried out by dilution.

Two-step chromatographic procedure for purification of basic fibroblast growth factor from recombinant Escherichia coli and characterization of the equilibrium parameters of adsorption.

A two-step chromatographic procedure for purification of basic fibroblast growth factor (bFGF) from high-cell-density cultures of recombinant E. coli is described. Heparin-Sepharose as a material which shows a high affinity to endothelial growth factors was used as sorbent for purification of bFGF from the soluble cell fraction. A one-step affinity chromatographic procedure resulted in very pure bFGF. However, this one-step affinity isolation of bFGF caused the loss of around 60% of the recombinant protein. A combination of ion-exchange chromatography with heparin-Sepharose affinity chromatography was favored for bFGF purification. A first cation-exchange chromatographic step resulted in a solution of bFGF with a purity of around 70%. The weak cation exchanger CM Sepharose C50 was preferred in comparison to the strong cation exchanger S-Sepharose because of the higher recovery of bFGF. With the ion-exchange chromatographic step prior to the heparin-Sepharose affinity chromatography, the total yield of recovery of bFGF increased to 56% compared to 40% using the one-step purification procedure with heparin-Sepharose. To characterize the equilibrium parameters of adsorption, batch experiments for the calculation of maximum capacities and dissociation constants for CM-Sepharose C50 and heparin-Sepharose were carried out. The equilibrium experiments revealed that adsorption of bFGF to the ion-exchange sorbent followed single-site interaction according to the Langmuir model of adsorption. The adsorption of bFGF to heparin-Sepharose was described by a double Langmuir approach of two independent binding sites with different maximum capacities and dissociation constants. The purified bFGF showed a high biological activity and circular dichroic spectra of a proper folded molecule. The analysis of the N-terminal amino acid sequence revealed a mixture of two fractions of bFGF, which both are characterized by the cleavage of the first amino acid methionine. In addition, half of the bFGF molecules lacked the second amino acid alanine.

Purification of recombinant human basic fibroblast growth factor: stability of selective sorbents under cleaning in place conditions.

Human basic fibroblast growth factor (bFGF) was produced from recombinant Escherichia coli by high-cell-density cultivation. In order to develop a purification strategy for large-scale purification, chromatographic sorbents with different anionic functional groups were compared in terms of selectivity for bFGF and stability under cleaning in place (CIP) conditions. Heparin-Sepharose CL-6B, Fractogel EMD-SO3- 650 (S) and SP-Sepharose (high performance) were found suitable for this purpose with decreasing selectivity in that order. Each sorbent was treated eight times under CIP conditions employing both 0.2 and 1.0 M NaOH, in order to study modifications of these sorbents. Heparin-Sepharose displayed more than 50% loss of capacity after the first CIP treatment and decreasing selectivity with each cycle. Both cation exchangers displayed almost constant results regarding selectivity and capacity. The Fractogel EMD-SO3- exhibited only slightly lower selectivity for bFGF than Heparin-Sepharose and the highest capacity of all sorbents tested. Agglomeration of bFGF at low salt concentrations was a serious problem. By direct application of pooled fractions from Fractogel EMD-SO3- onto Heparin-Sepharose a highly pure product was obtained; however, the recovery after Heparin-Sepharose was only 30%.

Synthesis rates of cellular proteins involved in translation and protein folding are strongly altered in response to overproduction of basic fibroblast growth factor by recombinant Escherichia coli.

The cellular response to temperature-induced production of human basic fibroblast growth (bFGF) factor by recombinant E. coli (bacteriophage lambda PRPL promoter/cI857 repressor expression system) was studied by one- and two-dimensional gel electrophoresis. Temperature shift from 30 to 42 degrees C caused the induction of heat-shock protein synthesis and the repression of synthesis of ribosomal proteins and the protein folding catalyst trigger factor. Compared to control cells, carrying the expression vector without structural bFGF gene cells, producing the heterologous protein exhibited a stronger increase in the synthesis rate of heat-shock proteins ClpB (HtpM), DnaK, HtpG, GroEL, GrpE, and IbpB (HtpE) in response to temperature upshift. Unexpectedly, formation of the chaperone heat-shock protein GroES was not detected after temperature shift to 42 degrees C in cells producing bFGF. In addition to amplified heat-shock protein formation, the syntheses of ribosomal proteins and of the protein folding catalyst trigger factor were more severely repressed after temperature upshift in cells producing bFGF. In conclusion, the normal cellular stress response caused by the high inducing temperature was strongly amplified by heterologous protein synthesis. In particular, syntheses of proteins involved in translation and protein folding were affected by the overproduction of the heterologous protein.

Kinetics of heat-shock response and inclusion body formation during temperature-induced production of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli.

The kinetics of the heat-shock response and the formation of inclusion bodies in recombinant Escherichia coli TG1 were studied in glucose-limited high-cell-density cultures in response to temperature-induced production of human basic fibroblast growth factor (hFGF-2), a protein which partially aggregates into inclusion bodies. The maximum synthesis rates of heat-shock proteins were similar to those in a control cultivation with a strain carrying an expression vector without inducible structural gene. However, the maximum of induction for many heat-shock proteins including DnaK, ClpB, and HtpG was reached at least 30 min later when synthesis of hFGF-2 was simultaneously induced by the temperature upshift. During this first production phase, hFGF-2 was exclusively deposited in the insoluble cell fraction. Thereafter, accumulation of soluble hFGF-2 was observed, too, indicating that the recombinant protein needs heat-shock chaperones for proper folding at elevated temperatures. Strong recombinant protein production prolonged the synthesis of the majority of heat-shock proteins (including GroELS, DnaK, ClpB, and HtpG) even in a wildtype dnaK(+) background. In contrast, the synthesis rates of the small heat-shock proteins IbpA and IbpB declined within 1 h to preinduction values in control and hFGF-2 producing cultures. In the producing cultivation, IbpA and IbpB synthesis ceased to an undetectable level when soluble hFGF-2 started to accumulate, whereas the synthesis rates of the other heat-shock proteins including those belonging to the DnaK and GroEL families remained high throughout the entire production phase.

Cultivation of recombinant E. coli and production of fusion protein in 60-1 bubble column and airlift tower loop reactors.

E. coli K12 with multicopy plasmid (lambda PR-promoter and temperature-sensitive lambda cI 857 repressor) was cultivated in 60-l bubble column and airlift tower loop reactors. The medium composition, cell concentration, and intracellulary enzyme activity were monitored on-line during batch, fed-batch, and continuous cultivations. The specific growth rates, cell mass yield coefficients, plasmid stabilities, productivities of the amount of active fusion protein (beta-galactosidase activity), concentrations and yields of acetic acid, and volumetric oxygen transfer coefficient were evaluated for different medium compositions and cultivation conditions. The enzyme activity was also monitored during the temperature induction. The results evaluated in the 60-l bubble column and airlift tower loop reactors are compared with those evaluated in a 1-1 stirred-tank reactor.

GpdA-promoter-controlled production of glucose oxidase by recombinant Aspergillus niger using nonglucose carbon sources.

The gpdA-promoter-controlled exocellular production of glucose oxidase (GOD) by recombinant Aspergillus niger NRRL-3 (GOD3-18) during growth on glucose and nonglucose carbon sources was investigated. Screening of various carbon substrates in shake-flask cultures revealed that exocellular GOD activities were not only obtained on glucose but also during growth on mannose, fructose, and xylose. The performance of A. niger NRRL-3 (GOD3-18) using glucose, fructose, or xylose as carbon substrate was compared in more detail in bioreactor cultures. These studies revealed that gpdA-promoter-controlled GOD synthesis was strictly coupled to cell growth. The gpdA-promoter was most active during growth on glucose. However, the unfavorable rapid GOD-catalyzed transformation of glucose into gluconic acid, a carbon source not supporting further cell growth and GOD production, resulted in low biomass yields and, therefore, reduced the advantageous properties of glucose. The total (endo- and exocellular) specific GOD activities were lowest when growth occurred on fructose (only a third of the activity that was obtained on glucose), whereas utilization of xylose resulted in total specific GOD activities nearly as high as reached during growth on glucose. Also, the portion of GOD excreted into the culture fluid reached similar high levels (approximately equal to 90%) by using either glucose or xylose as substrate, whereas growth on fructose resulted in a more pelleted morphology with more than half the total GOD activity retained in the fungal biomass. Finally, growth on xylose resulted in the highest biomass yield and, consequently, the highest total volumetric GOD activity. These results show that xylose is the most favorable carbon substrate for gpdA-promoter-controlled production of exocellular GOD.

Fungal morphology in submerged cultures and its relation to glucose oxidase excretion by recombinant Aspergillus niger.

The effect of culture conditions such as medium composition and shear stress on the fungal pellet morphology in shake-flask cultures and its relation to glucose oxidase (GOD) excretion by recombinant Aspergillus niger NRRL 3 (GOD 3-18) was investigated. It was shown that culture conditions resulting in the formation of smaller fungal pellets with an increased mycelial density result in higher yields of exocellular GOD. The pellets obtained in shake-flask cultures showed distinct layers of mycelial density with only the thin outer layer consisting of a dense mycelial network. The performance of the recombinant strain and the process of pellet formation was also analyzed during batch cultivation in a stirred-tank bioreactor. It was shown that the process of pellet formation occurred in two steps: (1) aggregation of free spores to spore clusters with subsequent germination and formation of small aggregates surrounded by a loose hyphal network, and (2) aggregation of the primary aggregates to the final full-size pellets. The fungal pellets formed during bioreactor cultivation were smaller, did not show large differences in mycelial density, and were more efficient with respect to the production of exocellular GOD. The decreasing pellet size also correlated with an increased mycelial density, indicating an improvement of the transport of nutrients to the inner parts of the pellet.

Characterization of recombinant factor XIIIa produced in Saccharomyces cerevisiae.

Recombinant factor XIIIa (FXIIIa), produced in Saccharomyces cerevisiae, was recovered as a fully active cytosolic component and rigorously compared to natural F XIIIa from human placenta with respect to physicochemical and functional properties. Identical parameters were found in SDS polyacrylamide gel electrophoresis, analytical ultracentrifugation and HPLC gel filtration, and all spectral characteristics including derivative UV absorbance, fluorescence and circular dichroism were identical. Similarly, the interaction of both proteins with polyclonal antibodies directed against the entire FXIIIa or its N-terminal 4 kD activation peptide were identical. Furthermore, thrombin cleavage and fibrin cross-linking showed indistinguishable patterns. The only difference we observed was with respect to endgroup analysis. The recombinant protein is homogeneous, whereas placental FXIIIa shows multiple electrophoretic bands caused by microheterogeneity in the C-terminal part of the protein.

Expression of intracellular hemoglobin improves protein synthesis in oxygen-limited Escherichia coli.

We have previously cloned the Vitreoscilla hemoglobin gene (VHb) and expressed the protein in Escherichia coli in its active form. Under oxygen-limited conditions the presence of VHb improves protein synthesis as indicated by both total protein content and the activity of an enzyme expressed from a cloned gene present on a multicopy plasmid. Measurements of nitrogen utilization rates corroborate the observation of enhanced protein synthesis; however, the rates of carbon consumption and acid synthesis remain unchanged. This suggests that the net effect of VHb in E. coli is to improve the efficiency, rather than the kinetics, of oxygen-limited aerobic metabolism. We propose two possible models for the mechanism of action of VHb: the facilitated diffusion hypothesis and the intracellular redox effector hypothesis. These suggest other systems in which cloned VHb may enhance bioprocess productivity.

Cysteine to serine substitutions in basic fibroblast growth factor: effect on inclusion body formation and proteolytic susceptibility during in vitro refolding.

We have investigated the effect of cysteine to serine substitutions in human basic fibroblast growth factor (bFGF) on the formation of inclusion bodies in Escherichia coli. Using a temperature-sensitive expression, system, about 30% of human bFGF, which contains four cysteines at positions 26, 70, 88, and 93, is deposited into inclusion bodies. A single mutation at position 88 and a double mutation at positions 70 and 88 do not greatly alter the partition of bFGF into soluble and insoluble cell fractions. However, a single substitution of cysteine 70 by serine decreases the fraction of soluble bFGF significantly. When cysteines 26 and 93 (conserved among related growth factors) are replaced by serines, no soluble bFGF is formed in E. coli. Cysteine to serine substitutions also affect proteolytic susceptibility of bFGF during in vitro refolding from crude inclusion bodies. About 60% of human bFGF is lost to proteolytic degradation during in vitro refolding. Replacement of cysteines by serines increases the total recovery of bFGF, although more aggregates are formed during refolding. Ser-88-bFGF was expressed at the highest level, gave the highest soluble fraction in vivo, and exhibited the greatest fractional recovery and was recovered with the largest insoluble fraction after in vitro refolding. Thermal stability experiments at 42 degrees C and 70 degrees C revealed that cysteine to serine substitutions did not cause aggregation of the folded protein in vitro.