Probiotic Escherichia coli Nissle inhibits IL-6 and MAPK-mediated cardiac hypertrophy during STZ-induced diabetes in rats.

Escherichia coli Nissle (EcN), a probiotic bacterium protects against several disorders. Multiple reports have studied the pathways involved in cardiac hypertrophy. However, the effects of probiotic EcN against diabetes-induced cardiac hypertrophy remain to be understood. We administered five weeks old Wistar male (271±19.4 g body weight) streptozotocin-induced diabetic rats with 109 cfu of EcN via oral gavage every day for 24 days followed by subjecting the rats to echocardiography to analyse the cardiac parameters. Overexpressed interleukin (IL)-6 induced the MEK5/ERK5, JAK2/STAT3, and MAPK signalling cascades in streptozotocin-induced diabetic rats. Further, the upregulation of calcineurin, NFATc3, and p-GATA4 led to the elevation of hypertrophy markers, such as atrial and B-type natriuretic peptides. In contrast, diabetic rats supplemented with probiotic EcN exhibited significant downregulated IL-6. Moreover, the MEK5/ERK5 and JAK2/STAT3 cascades involved during eccentric hypertrophy and MAPK signalling, including phosphorylated MEK, ERK, JNK, and p-38, were significantly attenuated in diabetic rats after supplementation of EcN. Western blotting and immunofluorescence revealed the significant downregulation of NFATc3 and downstream mediators, thereby resulting in the impairment of cardiac hypertrophy. Taken together, the findings demonstrate that supplementing probiotic EcN has the potential to show cardioprotective effects by inhibiting diabetes-induced cardiomyopathies.

Antioxidant molecular targets of wheat bran fermented by white rot fungi and its potential modulation of antioxidative status in broiler chickens.

1. The study focused on antioxidant molecular targets of wheat bran fermented by white rot fungi (WRF) in poultry. After solid-state fermentation of wheat bran by WRF for 12 d, scanning electron microscopy found that the lignocellulose structure showed degradation and fragmentation. 2. A total of 300 1-d-old broilers were evenly divided by gender and randomly allocated into the following treatments: (1) maize-soybean meal (control group), (2) 10% of wheat bran replacing maize (10% WB group) or (3) 10% of fermented wheat bran replacing maize (10% FWB group). 3. The results indicated that the antioxidant gene expression, such as haem oxygenase-1 and glutathione-S-transferase of chicken peripheral blood mononuclear cells, of the 10% FWB group was significantly higher than that of the control group at d 35. For genes of Nicotinamide adenine dinucleotide phosphate oxygenase 1 and reactive oxygen species modulator protein 1, the expression of the 10% FWB group was lower than that of the control group at d 21 and 35. 4. In conclusion, wheat bran fermented by WRF could increase lignocellulolytic enzyme activities and the levels of active components that further regulate the expression of antioxidant molecular targets in poultry.

Mapping the Orientation of White Matter Fiber Bundles: A Comparative Study of Diffusion Tensor Imaging, Diffusional Kurtosis Imaging, and Diffusion Spectrum Imaging.

BACKGROUND AND PURPOSE: White matter fiber tractography relies on fiber bundle orientation estimates from diffusion MR imaging. However, clinically feasible techniques such as DTI and diffusional kurtosis imaging use assumptions, which may introduce error into in vivo orientation estimates. In this study, fiber bundle orientations from DTI and diffusional kurtosis imaging are compared with diffusion spectrum imaging as a criterion standard to assess the performance of each technique. MATERIALS AND METHODS: For each subject, full DTI, diffusional kurtosis imaging, and diffusion spectrum imaging datasets were acquired during 2 independent sessions, and fiber bundle orientations were estimated by using the specific theoretic assumptions of each technique. Angular variability and angular error measures were assessed by comparing the orientation estimates. Tractography generated with each of the 3 reconstructions was also examined and contrasted. RESULTS: Orientation estimates from all 3 techniques had comparable angular reproducibility, but diffusional kurtosis imaging decreased angular error throughout the white matter compared with DTI. Diffusion spectrum imaging and diffusional kurtosis imaging enabled the detection of crossing-fiber bundles, which had pronounced effects on tractography relative to DTI. Diffusion spectrum imaging had the highest sensitivity for detecting crossing fibers; however, the diffusion spectrum imaging and diffusional kurtosis imaging tracts were qualitatively similar. CONCLUSIONS: Fiber bundle orientation estimates from diffusional kurtosis imaging have less systematic error than those from DTI, which can noticeably affect tractography. Moreover, tractography obtained with diffusional kurtosis imaging is qualitatively comparable with that of diffusion spectrum imaging. Because diffusional kurtosis imaging has a shorter typical scan time than diffusion spectrum imaging, diffusional kurtosis imaging is potentially more suitable for a variety of clinical and research applications.

A poly(dimethylsiloxane) based prism for surface plasmon resonance imaging system and its application for gas detection.

This paper presents a surface plasmon resonance (SPR) imaging system based on a low-cost, convenient poly(dimethylsiloxane) (PDMS) prism featured with a close contact with the gold film. Compared to conventional glass prism, both numerical simulations and experimental studies indicated a deeper but wider absorption peak with a higher coupling angle for the PDMS based prism. System repeatability was quantified by the cycled detection of helium and air, with the effect of the flow rate investigated. Furthermore, five types of gases (nitrogen, air, oxygen, hydrogen, and helium) were detected and differentiated by the SPR system, with a calculated sensitivity of 5 × 10(-6) RIU.

Preoperative diagnosis and successful laparoscopic treatment of incarcerated obturator hernia.

Obturator hernia is rare and frequently delayed in surgical treatment and, thus, is associated with high morbidity and mortality. It is often found in thin, elderly, multiparous women. We reported a case of early diagnosis due to clinical alert and in-time computed tomography (CT). The patient received laparoscopic surgery and had good postoperative recovery. The regular use of CT for diagnosing nonspecific abdominal complaints associated with pain over the inner thigh, especially in thin, elderly, multiparous women, will result in increased preoperative detection of the obturator hernia.

High production of heterologous proteins in Escherichia coli using the thermo-regulated T7 expression system.

The exclusive use of isopropyl beta-D-thiogalactopyranoside to activate the T7 promoter for protein production has limited the general use of the expression system. We have sought an alternative by constructing a recombinant Escherichia coli strain, BL21 (G2), to carry a chromosomal copy of T7 gene 1 fused to the lambdaPL and lambdaP(R) tandem promoter. As a result, the recombinant strain harboring the carbamoylase gene from Agrobacterium radiobacter NRRL B11291 was shown to display various levels of.protein production in response to different degrees of heat shock. In particular, the system remained inactive at 30 degrees C and exhibited high sensitivity to heat such that a detectable carbamoylase activity could be measured after exposure to 33 degrees C. Moreover, heating in two steps - elevating the temperature from 30 degrees C to 39 degrees C and holding for a brief period, followed by reducing to 37 degrees C--was found to be the most potent method for protein production in this case. Using this approach, the recombinant protein accounted for 20% of total protein content of the cell. These results reveal the advantages of this expression system: responsiveness to thermal modulation and high-level production capability. In an attempt to enhance the total protein yield, a fed-batch fermentation process was carried out to control the cell growth rate by adjusting the substrate inflow. By applying the two-step temperature change. a carbamoylase yield with enzyme activity corresponding to 14,256 units was obtained. This production yield is a 10-fold increase in comparison with that at the batch-fermentation scale and 2,000-fold higher than that achieved at the shake-flask scale. Overall, it illustrates the promise of the newly constructed T7 system based on heat inducibility for industrial scale production of recombinant proteins.

Coupling the T7 A1 promoter to the runaway-replication vector as an efficient method for stringent control and high-level expression of lacZ.

An expression vector characterized by tight regulation and high expression of cloned genes appears to be indispensable for the engineering need. To achieve this goal, in association with lacI the T7 A1 promoter containing two synthetic lac operators was constructed into a runaway-replication vector. To further examine this vector system, lacZ was subcloned and placed under the control of the T7 A1 promoter on the plasmid. With the application of the thermal induction alone, the Escherichia coli strain harboring the recombinant plasmid was able to produce 15,000 Miller units of beta-galactosidase, while it yielded the recombinant protein with 45,000-50,000 Miller units upon both thermal and chemical induction. In sharp contrast, only 60-90 Miller units of beta-galactosidase was obtained for the cell at an uninduced state. As a result, the production yield of beta-galactosidase over the background level is amplified approximately 170-fold by thermal induction and 500-fold by thermal and chemical induction. To produce the recombinant protein on a large scale, an approach by connecting two fermenters in series was newly developed. By applying the three-stage temperature shift in this dual fermenter system, 55,000 Miller units of beta-galactosidase was obtained. Overall, it shows the potential use of the vector system developed here for its tight control and high production of recombinant proteins.

Overproduction of D-hydantoinase and carbamoylase in a soluble form in Escherichia coli.

The production of D-hydantoinase and carbamoylase from Agrobacterium radiobacter NRRL B11291 using T7 and trc promoters, respectively, was found to cause protein aggregates in Escherichia coli. We initiated a systematic study aimed at overproducting these two proteins in a soluble form. As a result, the protein aggregate from carbamoylase overproduction could be alleviated with the aid of GroEL/GroES. In contrast, the production of a high level of D-hydantoinase in an active form can be achieved at low temperature (25 degrees C) or by the coproduction of DnaJ/DnaK. Overall, with such approaches both recombinant proteins gain more than a four-fold increase in enzyme activity. In addition, by fusion with thioredoxin, D-hydantoinase activity can be increased 25% more than the unfused counterpart in the presence of DnaJ/DnaK. These results indicate the success of our approaches to overproducing D-hydantoinase and carbamoylase in a soluble form in E. coli.

One-step production of D-p-hydroxyphenylglycine by recombinant Escherichia coli strains.

The gene encoding D-hydantoinase from Agrobacterium radiobacter NRRL B11291 was successfully cloned by use of polymerase chain reaction. A positive clone was scored, and its nucleotide sequence was further analyzed. The analysis by deleting various lengths of nucleotides from the amino terminus of the open reading frame revealed the putative regions for promoter and RBS site. By highly expressing both D-hydantoinase and carbamoylase, recombinant Escherichia coli strains were able to convert DL-hydroxyphenyl hydantoin (DL-HPH) to D-p-hydroxyphenylglycine (D-HPG) with a conversion yield of 97%, accounting for productivity 5 times higher than that obtained by A. radiobacter NRRL B11291. Immobilizing the recombinant cells with kappa-carrageenan could also achieve a conversion of 93%, while A. radiobacter NRRL B11291 attained 20% within the same period of reaction time. These results illustrate the feasibility in employing recombinant E. coli to accomplish one-step conversion of DL-HPH to D-HPG. In the process of improving D-HPG production, D-hydantoinase activity was increased 2.57-fold but carbamoylase activity remained constant, which resulted in only a 30% increase in the reaction rate. It suggests that carbamoylase is the step setting the pace of the reaction. Since the reaction substrate is highly insoluble, achieving sufficient agitation appears to be an important issue in this heterogeneous system. This view is further supported by the study on repeated use of cells, which shows that to reach a conversion of more than 90% free cells can be recycled six times, whereas immobilized cells can be used only twice. In conclusion, the poor reusability of immobilized cells is due to the fouling on the gel surface.

Production of D-p-hydroxyphenylglycine by N-carbamoyl-D-amino acid amidohydrolase-overproducing Escherichia coli strains.

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene from Agrobacterium radiobacter NRRL B11291 has been successfully cloned and expressed in Escherichia coli. Subcloning of the D-carbamoylase gene into different types of vectors and backgrounds of E. coli strains showed that the optimal expression level of D-carbamoylase was achieved in a ColE1-derived plasmid with a 150-fold increase in specific enzyme activity compared to that in a pSC101-derived plasmid. In addition, the recombinant plasmids were very stable in the E. coli strain ATCC11303 but not in JCL1258 tested here. Employing the recombinant E. coli strain DH5alpha/pAH61 for D-p-hydroxyphenylglycine production showed that the cell was capable of transforming N-carbamoyl-D-hydroxylphenylglycine to D-p-hydroxyphenylglycine with a molar conversion yield of 100% and a production rate of 1.9 g/(L h). In comparison with A. radiobacter NRRL B11291, this productivity approximates a 55-fold increase in D-hydroxyphenylglycine production. This result suggests the potential application of recombinant E. coli strains for the transformation reaction.

Enhanced conversion rate of L-phenylalanine by coupling reactions of aminotransferases and phosphoenolpyruvate carboxykinase in Escherichia coli K-12.

In Escherichia coli, aspartate aminotransferase (encoded by aspC) and aromatic amino acid aminotransferase (encoded by tyrB) share overlapping substrate specificity in the syntheses of aromatic amino acids. Through the transamination reactions catalyzed by AspC or TyrB, L-phenylalanine (L-Phe) can be produced from phenylpyruvate with aspartic acid as the amino donor. To modulate and enhance the production levels of proteins, both aspC and tyrB were subcloned into a runaway-replication vector. As a result, the specific activities of AspC and TyrB obtained showed 65-fold and 50-fold increases, respectively, compared with the wild-type level. Employing resting cells of AspC- and TyrB-overproducing E. coli K-12 strains for L-Phe productions resulted in molar conversion yields of 70% and 55%, respectively. With an additional introduction of phosphoenolpyruvate carboxykinase (encoded by pck) into the transamination reactions, the conversion yields were improved to 93% from 70% and to 75% from 55% in a relatively short time. These results account for more than an 8-fold increase in productivity, as compared to the previous report (Calton et al., 1985). In addition, a four-run reuse of the recombinant cells for L-Phe production gave a total yield of 91 g/L with a 93% conversion.

Repeated fed-batch operations for microbial detoxification of mercury using wild-type and recombinant mercury-resistant bacteria.

A wild-type mercury-resistant strain Pseudomonas aeruginosa PU21 (Rip64), and an Escherichia coli PWS1 strain genetically engineered to harbor mercury resistance were examined for their capacity to detoxify soluble mercuric ions with repeated fed-batch operations. The specific mercury detoxification activity for the two strains at different initial mercury concentrations was determined by resting-cell experiments. The fed-batch operations were conducted with different initial culture volumes (Vo), inoculum sizes (Xo), and different mercury feeding rates (FHg) to investigate the effects of those operation parameters on the performance of mercury detoxification. The results showed that the wild-type and the recombinant strains had an optimal specific activity of 5 x 10(-7) and 8 x 10(-8) micrograms cell-1 h-1, respectively. In fed-batch operation for P. aeruginosa PU21, under the conditions of Vo = 400 ml and Xo = 4.5-4.8 x 10(9) cells ml-1 the overall mercury detoxification efficiency (eta) for FHg = 16.9 mg Hg h-1 was 5.26 mg Hg l-1 h-1, nearly 35% higher than that for a lower FHg (11.7 mg Hg h-1). Among the three initial culture volumes examined in this study, the highest eta (5.60 mg Hg l-1 h-1) was obtained when Vo = 1200 ml and FHg = 16.9 mg Hg h-1. It was also found that an inoculum size higher than 4.0 x 10(9) cells ml-1 enabled a stable fed-batch operation, while as the inoculum was reduced to around 1.6 x 10(9) cells ml-1, the mercury feeding caused severe cell death, leading to an unsuccessful fed-batch operation. In the fed-batch operation for E. coli PWS1 strain with Vo = 1200 ml and FHg = 16.9 mg Hg h-1, the mercury detoxification efficiency was 3.07 mg Hg l-1 h-1, only 54% of that for the wild-type P. aeruginosa PU21 strain under the same operating conditions. It was also noticed that the operation with E. coli PWS1 became less efficient at the second fed-batch cycle due to plasmid instability of the recombinant strain.

Pathway analysis, engineering, and physiological considerations for redirecting central metabolism.

The rate and yield of producing a metabolite is ultimately limited by the ability to channel metabolic fluxes from central metabolism to the desired biosynthesis pathway. Redirection of central metabolism thus is essential to high-efficiency production of biochemicals. This task begins with pathway analysis, which considers only the stoichiometry of the reaction networks but not the regulatory mechanisms. An approach extended from convex analysis is used to determine the basic reaction modes, which allows the determination of optimal and suboptimal flux distributions, yield, and the dispensable sets of reactions. Genes responsible for reactions in the same dispensable set can be deleted simultaneously. This analysis serves as an initial guideline for pathway engineering. Using this analysis, we successfully constructed an Escherichia coli strain that can channel the metabolic flow from carbohydrate to the aromatic pathway with theoretical yield. This analysis also predicts a novel cycle involving phosphoenolpyruvate (PEP) carboxykinase (Pck) and the glyoxylate shunt, which can substitute the tricarboxylic acid cycle with only slightly less efficiency. However, the full cycle could not be confirmed in vivo, possibly because of the regulatory mechanism not considered in the pathway analysis.In addition to the kinetic regulation, we have obtained evidence suggesting that central metabolites are involved in specific regulons in E. coli. Overexpression of PEP-forming enzymes (phosphoenolpyruvate synthase [Pps] and Pck) stimulates the glucose consumption rate, represses the heat shock response, and negatively regulates the Ntr regulon. These results suggest that some glycolytic intermediates may serve as a signal in the regulation of the phosphotransferase system, heat shock response, and nitrogen regulation. However, the role of central metabolites in these regulations has not been determined conclusively. (c) 1996 John Wiley & Sons, Inc.

Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli.

Fermentative production of succinic acid from glucose by Escherichia coli was significantly increased by overexpression of phosphoenolpyruvate carboxylase. In contrast, overexpression of phosphoenolpyruvate carboxykinase had no effect. Under optimized conditions, induction of the carboxylase resulted in a 3.5-fold increase in the concentration of succinic acid, making succinic acid the major fermentation product by weight.

A mutant phosphoenolpyruvate carboxykinase in Escherichia coli conferring oxaloacetate decarboxylase activity.

The phosphoenolpyruvate carboxykinase in Escherichia coli (encoded by pck) catalyzes the conversion from oxaloacetate (OAA) to phosphoenolpyruvate under gluconeogenic conditions. We report here the characterization of two mutant alleles, pck-51 and pck-53, both of which are point mutations leading to single amino acid changes (D to N at position 268 and G to S at position 284, respectively). Pck51 is an altered-activity mutant that catalyzes the conversion from OAA to pyruvate (OAA decarboxylase activity). This new activity was not detected from the wild-type Pck, and it complements the pck null mutation only in a pps+ background. Pck53 is a reduced-activity mutant that complements the pck null mutation in a strain-dependent fashion.

Alteration of the biochemical valves in the central metabolism of Escherichia coli.

Although E. coli central metabolism has been studied for several decades, many regulatory features are still unknown. To achieve the goal of rational manipulation of cellular metabolism, it is important to understand how E. coli responds to overexpressed enzymes. By studying the biochemical control of fluxes between PEP, pyruvate, and OAA, we have addressed some fundamental questions that may prove to be essential for applications in metabolic engineering. First, we found that simultaneous overexpression of Pck and Ppc, or Pps alone in the presence of glucose leads to phenotypes consistent with futile cycline. In contrast to our expectation, futile cycling per se does not affect the growth rate significantly. However, excessive futile cycling may cause competitive disadvantage in the natural environment. Overexpression of Pck caused growth inhibition but no futile cycling. Therefore, E. coli controls the expression of gluconeogenic enzymes not only to avoid excessive futile cycling, but also to prevent toxicity effects. In metabolic engineering, futile cycling may be used as a strategy to stimulate metabolism for either production of metabolites or digestion of toxic wastes. Second, we found that the expression levels of Pps and Pck in E. coli are not optimal for growth on pyruvate and succinate, respectively. Overexpression of these enzymes increases the growth rate on pyruvate and on succinate, respectively, indicating that the slow growth rates on these substrates are at least partially caused by the insufficient supply of PEP and its derivatives. Moreover, E. coli also has not optimized the Ppc level for optimal growth yield on glucose in uncontrolled batch cultures. These results demonstrate that the central metabolism is not optimized for growth under defined laboratory conditions. Thus, the possibility exists that adjustment of native enzyme levels in the central metabolism can improve bioreactor performance. Third, we found that overexpression of Pck affects the transcriptional levels of unrelated genes. This example indicates that physiological responses to enzyme (over)expression should be interpreted cautiously, as changing the expression level of a specific enzyme may affect many unlinked genes. Similar results have also been obtained by use of two-dimensional electrophoresis of proteins from E. coli. Although more questions remain to be answered, fast progress in the area of metabolic engineering can be expected in the near future.

Metabolic responses to substrate futile cycling in Escherichia coli.

A cyclic pathway between phosphoenolpyruvate and oxaloacetate was created in Escherichia coli by simultaneous overexpression of phosphoenolpyruvate carboxykinase (encoded by pck) and phosphoenopyruvate carboxylase (encoded by ppc) from a multicopy plasmid under the control of the tac promoter. The simultaneous overexpression of these two enzymes stimulated oxygen and glucose consumption, reduced growth yields, and resulted in high level excretion of pyruvate and acetate. These responses were abolished when either pck or ppc was deleted from the plasmid or when both enzymes were inactivated by mutation. Therefore, the observed effects imply the existence of futile cycling. Incremental induction of futile cycling showed that stimulation of oxygen consumption was the first response, followed by the increased glucose consumption and the excretion of fermentation products. The specific growth rate of E. coli was insensitive to futile cycling per se, because the growth rate was also reduced by the overexpression of inactive enzymes at high levels, and the activity of the two enzymes did not inhibit growth further. Wild-type cells appear to be capable of compensating for the increased ATP drain due to futile cycling but cannot be as effective when a tricarboxylic acid cycle enzyme, alpha-ketoglutarate dehydrogenase, is defective.

Alteration of growth yield by overexpression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase in Escherichia coli.

Phosphoenolpyruvate and oxaloacetate are key intermediates at the junction between catabolism and biosynthesis. Alteration of carbon flow at these branch points will affect the growth yield and the formation of products. We attempted to modulate the metabolic flow between phosphoenolpyruvate and oxaloacetate by overexpressing phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase from a multicopy plasmid under the control of the tac promoter. It was found that overexpression of phosphoenolpyruvate carboxylase decreased the rates of glucose consumption and organic acid excretion, but the growth and respiration rates remained unchanged. Consequently, the growth yield on glucose was improved. This result indicates that the wild-type level of phosphoenolpyruvate carboxylase is not optimal for the most efficient glucose utilization in batch cultures. On the other hand, overexpression of phosphoenolpyruvate carboxykinase increased glucose consumption and decreased oxygen consumption relative to those levels required for growth. Therefore, the growth yield on glucose was reduced because of a higher rate of fermentation product excretion. These data provide useful insights into the regulation of central metabolism and facilitate further manipulation of pathways for metabolite production.

Control of gluconeogenic growth by pps and pck in Escherichia coli.

It is well-known that Escherichia coli grows more slowly on gluconeogenic carbon sources than on glucose. This phenomenon has been attributed to either energy or monomer limitation. To investigate this problem further, we varied the expression levels of pck, encoding phosphoenolpyruvate carboxykinase (Pck), and pps, encoding phosphoenolpyruvate synthase (Pps). We found that the growth rates of E. coli in minimal medium supplemented with succinate and with pyruvate are limited by the levels of Pck and Pps, respectively. Optimal overexpression of pck or pps increases the unrestricted growth rates on succinate and on pyruvate, respectively, to the same level attained by the wild-type growth rate on glycerol. Since Pps is needed to supply precursors for biosyntheses, we conclude that E. coli growing on pyruvate is limited by monomer supply. However, because pck is required both for biosyntheses and catabolism for cells growing on succinate, it is possible that growth on succinate is limited by both monomer and energy supplies. The growth yield with respect to oxygen remains approximately constant, even though the overproduction of these enzymes enhances gluconeogenic growth. It appears that the constant yield for oxygen is characteristic of efficient growth on a particular substrate and that the yield is already optimal for wild-type strains. Further increases in either Pck or Pps above the optimal levels become growth inhibitory, and the growth yield for oxygen is reduced, indicating less efficient growth.