Bioavailability of vitamin C from mashed potatoes and potato chips after oral administration in healthy Japanese men.

Potato (Solanum tuberosum) tubers contain vitamin C (VC) and commercial potato chips have more VC content per wet weight by dehydration during frying. However, intestinal absorption of VC from orally ingested potatoes and its transfer to the blood remains questionable. The present study was designed to determine whether the dietary consumption of potatoes affects VC concentration in plasma and urinary excretion of VC in human subjects. After overnight fasting, five healthy Japanese men between 22 and 27 years of age consumed 87 g mashed potatoes and 282 g potato chips. Each portion contained 50 mg of VC, 50 mg VC in mineral water and mineral water. Before and after a single episode of ingestion, blood and urine samples were collected every 30 min or 1 h for 8 h. When measured by subtraction of the initial baseline value before administration of potatoes from the values measured throughout the 8 h test period, plasma VC concentrations increased almost linearly up to 3 h. Subsequently, the values of potato-fed subjects were higher than those of water, but did not differ significantly from those of VC in water (P = 0·14 and P = 0·5). Less VC tended to be excreted in urine during the 8 h test than VC in water alone (17·0 (sem 7·5) and 25·9 (sem 8·8) v. 47·9 (sem 17·9) µmol/mmol creatinine). Upon human consumption, mashed potatoes and potato chips provide VC content that is effectively absorbed in the intestine and transferred to the blood. Clearly, potatoes are a readily available source of dietary VC.

Improvement of multiple-stress tolerance and lactic acid production in Lactococcus lactis NZ9000 under conditions of thermal stress by heterologous expression of Escherichia coli DnaK.

The effects of nisin-induced dnaK expression in Lactococcus lactis were examined, and this expression was shown to improve stress tolerance and lactic acid fermentation efficiency. Using a nisin-inducible expression system, DnaK proteins from L. lactis (DnaK(Lla)) and Escherichia coli (DnaK(Eco)) were produced in L. lactis NZ9000. In comparison to a strain harboring the empty vector pNZ8048 (designated NZ-Vector) and one expressing dnaK(Lla) (designated NZ-LDnaK), the dnaK(Eco)-expressing strain, named NZ-EDnaK, exhibited more tolerance to heat stress at 40 degrees C in GM17 liquid medium. The cell viability of NZ-Vector was reduced 4.6-fold after 6 h of heat treatment. However, NZ-EDnaK showed 13.5-fold increased viability under these conditions, with a very low concentration of DnaK(Eco) production. Although the heterologous expression of dnaK(Eco) did not effect DnaK(Lla) production, heat treatment increased the DnaK(Lla) level 3.5- and 3.6-fold in NZ-Vector and NZ-EDnaK, respectively. Moreover, NZ-EDnaK showed tolerance to multiple stresses, including 3% NaCl, 5% ethanol, and 0.5% lactic acid (pH 5.47). In CMG medium, the lactate yield and the maximum lactate productivity of NZ-EDnaK were higher than the corresponding values for NZ-Vector at 30 degrees C. Interestingly, at 40 degrees C, these values of NZ-EDnaK were not significantly different from the corresponding values for the control strain at 30 degrees C. Lactate dehydrogenase (LDH) activity was also found to be stable at 40 degrees C in the presence of DnaK(Eco). These findings suggest that the heterologous expression of dnaK(Eco) enhances the quality control of proteins and enzymes, resulting in improved growth and lactic acid fermentation at high temperature.

The proper ratio of GrpE to DnaK is important for protein quality control by the DnaK-DnaJ-GrpE chaperone system and for cell division.

A balance of the intracellular concentrations of molecular chaperones in response to environmental conditions is of considerable importance for cellular homeostasis. Here, the physiological consequences of overexpression of GrpE in wild-type Escherichia coli MC4100 were examined. Overexpression of GrpE resulted in defects in cell division and growth, but overexpression of GrpE-G122D, which carries the G122D point mutation resulting in impaired interaction with DnaK, did not; this indicated that the effect of GrpE overexpression could be related to the DnaK chaperone function. Phase-contrast and fluorescence micrographs suggested that the N-terminal GFP-fused GrpE was colocalized with DnaK on the surface of inclusion bodies. An in vitro luciferase-refolding activity assay using purified DnaK, DnaJ and GrpE proteins demonstrated that high concentrations of GrpE significantly inhibited DnaK-mediated refolding. Furthermore, cell-free extracts from wild-type cells and GrpE-G122D-overexpressing cells significantly enhanced the refolding of luciferase. In the GrpE-overexpressing cells, abnormal localization of the cell-division protein FtsZ was observed by indirect immunofluorescence microscopy. In conclusion, the overexpression of GrpE caused a defect in the functionality of the DnaK chaperone system; this would result in filamentous morphology via abnormalities in the cell-division machinery.

Construction of Escherichia coli dnaK-deletion mutant infected by lambdaDE3 for overexpression and purification of recombinant GrpE proteins.

Escherichia coli is widely employed to produce recombinant proteins because this microorganism is simple to manipulate, inexpensive to culture, and of short duration to produce a recombinant protein. However, contamination of molecular chaperone DnaK during purification of the recombinant protein is sometimes a problem, since DnaK sometimes has a negative effect on subsequent experiments. Previously, several efforts have been done to remove the DnaK contaminants by several sequential chromatography or washing with some expensive chemicals such as ATP. Here, we developed a simple and inexpensive method to express and purify recombinant proteins based on an E. colidnaK-deletion mutant. The E. coli DeltadnaK52 mutant was infected by lambdaDE3 phage to overexpress desired recombinant proteins under the control of T7 promoter. Using this host cell, recombinant hexa histidine-tag fused GrpE, which is well known as a co-chaperone for DnaK and to strongly interact with DnaK, was overexpressed and purified by one-step nickel affinity chromatography. As a result, highly purified recombinant GrpE was obtained without washing with ATP. The purified recombinant GrpE showed a folded secondary structure and a dimeric structure as previous findings. In vitro ATPase activity assay and luciferase-refolding activity assay demonstrated that the recombinant GrpE worked together with DnaK. Thus, this developed method would be rapid and useful for expression and purification of recombinant proteins which is difficult to remove DnaK contaminants.

In vivo and in vitro complementation study comparing the function of DnaK chaperone systems from halophilic lactic acid bacterium Tetragenococcus halophilus and Escherichia coli.

In this study, we characterized the DnaK chaperone system from Tetragenococcus halophilus, a halophilic lactic acid bacterium. An in vivo complementation test showed that under heat stress conditions, T. halophilus DnaK did not rescue the growth of the Escherichia coli dnaK deletion mutant, whereas T. halophilus DnaJ and GrpE complemented the corresponding mutations of E. coli. Purified T. halophilus DnaK showed intrinsic weak ATPase activity and holding chaperone activity in vitro, but T. halophilus DnaK did not cooperate with the purified DnaJ and GrpE from either T. halophilus or E. coli in ATP hydrolysis or luciferase-refolding reactions under the conditions tested. E. coli DnaK, however, cross-reacted with those from both bacteria. This difference in the cooperation with DnaJ and GrpE appears to result in an inability of T. halophilus DnaK to replace the in vivo function of the DnaK chaperone of E. coli.

A gram-negative characteristic segment in Escherichia coli DnaK is essential for the ATP-dependent cooperative function with the co-chaperones DnaJ and GrpE.

We describe importance of the characteristic segment in ATPase domain of DnaK chaperone which is present in all gram-negative bacteria but is absent in all gram-positive bacteria. In vitro studies, ATPase activity, luciferase-refolding activity, and surface plasmon resonance analyses, demonstrated that a segment-deletion mutant DnaKDelta74-96 became defective in the cooperation with the co-chaperones DnaJ and GrpE. In addition, in vivo complementation assay showed that expression of DnaKDelta74-96 could not rescue the viability of Escherichia coli DeltadnaK mutant at 43 degrees C. Consequently, we suggest evolutionary significance for this DnaK ATPase domain segment in gram-negative bacteria towards the DnaK chaperone system.