The precise effects of protein intake on fractional synthesis rate (FSR) of muscle protein are still under debate. The sample size of these studies was small and the conclusions in young and elderly subjects were inconsistent. To assess the effect of dietary protein intake on the FSR level, we conducted a meta-analysis of controlled protein intake trials. Random-effects models were used to calculate the weighted mean differences (WMDs). Ten studies were included and effects of short-term protein intake were evaluated. In an overall pooled estimate, protein intake significantly increased the FSR (20 trials, 368 participants; WMD: 0.025%/h; 95%CI: 0.019-0.031; P < 0.0001). Meta-regression analysis suggested that the protein dose was positively related to the effect size (regression coefficient = 0.108%/h; 95%CI: 0.035, 0.182; P = 0.009). A subgroup analysis indicated that protein intake significantly increased FSR when the protein dose was ≤ 0.80 g/kg BW (16 trials, 308 participants; WMD: 0.027%/h; 95%CI: 0.019-0.031; P < 0.0001), but did not affect FSR when the protein dose was > 0.80 g/kg BW (4 trials, 60 participants; WMD: 0.016%/h; 95%CI: 0.004-0.029; P = 0.98). In conclusion, this study is the first integrated results showing that a short-term protein intake is effective at improving the FSR of muscle protein in the healthy elderly as well as young subjects. This beneficial effect seems to be dose-dependent when the dose levels of protein range from 0.08 to 0.80 g/kg BW.

Bifidobacterium bifidum, a common endosymbiotic inhabitant of the human gut, is considered a prominent probiotic microorganism that may promote health. We completely decrypted the 2.2-Mb genome sequence of B. bifidum BGN4, a strain that had been isolated from the fecal sample of a healthy breast-fed infant, and annotated 1,835 coding sequences.

Enzymes are thought to have evolved highly specific catalytic activities from promiscuous ancestral proteins. By analyzing a genome-scale model of Escherichia coli metabolism, we found that 37% of its enzymes act on a variety of substrates and catalyze 65% of the known metabolic reactions. However, it is not apparent why these generalist enzymes remain. Here, we show that there are marked differences between generalist enzymes and specialist enzymes, known to catalyze a single chemical reaction on one particular substrate in vivo. Specialist enzymes (i) are frequently essential, (ii) maintain higher metabolic flux, and (iii) require more regulation of enzyme activity to control metabolic flux in dynamic environments than do generalist enzymes. Furthermore, these properties are conserved in Archaea and Eukarya. Thus, the metabolic network context and environmental conditions influence enzyme evolution toward high specificity.

Background

Elucidation of a genotype-phenotype relationship is critical to understand an organism at the whole-system level. Here, we demonstrate that comparative analyses of multi-omics data combined with a computational modeling approach provide a framework for elucidating the phenotypic characteristics of organisms whose genomes are sequenced.

Results

We present a comprehensive analysis of genome-wide measurements incorporating multifaceted holistic data - genome, transcriptome, proteome, and phenome - to determine the differences between Escherichia coli B and K-12 strains. A genome-scale metabolic network of E. coli B was reconstructed and used to identify genetic bases of the phenotypes unique to B compared with K-12 through in silico complementation testing. This systems analysis revealed that E. coli B is well-suited for production of recombinant proteins due to a greater capacity for amino acid biosynthesis, fewer proteases, and lack of flagella. Furthermore, E. coli B has an additional type II secretion system and a different cell wall and outer membrane composition predicted to be more favorable for protein secretion. In contrast, E. coli K-12 showed a higher expression of heat shock genes and was less susceptible to certain stress conditions.

Conclusions

This integrative systems approach provides a high-resolution system-wide view and insights into why two closely related strains of E. coli, B and K-12, manifest distinct phenotypes. Therefore, systematic understanding of cellular physiology and metabolism of the strains is essential not only to determine culture conditions but also to design recombinant hosts.

The aim of this study was to investigate the effect of quercetin, a flavonoid, on the apoptotic pathway in a human prostate cell line (LNCaP). We observed that treatment of cells for 24 h with quercetin induced cell death in a dose-dependent manner. A sustained inhibition of the major survival signal, Akt, occurred in quercetin-treated cells. Treatment of LNCaP cells with an apoptosis inducing concentration of quercetin (100 μM) resulted in a rapid decrease in the inhibitory Ser(473) phosphorylation of Akt leading to inhibition of its kinase activity. Quercetin treatment (100 μM) also caused a decrease in Ser(136) phosphorylation of Bad, which is a downstream target of Akt. Protein interaction assay revealed that during treatment with quercetin, Bcl-xL dissociated from Bax and then associated with Bad. Our results also show that quercetin decreases the Bcl-xL:Bax ratio and increases translocation and multimerization of Bax to the mitochondrial membrane. The translocation is accompanied by cytochrome c release, and procaspases-3, -8 and -9 cleavage and increased poly (ADP-ribose) polymerase (PARP) cleavage. Similar results were observed in human colon cancer HCT116Bax+/+ cell line, but not HCT116Bax−/− cell line. Interestingly, at similar concentrations (100 μM), quercetin treatment did not affect the viability or rate of apoptosis in normal human prostate epithelial cell line (PrEC) and rat prostate epithelial cell line (YPEN-1). Our results indicate that the apoptotic processes caused by quercetin are mediated by the dissociation of Bax from Bcl-xL and the activation of caspase families in human prostate cancer cells.

Astaxanthin (AST) is a powerful antioxidant that occurs naturally in a wide variety of living organisms. We have investigated the role of AST in preventing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis of the substantia nigra (SN) neurons in the mouse model of Parkinson’s disease (PD) and 1-methyl-4-phenylpyridinium (MPP+)-induced cytotoxicity of SH-SY5Y human neuroblastoma cells. In in vitro study, AST inhibits MPP+-induced production of intracellular reactive oxygen species (ROS) and cytotoxicity in SH-SY5Y human neuroblastoma cells. Preincubation of AST (50 μM) significantly attenuates MPP+-induced oxidative damage. Furthermore, AST is able to enhance the expression of Bcl-2 protein but reduce the expression of α-synuclein and Bax, and suppress the cleavage of caspase-3. Our results suggest that the protective effects of AST on MPP+-induced apoptosis may be due to its anti-oxidative properties and anti-apoptotic activity via induction of expression of superoxide dismutase (SOD) and catalase and regulating the expression of Bcl-2 and Bax. Pretreatment with AST (30mg /kg) markedly increases tyrosine hydroxylase (TH)-positive neurons and decreases the argyrophilic neurons compared with the MPTP model group. In summary, AST shows protection from MPP+/MPTP-induced apoptosis in the SH-SY5Y cells and PD model mouse SN neurons, and this effect may be attributable to upregulation of the expression of Bcl-2 protein, downregulation of the expression of Bax and α-synuclein, and inhibition of the activation of caspase-3. These data indicate that AST may provide a valuable therapeutic strategy for the treatment of progressive neurodegenerative disease such as Parkinson’s disease.

Adaptive laboratory evolution (ALE) under controlled conditions has become a valuable approach for the study of the genetic and biochemical basis for microbial adaptation under a given selection pressure. Conventionally, the timescale in ALE experiments has been set in terms of number of generations. As mutations are believed to occur primarily during cell division in growing cultures, the cumulative number of cell divisions (CCD) would be an alternative way to set the timescale for ALE. Here we show that in short-term ALE (up to 40–50 days), Escherichia coli, under growth rate selection pressure, was found to undergo approximately 1011.2 total cumulative cell divisions in the population to produce a new stable growth phenotype that results from 2 to 8 mutations. Continuous exposure to a low level of the mutagen N-methyl-N′-nitro-N-nitrosoguanidine was found to accelerate this timescale and led to a superior growth rate phenotype with a much larger number of mutations as determined with whole-genome sequencing. These results would be useful for the fundamental kinetics of the ALE process in designing ALE experiments and provide a basis for its quantitative description.

Laboratory adaptive evolution studies can provide key information to address a wide range of issues in evolutionary biology. Such studies have been limited thus far by the inability of workers to readily detect mutations in evolved microbial strains on a genome scale. This limitation has now been overcome by recently developed genome sequencing technology that allows workers to identify all accumulated mutations that appear during laboratory adaptive evolution. In this study, we evolved Escherichia coli K-12 MG1655 with a nonnative carbon source, l-1,2-propanediol (l-1,2-PDO), for ∼700 generations. We found that (i) experimental evolution of E. coli for ∼700 generations in 1,2-PDO-supplemented minimal medium resulted in acquisition of the ability to use l-1,2-PDO as a sole carbon and energy source so that the organism changed from an organism that did not grow at all initially to an organism that had a growth rate of 0.35 h−1; (ii) six mutations detected by whole-genome resequencing accumulated in the evolved E. coli mutant over the course of adaptive evolution on l-1,2-PDO; (iii) five of the six mutations were within coding regions, and IS5 was inserted between two fuc regulons; (iv) two major mutations (mutations in fucO and its promoter) involved in l-1,2-PDO catabolism appeared early during adaptive evolution; and (v) multiple defined knock-in mutant strains with all of the mutations had growth rates essentially matching that of the evolved strain. These results provide insight into the genetic basis underlying microbial evolution for growth on a nonnative substrate.

Approximately 25% of patients with colorectal cancer develop metastases to the liver, and surgery is currently the best treatment available. But there are several patients who are unresectable, and isolated hepatic perfusion (IHP) offers a different approach in helping to treat these patients. IHP is a method used for isolating the liver and delivering high doses of chemotherapeutic agents. The efficacy of IHP has been improved by combining hyperthermia not only with chemotherapeutics but with other deliverable agents such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we used human colorectal cancer CX-1 cells and treated them with hyperthermia and TRAIL, causing cytotoxicity. We were able to demonstrate that the numbers of live cells were significantly reduced with hyperthermia and 10 ng/ml of TRAIL combined. We also showed that the effect of hyperthermia on TRAIL in our studies was enhancement of the apoptotic pathway by the promotion of JNK and BimEL activity as well as PARP cleavage. We have also used our CX-1 cells to generate tumors in Balb/c nude mice. With intra-tumoral injections of TRAIL combined with hyperthermia at 42°C, we were able to show a delayed onset of tumor growth in our xenograft model.

Astaxanthin (ATX), a naturally occurring carotenoid pigment, is a powerful biological antioxidant. In the present study, we investigated whether ATX pharmacologically offers neuroprotection against oxidative stress by cerebral ischemia. We found that the neuroprotective efficacy of ATX at the dose of 30 mg/kg (n = 8) was 59.5% compared with the control group (n = 3). In order to make clear the mechanism of ATX neuroprotection, the up-regulation inducible nitric oxide synthase (iNOS) and heat shock proteins (HSPs) together with the oxygen glucose deprivation (OGD) in SH-SY5Y cells were also investigated. The induction of various factors involved in oxidative stress processes such as iNOS was suppressed by the treatment of ATX at 25 and 50 µM after OGD-induced oxidative stress. In addition, Western blots showed that ATX elevated of heme oxygenase-1 (HO-1; Hsp32) and Hsp70 protein levels in in vitro. These results suggest that the neuroprotective effects of ATX were related to anti-oxidant activities in global ischemia.

Background

Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. Candida magnoliae JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from C. magnoliae JH110.

Results

The gene encoding a novel ER was isolated from an osmophilic yeast C. magnoliae JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from Trichosporonoides megachiliensis SNG-42. The intact coding region of ER from C. magnoliae JH110 was cloned, functionally expressed in Escherichia coli using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42°C and 5.5, respectively. Among various aldoses, the C. magnoliae JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the C. magnoliae JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme's absolute requirement of NADPH over NADH.

Conclusions

A novel ER enzyme and its corresponding gene were isolated from C. magnoliae JH110. The C. magnoliae JH110 ER with high activity and catalytic efficiency would be very useful for in vitro erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.

Study Design

Prospective controlled study.

Purpose

The results of conventional open surgery was compared with those from minimally invasive transforaminal lumbar interbody fusion (MI-TLIF) for lumbar fusion to determine which approach resulted in less postoperative paraspinal muscle degeneration.

Overview of Literature

MI TLIF is new surgical technique that appears to minimize iatrogenic injury. However, there aren't any reports yet that have quantitatively analyzed and proved whether there's difference in back muscle injury and degeneration between the minimally invasive surgery and conventional open surgery in more than 1 year follow-up after surgery.

Methods

This study examined a consecutive series of 48 patients who underwent lumbar fusion in our hospital during the period, March 2006 to March 2008, with a 1-year follow-up evaluation using MRI. There were 17 cases of conventional open surgery and 31 cases of MI-TLIF (31 cases of single segment fusion and 17 cases of multi-segment fusion). The digital images of the paravertebral back muscles were analyzed and compared using the T2-weighted axial images. The point of interest was the paraspinal muscle of the intervertebral disc level from L1 to L5. Picture archiving and communication system viewing software was used for quantitative analysis of the change in fat infiltration percentage and the change in cross-sectional area of the paraspinal muscle, before and after surgery.

Results

A comparison of the traditional posterior fusion method with MI-TLIF revealed single segment fusion to result in an average increase in fat infiltration in the paraspinal muscle of 4.30% and 1.37% and a decrease in cross-sectional area of 0.10 and 0.07 before and after surgery, respectively. Multi-segment fusion showed an average 7.90% and 2.79% increase in fat infiltration and a 0.16 and 0.10 decrease in cross-sectional area, respectively. Both single and multi segment fusion showed less change in the fat infiltration percentage and cross-sectional area, particularly in multi segment fusion. There was no significant difference between the two groups in terms of the radiologic results.

Conclusions

A comparison of conventional open surgery with MI-TLIF upon degeneration of the paraspinal muscle with a 1 year follow-up evaluation revealed that both single and multi segment fusion showed less change in fat infiltration percentage and cross-sectional area in the MI-TLIF but there was no significant difference between the two groups. This suggests that as time passes after surgery, there is no significant difference in the level of degeneration of the paraspinal muscle between surgical techniques.

Combined treatment with quercetin and TRAIL induced cytotoxicity and enhanced annexin V staining and poly (ADP-ribose) polymerase (PARP) cleavage in human prostate cancer cell lines DU-145 and PC-3. These indicators of apoptosis resulted from the activation of caspase-8, -9, and -3. Although the expression levels of FLIPs, cIAP1, cIAP2, and the Bcl-2 family were not changed in quercetin-treated cells, significant downregulation of survivin occurred. Knockdown survivin by siRNA significantly increased TRAIL-induced apoptosis. We hypothesized that quercetin-induced activation of MAPK (ERK, p38, JNK) is responsible for downregulation of survivin gene expression. To test this hypothesis, we selectively inhibited MAPK during treatment with quercetin. Our data demonstrated that inhibitor of ERK (PD98059), but not p38 MAPK (SB203580) or JNK (SP600125), significantly maintained the intracellular level of survivin during treatment with quercetin. Interestingly, PD98059 also prevented quercetin-induced deacetylation of histone H3. Data from survivin promoter activity assay suggest that the Sp1 transcription factor binds to the survivin promoter region and quercetin inhibits its binding activity through deacetylation of histone H3. Quercetin-induced activation of the ERK-MSK1 signal transduction pathway may be responsible for deacetylation of histone H3. Taken together, our findings suggest that quercetin enhances TRAIL induced apoptosis by inhibition of survivin expression, through ERK-MSK1-mediated deacetylation of H3.

We observed that treatment of prostate cancer cells for 24 h with wogonin, a naturally occurring monoflavonoid, induced cell death in a dose- and time-dependent manner. Exposure of wogonin to LNCaP cells was associated with increased intracellular levels of p21Cip-1, p27Kip-1, p53, and PUMA, oligomerization of Bax, release of cytochrome c from the mitochondria, and activation of caspases. We also confirmed the role of p53 by noting that knock-in in p53 expression by transfecting p53 DNA increased wogonin-induced apoptosis in p53-null PC-3 cells. To study the mechanism of PUMA upregulation, we determined the activities of PUMA promoter in the wogonin treated and untreated cells. Increase of the intracellular levels of PUMA protein was due to increase in transcriptional activity. Data from chromatin immunoprecipitation (ChIP) analyses revealed that wogonin activated the transcription factor p53 binding activity to the PUMA promoter region. We observed that the upregulation of PUMA mediated wogonin cytotoxicity. Further characterization of the transcriptional response to wogonin in HCT116 human colon cancer cells demonstrated that PUMA induction was p53-dependent; deficiency in either p53 or PUMA significantly protected HCT116 cells against wogonin-induced apoptosis. Also, wogonin promoted mitochondrial translocation and multimerization of Bax. Interestingly, wogonin (100 μM) treatment did not affect the viability of normal human prostate epithelial cells (PrEC). Taken together, these results indicate that p53-dependent transcriptional induction of PUMA and oligomerization of Bax play important roles in the sensitivity of cancer cells to apoptosis induced by caspase activation through wogonin.

Background

Escherichia coli has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in E. coli frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant E. coli. Here, we describe the successful use of the immobilized folding machineries for in vitro refolding with the examples of high yield refolding of a ribonuclease A (RNase A) and cyclohexanone monooxygenase (CHMO).

Results

We have generated refolding-facilitating media immobilized with three folding machineries, mini-chaperone (a monomeric apical domain consisting of residues 191–345 of GroEL) and two foldases (DsbA and human peptidyl-prolyl cis-trans isomerase) by mimicking oxidative refolding chromatography. For efficient and simple purification and immobilization simultaneously, folding machineries were fused with the positively-charged consecutive 10-arginine tag at their C-terminal. The immobilized folding machineries were fully functional when assayed in a batch mode. When the refolding-facilitating matrices were applied to the refolding of denatured and reduced RNase A and CHMO, both of which contain many cysteine and proline residues, RNase A and CHMO were recovered in 73% and 53% yield of soluble protein with full enzyme activity, respectively.

Conclusion

The refolding-facilitating media presented here could be a cost-efficient platform and should be applicable to refold a wide range of E. coli inclusion bodies in high yield with biological function.