Ruminal Prevotella spp. May Play an Important Role in the Conversion of Plant Lignans into Human Health Beneficial Antioxidants

Secoisolariciresinol diglucoside (SDG), the most abundant lignan in flaxseed, is metabolized by the ruminal microbiota into enterolignans, which are strong antioxidants. Enterolactone (EL), the main mammalian enterolignan produced in the rumen, is transferred into physiological fluids, with potentially human health benefits with respect to menopausal symptoms, hormone-dependent cancers, cardiovascular diseases, osteoporosis and diabetes. However, no information exists to our knowledge on bacterial taxa that play a role in converting plant lignans into EL in ruminants. In order to investigate this, eight rumen cannulated cows were used in a double 4×4 Latin square design and fed with four treatments: control with no flax meal (FM), or 5%, 10% and 15% FM (on a dry matter basis). Concentration of EL in the rumen increased linearly with increasing FM inclusion. Total rumen bacterial 16S rRNA concentration obtained using Q-PCR did not differ among treatments. PCR-T-RFLP based dendrograms revealed no global clustering based on diet indicating between animal variation. PCR-DGGE showed a clustering by diet effect within four cows that had similar basal ruminal microbiota. DNA extracted from bands present following feeding 15% FM and absent with no FM supplementation were sequenced and it showed that many genera, in particular Prevotella spp., contributed to the metabolism of lignans. A subsequent in vitro study using selected pure cultures of ruminal bacteria incubated with SDG indicated that 11 ruminal bacteria were able to convert SDG into secoisolariciresinol (SECO), with Prevotella spp. being the main converters. These data suggest that Prevotella spp. is one genus playing an important role in the conversion of plant lignans to human health beneficial antioxidants in the rumen.     

Structural characterization and interaction of periostin and bone morphogenetic protein for regulation of collagen cross-linking

Periostin appears to be a unique extracellular protein secreted by fibroblasts that is upregulated following injury to the heart or changes in the environment. Periostin has the ability to associate with other critical extracellular matrix (ECM) regulators such as TGF-β, tenascin, and fibronectin, and is a critical regulator of fibrosis that functions by altering the deposition and attachment of collagen. Periostin is known to be highly expressed in carcinoma cells, but not in normal breast tissues. The protein has a structural similarity to insect fasciclin-1 (Fas 1) and can be induced by transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP)-2. To investigate the molecular interaction of periostin and bone morphogenetic protein, we modeled these three-dimensional structures and their binding sites. We demonstrated direct interaction between periostin and BMP1/2 in vitro using several biochemical and biophysical assays. We found that the structures of the first, second, and fourth Fas1 domains in periostin are similar to that of the fourth Fas 1 domain of TGFBIp. However, the structure of the third Fas 1 domain in periostin is different from those of the first, second, and fourth Fas1 domains, while it is similar to the NMR structure of Fasciclin-like protein from Rhodobacter sphaeroides. These results will useful in further functional analysis of the interaction of periostin and bone morphogenetic protein.     

Angiotensin-(1-7) Attenuates Kidney Injury Due to Obstructive Nephropathy in Rats

Background

Angiotensin-(1–7) [Ang-(1–7)] counteracts many actions of the renin-angiotensin-aldosterone system. Despite its renoprotective effects, extensive controversy exists regarding the role of Ang-(1–7) in obstructive nephropathy, which is characterized by renal tubulointerstitial fibrosis and apoptosis.

Methods

To examine the effects of Ang-(1–7) in unilateral ureteral obstruction (UUO), male Sprague-Dawley rats were divided into three groups: control, UUO, and Ang-(1–7)-treated UUO rats. Ang-(1–7) was continuously infused (24 μg/[kg·h]) using osmotic pumps. We also treated NRK-52E cells in vitro with Ang II (1 μM) in the presence or absence of Ang-(1–7) (1 μM), Mas receptor antagonist A779 (1 μM), and Mas receptor siRNA (50 nM) to examine the effects of Ang-(1–7) treatment on Ang II-stimulated renal injury via Mas receptor.

Results

Angiotensin II (Ang II) and angiotensin type 1 receptor (AT₁R) protein expression was higher in UUO kidneys than in controls. Ang-(1–7) treatment also decreased proapoptotic protein expression in UUO kidneys. Ang-(1–7) also significantly ameliorated TUNEL positive cells in UUO kidneys. Additionally, Ang-(1–7) reduced profibrotic protein expression and decreased the increased tumor growth factor (TGF)-β1/Smad signaling present in UUO kidneys. In NRK-52E cells, Ang II induced the expression of TGF-β1/Smad signaling effectors and proapoptotic and fibrotic proteins, as well as cell cycle arrest, which were attenuated by Ang-(1–7) pretreatment. However, treatment with A779 and Mas receptor siRNA enhanced Ang II-induced apoptosis and fibrosis. Moreover, Ang II increased tumor necrosis factor-α converting enzyme (TACE) and decreased angiotensin-converting enzyme 2 (ACE2) expression in NRK-52E cells, while pretreatment with Ang-(1–7) or A779 significantly inhibited or enhanced these effects, respectively.

Conclusion

Ang-(1–7) prevents obstructive nephropathy by suppressing renal apoptosis and fibrosis, possibly by regulating TGF-β1/Smad signaling and cell cycle arrest via suppression of AT₁R expression. In addition, Ang-(1–7) increased and decreased ACE2 and TACE expression, respectively, which could potentially mediate a positive feedback mechanism via the Mas receptor.     

Construction and evaluation of shuttle vector, pGYC4α, based on pYC2 from Lactobacillus sakei

The shuttle vector pGYC4α (6,157 bp) was constructed based on the sigma-replicon plasmid pYC2 from Lactobacillus sakei BM5 isolated from kimchi. The vector contained inserts of the ColE1 replicon, α-amylase gene from Bacillus licheniformis containing its own signal peptide, and lactococcal promoter P32. Transformation and expression of a selection marker gene (α-amylase) with pGYC4α were demonstrated in Escherichia coli and several lactic acid bacteria (LAB). The highest α-amylase activity in LAB transformants was obtained in M17/0.25% glucose media with 0.5% CaCO(3). The segregational stability of the shuttle vector in LAB was 100% for more than 100 generations in the absence of antibiotic pressure. The developed vector might be useful as a genetic tool for food industries.     

Silencing of cytosolic NADP+-dependent isocitrate dehydrogenase by small interfering RNA enhances the sensitivity of HeLa cells toward staurosporine

Staurosporine induces the production of reactive oxygen species, which play an important causative role in apoptotic cell death. Recently, it was demonstrated that the control of cellular redox balance and the defense against oxidative damage is one of the primary functions of cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) by supplying NADPH for antioxidant systems. The present report shows that silencing of IDPc expression in HeLa cells greatly enhances apoptosis induced by staurosporine. Transfection of HeLa cells with an IDPc small interfering RNA (siRNA) markedly decreased activity of IDPc, enhancing the susceptibility of staurosporine-induced apoptosis reflected by DNA fragmentation, cellular redox status and the modulation of apoptotic marker proteins. These results indicate that IDPc may play an important role in regulating the apoptosis induced by staurosporine and the sensitizing effect of IDPc siRNA on the apoptotic cell death of HeLa cells offers the possibility of developing a modifier of cancer chemotherapy.     

A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.     

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

Pro-apoptotic effect and cytotoxicity of genistein and genistin in human ovarian cancer SK-OV-3 cells

We investigated the effects of genistein and genistin on proliferation and apoptosis of human ovarian SK-OV-3 cells and explored the mechanism for these effects. SK-OV-3 cells were treated with genistein and genistin at various concentrations (ranging from 1 to 100 muM) either alone or in combination for 24 and 48 h. Cell proliferation was estimated using an MTT assay, and cell cycle arrest was evaluated using FACS. Caspase-3 activity and annexin-based cell cycle analysis were used as measures of apoptosis. In addition, genistein- and genistin-induced cytotoxicity was determined by measuring release of LDH. Genistein treatment for 24 or 48 h substantially inhibited SK-OV-3 cell proliferation in a dose-dependent manner, and genistin treatment for 48 h also inhibited cell proliferation. Genistein caused cell cycle arrest at G2/M phase in dose- and time-dependent manner, and genistin caused cell cycle arrest not only at G2/M phase but also at G1 phase. Genistein markedly induced apoptosis and significantly increased LDH release, whereas genistin did not affect LDH release. Moreover, exposure to both genistein and genistin in combination for 48 h induced apoptosis without increasing LDH release. Genistein and genistin inhibit cell proliferation by disrupting the cell cycle, which is strongly associated with the arrest induction of either G1 or G2/M phase and may induce apoptosis. Based on our findings, we speculate that both genistein and genistin may prove useful as anticancer drugs and that the combination of genistein and genistin may have further anticancer activity.