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Vibrio vulnificus, a marine bacterium capable of causing wound infection and septicemia, secretes a 45-kDa metalloprotease (vEP) with many biological activities. The precursor of vEP consists of four regions: a signal peptide, an N-terminal propeptide (nPP), a C-terminal propeptide, and the mature protease. Two forms of vEP--vEP-45, which contains the mature protease plus the C-terminal propeptide, and vEP-34, which contains only the mature protease--were expressed in Escherichia coli and purified. vEP-45 and vEP-34 had similar activities with azocasein as a substrate, but vEP-34 had reduced activity toward insoluble proteins. The nPP of vEP was expressed as a His tag fusion protein, and its effect on vEP activity was investigated. nPP inhibited the activities of both vEP-45 and vEP-34 but not that of thermolysin, a different but related zinc-dependent protease. The inhibition of vEP by nPP was further examined using vEP-34 as a representative enzyme. The inhibition could be completely reversed under conditions of low enzyme and propeptide concentrations and with prolonged incubation, which resulted from the degradation of nPP by vEP. However, even at high nPP and vEP concentrations, inhibition of vEP by nPP at high temperatures was not effective, resulting in the degradation of both nPP and vEP. These results demonstrate that the nPP of vEP could bind to vEP and inhibit its activity, resulting in the degradation of the propeptide.

Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to the consumption of raw seafood. It secretes a metalloprotease that is associated with skin lesions and serious hemorrhagic complications. In this study, we purified and characterized an extracellular metalloprotease (designated as vEP) having prothrombin activation and fibrinolytic activities from V. vulnificus ATCC 29307. vEP could cleave various blood clotting-associated proteins such as prothrombin, plasminogen, fibrinogen, and factor Xa, and the cleavage could be stimulated by addition of 1 mM [Mn.sup.2+] in the reaction. The cleavage of prothrombin produced active thrombin capable of converting fibrinogen to fibrin. The formation of active thrombin appeared to be transient, with further cleavage resulting in a loss of activity. The cleavage of plasminogen, however, did not produce an active plasmin, vEP could cleave all three major chains of fibrinogen without forming a clot. It could cleave fibrin polymer formed by thrombin as well as the cross-linked fibrin formed by factor XIIIa. In addition, vEP could also cleave plasma proteins such as bovine serum albumin and gamma globulin, and its broad specificity is reflected in the cleavage sites, which include [Asp.sup.207]-[Phe.sup.208] and [Thr.sup.272]-[Ala.sup.273] bonds in prothrombin and a [Tyr.sup.80]-[Leu.sup.81] bond in plasminogen. Taken together, the data suggest that vEP is a broad-specificity protease that could function as a prothrombin activator and a fibrinolytic enzyme to interfere with blood homeostasis as part of the mechanism associated with the pathogenicity of V. vulnificus in humans and thereby facilitate the development of systemic infection.

The side chain of residue 561 and the backbone atoms of residue 560 are critical for the proper function of pyruvate decarboxylase from Zymomonas mobilis. The carboxy-terminus locks in the cofactors and closes the active site in preparation for decarboxylation.

BACKGROUND: Increasing evidence has established neuroinflammation as the hallmark of neurodegenerative disorders such as Alzheimer's disease (AD). However, despite the underlying immunological mechanisms are far from being understood, the involvement of excessive activation of microglia is attracting more and more attention. OBJECTIVE: In the present study, we investigated the protective effect of black raspberry (BRB) anthocyanins on LPS-induced neuroinflammation in BV2 microglia. METHODS: LPS-induced mouse BV2 microglia were treated with black raspberry anthocyanins and the levels of NO, ROS, IL-1β and IL-18 produced by the cells were measured to determine the extent of oxidative stress and inflammatory response. RESULTS: The results showed that BRB anthocyanins reduced the production of ROS in LPS-induced BV2 microglia by down-regulating the level of NOX2 and its downstream factors, including thioredoxin-interacting protein (TXNIP) and NOD-like receptor protein 3 (NLRP3) inflammasome. Furthermore, BRB anthocyanins inhibited the secretion of Interleukin-18 (IL-18) and Interleukin-1β (IL-1β), eventually attenuating the LPS-induced inflammatory response of BV2 microglia. CONCLUSIONS: BRB anthocyanins might play an important neuroprotective role in inflammation-related neurodegenerative disease, potentially, by down-regulating the NOX2 /TXNIP/ NLRP3 signaling axis in brain microglia. [ABSTRACT FROM AUTHOR]

Background: The success of cuckoo parasitism is thought to depend largely on the extent of egg matching between cuckoo and host eggs, since poor-matching cuckoo egg would lead to more frequent egg rejection by the host. In this study, we investigated how egg-spot matching between the Common Cuckoo (Cuculus canorus) and its host, the Oriental Reed Warbler (Acrocephalus orientalis) is affected by the local parasitism rate, nest availability in breeding synchronization and egg rejection. Methods: We used the paired design of parasitized and their nearest non-parasitized nests where breeding occurred simultaneously to compare egg-spot matching. The image analysis was used to compare four eggshell pattern variables, namely spot size, density, coverage on the different areas of egg surface, and the distribution on the whole egg surface. Egg recognition experiments were conducted to test the effect of egg spots on egg rejection by the host. Results: Our results show that much better matching in almost all spot parameters tested on the side of the egg and the spot distribution on the whole egg occurred in parasitized nests than in non-parasitized nests. Matching of spot density between cuckoo and host eggs in parasitized nests increased with the synchronization between temporal availability of nests and the egg-laying period of female cuckoos. Egg recognition experiments in which the warbler eggs were deliberately painted with extra spots led to a significantly higher egg rejection rate (78.3%) than of unpainted eggs. Conclusion: Our data suggest that both the high temporal encounter rate between cuckoo and warbler nests as well as the high egg rejection ability of the host are important factors for egg-spot matching of the cuckoos. [ABSTRACT FROM AUTHOR]

The effect of dietary substitution of silkworm ( Bombyx mori L) meal ( SM) for fishmeal ( FM) on the growth performance and non-specific immunity of sea cucumber ( Apostichopus japonicus) (initial weight: 12.8 ± 0.16 g) was determined. Four isonitrogenous and isocaloric diets were formulated: Diet 1, which served as the control diet, contained 5% FM; Diet 2 contained 3.75% FM and 1.25% SM; Diet 3 contained 2.5% FM and 2.5% SM; and Diet 4 contained 5% SM. Other ingredients in each of the four diets were kept in the same proportion. After 8 weeks of feeding, the results showed that sea cucumbers fed Diet 2 had 18.7% increases in weight over those fed the control diet, but no significant difference was observed. No obvious difference in body wall composition was detected among the sea cucumbers fed the four different diets. Immunity analysis indicated that phagocytosis and serum alkaline phosphatase activity were not significantly ( P > 0.05) affected when FM was partially or completely replaced with SM. Serum lysozyme activity of sea cucumbers fed Diet 4 showed a significant ( P < 0.05) growth increase compared with those fed control diet. The results revealed that SM could be an effective substitute for FM in sea cucumber diet. [ABSTRACT FROM AUTHOR]

Growing genetic and epidemiological evidence suggests a direct connection between the disruption of circadian rhythm and breast cancer. Moreover, the expression of several molecular components constituting the circadian clock machinery has been found to be modulated by estrogen-estrogen receptor α (E2-ERα) signaling in ERα-positive breast cancer cells. In this study, we investigated the regulation of CLOCK expression by ERα and its roles in cell proliferation. Immunohistochemical analysis of human breast tumor samples revealed high expression of CLOCK in ERα-positive breast tumor samples. Subsequent experiments using ERα-positive human breast cancer cell lines showed that both protein and mRNA levels of CLOCK were up-regulated by E2 and ERα. In these cells, E2 promoted the binding of ERα to the EREs (estrogen-response elements) of CLOCK promoter, thereby up-regulating the transcription of CLOCK. Knockdown of CLOCK attenuated cell proliferation in ERα-positive breast cancer cells. Taken together, these results demonstrated that CLOCK could be an important gene that mediates cell proliferation in breast cancer cells. [ABSTRACT FROM AUTHOR]

3β-Hydroxysteroid-Δ24 reductase (DHCR24) is an endoplasmic reticulum (ER)-localized multifunctional enzyme that possesses anti-apoptotic and cholesterol-synthesizing activities. Accumulating evidence suggests that ER stress is involved in the pathogenesis of neurodegenerative disease. In this study, we investigated whether DHCR24 may function as a neuroprotective protein under ER stress. Neuroblastoma N2A cells were infected with adenovirus expressing myc-tagged DHCR24 (Ad-DHCR24) or lacZ (Ad-lacZ, serving as a control) and subjected to ER-stress, induced with Tunicamycin (TM). Cells infected with Ad-DHCR24-myc were resistant to TM-induced apoptosis, and showed weaker level of caspase-12 activity. These cells also exhibited lower levels of Bip and CHOP proteins than Ad-LacZ-infected cells. Moreover, a stronger and rapid activation of PERK, and a prolonged activation of JNK and p38 were observed in Ad-LacZ–infected cells. The generation of intracellular reactive oxygen species from ER stress was also diminished by the overexpression of DHCR24. Additionally, intracellular cholesterol level was also elevated in the Ad-DHCR24-infected cells, accompanied by a well-organized formation of caveolae (cholesterol-rich microdomain) on the plasma membrane, and improved colocalization of caveolin-1 and insulin-like growth factor 1 receptor. These results demonstrated for the first time that DHCR24 could protect neuronal cells from apoptosis induced by ER stress. [ABSTRACT FROM AUTHOR]

Epithelial Mesenchymal Transition (EMT) plays a major role in cancer metastasis. Several genes have been shown to play a role in EMT, and one of these is Amplified-in-breast cancer 1 (AIB1), which has oncogenic function and is known to be amplified in breast cancer. However, the role of AIB1 in EMT remains largely undefined at the molecular level. In this study, the effect of AIB1 overexpression on the EMT of the breast cancer cell line T47D was investigated. Overexpression of AIB1 disrupted the epithelial morphology of the cells. At the same time, the cells displayed a strong metastasis and reduced level of the epithelial marker E-cadherin. In contrast, knockdown of AIB1 in T47D cells increased cell-cell adhesion and produced weak metastasis, as well as a higher level of E-cadherin expression. We proposed that the regulation of EMT by AIB1 occurred through the action of the transcription factor SNAI1, and demonstrated that such interaction required the participation of ERα and the presence of ERα-binding site on SNAI1 promoter. The expression level of E-cadherin and the extent of cell migration and invasion in SNAI1-knocked down T47D cells that overexpressed AIB1 were similar to those of T47D cells that did not overexpress AIB1 and had no SNAI1 knockdown. Taken together, these results suggested that AIB1 exerted its effect on EMT through its interaction with ERα, which could directly bind to the ERα-binding site on the SNAI1 promoter, allowing the AIB1-ERα complex to promote the transcription of SNAI1 and eventually led to repression of E-cadherin expression, consistent with the loss of E-cadherin being a hallmark of EMT. [ABSTRACT FROM AUTHOR]

Background: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a phosphatase having both protein and lipid phosphatase activities, and is known to antagonize the phosphoinositide 3-kinase/AKT (PI3K/ AKT) signaling pathway, resulting in tumor suppression. PTEN is also known to play a role in the regulation of numerous transcription factors. Amplified in breast cancer 1 (AIB1) is a transcriptional coactivator that mediates the transcriptional activities of nuclear receptors and other transcription factors. The present study investigated how PTEN may regulate AIB1, which is amplified and/or overexpressed in many human carcinomas, including breast cancers. Results: PTEN interacted with AIB1 via its phophatase domain and regulated the transcriptional activity of AIB1 by enhancing the ubiquitin-mediated degradation of AIB1. This process did not appear to require the phosphatase activity of PTEN, but instead, involved the interaction between PTEN and F-box and WD repeat domain-containing 7 alpha (Fbw7a), the E3 ubiquitin ligase involved in the ubiquitination of AIB1. PTEN interacted with Fbw7a via its C2 domain, thereby acting as a bridge between AIB1 and Fbw7a, and this led to enhanced degradation of AIB1, which eventually accounted for its decreased transcriptional activity. At the cell level, knockdown of PTEN in MCF-7 cells promoted cell proliferation. However when AIB1 was also knocked down, knockdown of PTEN had no effect on cell proliferation. Conclusions: PTEN might act as a negative regulator of AIB1 whereby the association of PTEN with both AIB1 and Fbw7a could lead to the downregulation of AIB1 transcriptional activity, with the consequence of regulating the oncogenic function of AIB1. [ABSTRACT FROM AUTHOR]

Aryl hydrocarbon receptor (AhR) is a transcription factor that belongs to the basic helix-loop-helix (bHLH) Per-Arnt-Sim homology domain (PAS) family. AhR can be activated by 2, 3, 7, 8-tetrachlorodibenzo- p-dioxin (2, 3, 7, 8-TCDD) and once activated, it promotes the abnormal expression of cytochrome P450, leading to several diseases, including cancer. In this study, we showed that AhR is subjected to post-translational modification by SUMOylation and this modification could be reversed by SENP1. Two SUMOylation sites were identified, one in the bHLH domain (K63) and the other in the TAD domain (K510) of AhR. Substitution of either K63 or K510 with arginine resulted in reduced SUMOylation for AhR. Treatment of MCF-7 cells with TCDD led to a reduced level of SUMOylated AhR in a time-dependent manner, and this occurred mainly in the nucleus. SUMOylation of AhR enhanced its stability through inhibiting its ubiquitination. Moreover, SUMOylation also repressed the transactivation activity of AhR and this could be reversed by TCDD. These results suggested that SUMOylation of AhR might play an important role in the regulation of its function, and TCDD may activate the transcriptional activity of AhR through downregulating its SUMOylation. J. Cell. Physiol. 227: 3812-3819, 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background information Amplified in breast cancer 1 (AIB1) is a transcriptional coactivator of nuclear receptors and other transcription factors. It is required for animal growth and reproductive development, and has also been implicated in breast carcinogenesis. Although AIB1 is known to be covalently modified by SUMO-1, which serves to regulate its stability and transcriptional activity, the exact SUMO E3 ligase involved in its sumoylation has not been determined. In order to resolve this question, we investigated the interaction between AIB1 and different members of PIAS proteins (all are SUMO E3 ligases) through immunoprecipiation. Results Among the five different PIAS proteins, only PIAS1 co-immunoprecipitated with AIB1 in extract prepared from breast cancer cells (MCF-7). Over-expression of PIAS1 together with AIB1 in MCF-7 cells led to increased sumoylation of AIB1, resulting in repression of its transcriptional activity. In contrast, the PIAS1 mutant (C350S) lacking E3 ligase activity appeared to have no effect on the sumoylation of AIB1. Through sumoylation of AIB1, PIAS1 also promoted the stability of AIB1 and attenuated its interaction with estrogen receptor α (ERα), resulting in repression of the transactivation activity of ERα. In addition, MCF-7 cells co-transfected with wild-type PIAS1 and AIB1 showed about 40% reduction in cell growth, while cells co-transfected with wild-type PIAS1 and mutant AIB1 resistant to sumoylation showed about 34% increase in cell growth compared to cells transformed with wild-type AIB1 only. Conclusions Taken together, these results suggested that PIAS1 may play a crucial role in the regulation of AIB1 transcriptional activity through sumoylation. [ABSTRACT FROM AUTHOR]

Examines the mutagenesis of pyruvate decarboxylase (PDC) from Zymomonas mobilis. Identification of acetaldehyde as a normal product of PDC; Ability of the enzyme to produce acetoin as a by-product from the hydroxyethyl-ThDP reaction; Reversibility of acetaldehyde formation in wild-type zymomonas mobilis.

[Display omitted] • Mangosteen is a good source of xanthones with antidiabetic effect. • Six xanthones with PTP1B inhibitory activity were identified in mangosteen extract. • Garcinone E was the most effective PTP1B inhibitor (IC50 of 0.43 μM). • Mangosteen might exert its antidiabetic effect via its PTP1B-inhibiting xanthones. • The distribution of xanthones in eleven tissues has certain differences. Garcinia mangostana L. (mangosteen) is a tropical fruit that is rich in xanthones and is thought to have an anti-diabetic effect. In this study, we screened for the xanthones in mangosteen that could inhibit the activity of protein tyrosine phosphatase 1B (PTP1B), an enzyme that is targeted by diabetic drugs. Mice were orally administered mangosteen extract and blood samples were screened for the presence of PTP1B-interacting xanthones. Six such compounds (1 – 6) were identified by UF-HPLC-QTOF-MS and their inhibition against PTP1B was confirmed by activity assay. Among them, garcinone E (5) was found to be the most effective PTP1B inhibitor (IC 50 = 0.43 μM). Tissue distribution analysis showed that the six compounds were distributed in eleven tissues, including the liver, muscle, fat, stomach, large intestine, small intestine, brain, kidney, heart, lung, and spleen. The results demonstrated that mangosteen might be a promising source of natural compounds with high PTP1B-inhibitory activity. [ABSTRACT FROM AUTHOR]