Evaluation of GABA Production and Probiotic Activities of Enterococcus faecium BS5

Probiotics and Antimicrobial Proteins - Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of...     

Role of p38 mitogen-activated protein kinase phosphorylation and Fas-Fas ligand interaction in morphine-induced macrophage apoptosis

In this study, we evaluated the molecular mechanisms involved in morphine-induced macrophage apoptosis. Both morphine and TGF-beta promoted P38 mitogen-activated protein kinase (MAPK) phosphorylation, and this phosphorylation was inhibited by SB 202190 as well as by SB 203580. Anti-TGF-beta Ab as well as naltrexone (an opiate receptor antagonist) inhibited morphine-induced macrophage P38 MAPK phosphorylation. Anti-TGF-beta Ab also attenuated morphine-induced p53 as well as inducible NO synthase expression; in contrast, N(G)-nitro-L-arginine methyl ester, an inhibitor of NO synthase, inhibited morphine-induced P38 MAPK phosphorylation and Bax expression. Morphine also enhanced the expression of both Fas and Fas ligand (FasL), whereas anti-FasL Ab prevented morphine-induced macrophage apoptosis. Moreover, naltrexone inhibited morphine-induced FasL expression. In addition, macrophages either deficient in FasL or lacking p53 showed resistance to the effect of morphine. Inhibitors of both caspase-8 and caspase-9 partially prevented the apoptotic effect of morphine on macrophages. In addition, caspase-3 inhibitor prevented morphine-induced macrophage apoptosis. These findings suggest that morphine-induced macrophage apoptosis proceeds through opiate receptors via P38 MAPK phosphorylation. Both TGF-beta and inducible NO synthase play an important role in morphine-induced downstream signaling, which seems to activate proteins involved in both extrinsic (Fas and FasL) and intrinsic (p53 and Bax) cell death pathways.     

Monolayer formation of human osteoblastic cells on vertically aligned multiwalled carbon nanotube scaffolds

Monolayer formation of SaOS‐2 (human osteoblast‐like cells) was observed on VACNT (vertically aligned multiwalled carbon nanotubes) scaffolds without purification or functionalization. The VACNT were produced by a microwave plasma chemical vapour deposition on titanium surfaces with nickel or iron as catalyst. Cell viability and morphology studies were evaluated by LDH (lactate dehydrogenase) release assay and SEM (scanning electron microscopy), respectively. The non‐toxicity and the flat spreading with monolayer formation of the SaOs‐2 on VACNT scaffolds surface indicate that they can be used for biomedical applications.     

Resveratrol ameliorates high glucose-induced protein synthesis in glomerular epithelial cells

High glucose-induced protein synthesis in the glomerular epithelial cell (GEC) is partly dependent on reduction in phosphorylation of AMP-activated protein kinase (AMPK). We evaluated the effect of resveratrol, a phytophenol known to stimulate AMPK, on protein synthesis. Resveratrol completely inhibited high glucose stimulation of protein synthesis and synthesis of fibronectin, an important matrix protein, at 3 days. Resveratrol dose-dependently increased AMPK phosphorylation and abolished high glucose-induced reduction in its phosphorylation. We examined the effect of resveratrol on critical steps in mRNA translation, a critical event in protein synthesis. Resveratrol inhibited high glucose-induced changes in association of eIF4E with eIF4G, phosphorylation of eIF4E, eEF2, eEF2 kinase and, p70S6 kinase, indicating that it affects important events in both initiation and elongation phases of mRNA translation. Upstream regulators of AMPK in high glucose-treated GEC were explored. High glucose augmented acetylation of LKB1, the upstream kinase for AMPK, and inhibited its activity. Resveratrol prevented acetylation of LKB1 and restored its activity in high glucose-treated cells; this action did not appear to depend on SIRT1, a class III histone deacetylase. Our data show that resveratrol ameliorates protein synthesis by regulating the LKB1–AMPK axis.     

Viral proteomics: global evaluation of viruses and their interaction with the host

Viruses constantly adapt to and modulate the host environment during replication and propagation. Both DNA and RNA viruses encode multifunctional proteins that interact with and modify host cell proteins. While viral genomes were the first complete sequences known, the corresponding proteomes are only now elucidated, with some surprising results. Even more daunting is the task to globally monitor the impact of viral infection on the proteome of the host cell and many technical hurdles must still be overcome in order to facilitate robust and reproducible measurements. Further complicating the picture is the dynamic nature of proteins, including post-translational modifications, enzymatic cleavage and activation or destruction by proteolytic events. Nevertheless, several promising studies have been published using high-throughput methods directly measuring protein abundance. Particularly, quantitative or semiquantitative mass spectrometry-based analysis of viral and cellular proteomes are now being used to characterize viruses and their host interaction. In addition, the full set of interactions between viral and host proteins, the interactome, is beginning to emerge, with often unexpected interactions that need to be carefully validated. In this review, we will discuss two major areas of viral proteomics: first, virion proteomics (such as the protein characterization of viral particles) and second, proteoviromics, including the viral protein interactomics and the quantitative analysis of host cell proteome during viral infection.     

Immunophenotyping of human HT29 colon cancer cell primary tumours and their metastases in severe combined immunodeficient mice

The immunophenotype of HT29 human colon cancer cells implanted into severe combined immunodeficient mice was assessed in primary tumours and their metastases in the lungs using an indirect immunohistochemical method. After primary tumours were surgically removed, the metastases were given time to develop, thus paralleling the clinical situation. While vimentin was negative in both primary and secondary tumours, E-cadherin was present as membrane-bound labelling in the primary tumours only. Whereas the markers p53, MIB1, PCNA and CEA were consistently positive in both primary and metastatic tumours, CD44 variant 6 and CA125 were negative in metastases but positive in the primary tumours. There was a significant increase in the percentage of cells labelled for p53 in the primary tumours compared with the metastases. For the proliferation markers, there was no significant difference in labelling between primary tumours and metastases for MIB1. Of the cytokeratins examined, CK 20 gave the strongest and most consistent reaction in both primary and secondary tumours. The results indicate that, for certain immunohistochemical markers, results are the same in both primary tumours and metastases. Hence, in these cases, antigens that are expressed on the primary tumour as well as on the metastases can serve as target molecules for immunologically based forms of treatment of metastases. This revised version was published online in November 2006 with corrections to the Cover Date.     

Molecular Mechanism of BST2/Tetherin Downregulation by K5/MIR2 of Kaposi's Sarcoma-Associated Herpesvirus

K3/MIR1 and K5/MIR2 of Kaposi''s sarcoma-associated herpesvirus (KSHV) are viral members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family and contribute to viral immune evasion by directing the conjugation of ubiquitin to immunostimulatory transmembrane proteins. In a quantitative proteomic screen for novel host cell proteins downregulated by viral immunomodulators, we previously observed that K5, as well as the human immunodeficiency virus type 1 (HIV-1) immunomodulator VPU, reduced steady-state levels of bone marrow stromal cell antigen 2 (BST2; also called CD317 or tetherin), suggesting that BST2 might be a novel substrate of K5 and VPU. Recent work revealed that in the absence of VPU, HIV-1 virions are tethered to the plasma membrane in BST2-expressing HeLa cells. By targeting BST2, K5 might thus similarly overcome an innate antiviral host defense mechanism. Here we establish that despite its type II transmembrane topology and carboxy-terminal glycosylphosphatidylinositol (GPI) anchor, BST2 represents a bona fide target of K5 that is downregulated during primary infection by and reactivation of KSHV. Upon exit of the protein from the endoplasmic reticulum, lysines in the short amino-terminal domain of BST2 are ubiquitinated by K5, resulting in rapid degradation of BST2. Ubiquitination of BST2 is required for degradation, since BST2 lacking cytosolic lysines was K5 resistant and ubiquitin depletion by proteasome inhibitors restored BST2 surface expression. Thus, BST2 represents the first type II transmembrane protein targeted by K5 and the first example of a protein that is both ubiquitinated and GPI linked. We further demonstrate that KSHV release is decreased in the absence of K5 in a BST2-dependent manner, suggesting that K5 contributes to the evasion of intracellular antiviral defense programs.Bone marrow stromal cell antigen 2 (BST2) was recently identified as a host cell restriction factor that prevents the release of retroviral and filoviral particles from infected host cells (23). Human immunodeficiency virus type 1 (HIV-1) counteracts this antiviral function of BST2 by expressing the viral auxiliary protein VPU (41, 53). In the absence of VPU, virus particles are prevented from budding off the cellular membrane in cells that express BST2, resulting in virions being tethered to the plasma membrane. BST2 was therefore renamed tetherin (41), although questions still remain as to whether BST2 acts as the actual tether and whether BST2-dependent tethering occurs in all BST2-expressing cell types (36). Independently, BST2 was shown to be induced by type I and type II interferons (IFNs) (7), suggesting that BST2 is part of the innate antiviral response triggered in infected cells.Using a quantitative membrane proteomic approach, we observed that BST2 is underrepresented in plasma membranes from cells expressing not only VPU (14) but also the K5 protein of Kaposi''s sarcoma-associated herpesvirus (KSHV) (4). K5 is a viral homologue of a family of cellular transmembrane ubiquitin ligases, termed membrane-associated RING-CH (MARCH) proteins (3), that mediate the ubiquitination of the cytoplasmic portion of transmembrane proteins (reviewed in reference 40). Each member of this family targets a subset of cellular membrane proteins with both unique and shared specificities (4, 56). One of the functions of cellular MARCH proteins is to modulate antigen presentation by mediating the ubiquitin-dependent turnover of major histocompatibility complex (MHC) class II molecules in dendritic cells, B cells, and monocytes/macrophages (43, 52). In contrast, viral homologues of MARCH proteins encoded by KSHV, murine herpesvirus 68, and the leporipoxvirus myxomavirus all share the ability to mediate the destruction of MHC-I (reviewed in reference 16) but not MHC-II molecules. Thus, one of the functions of the viral proteins is to promote viral escape from immune clearance by CD8⁺ T lymphocytes (50). Furthermore, each viral MARCH homologue specifically eliminates additional host cell proteins, so each plays multiple roles in viral pathogenesis. KSHV carries two viral MARCH proteins, K3 and K5, also known as MIR1 and MIR2, which both support viral escape from T-cell, NK-cell, and NKT-cell recognition by eliminating the corresponding ligands from the surfaces of infected cells (reviewed in reference 10). In endothelial cells (ECs), K5 additionally downregulates EC-specific adhesion molecules that play an essential role in the formation of adhesive platforms and adherens junctions (31, 32). Since Kaposi''s sarcoma is a tumor of EC origin, K5 might thus also contribute to tumorigenesis by disrupting normal EC barrier function and by modulating the interaction of ECs with inflammatory leukocytes.The downregulation of BST2 by K5 further suggests that K5 also counteracts innate antiviral responses, which might benefit KSHV. However, most transmembrane proteins targeted by viral or cellular MARCH proteins are type I transmembrane proteins that belong to the immunoglobulin superfamily. In contrast, BST2 is a type II transmembrane protein that is also glycosylphosphatidylinositol (GPI) anchored (25). Thus, BST2 has a short cytoplasmic amino terminus followed by an outside-in transmembrane domain, a large glycosylated extracellular portion, and a GPI anchor. The additional propensity of BST2 to form homodimers (44) was speculated to be crucial for the tethering function of BST2 in that self-association of BST2 molecules in the viral envelope with plasma membrane BST2 could prevent viral exit (19). The unusual topology of BST2 and its multimerization raised the question of whether BST2 is a bona fide target of K5 or whether its downregulation is a downstream effect of K5 eliminating other transmembrane proteins. Additionally, it is not clear whether BST2 would be downregulated in the context of a normal viral infection and, particularly, whether virally expressed K5 would be able to overcome the high expression levels of BST2 observed upon IFN induction. We now demonstrate that KSHV efficiently downregulates IFN-induced BST2 both during primary infection and upon reactivation from latency in ECs. IFN-induced BST2 is ubiquitinated by K5 upon exiting the endoplasmic reticulum (ER) and is rapidly degraded by a pathway that is sensitive to proteasome inhibitors but resistant to inhibitors of lysosomal acidification. These data suggest that despite its unusual topology, BST2 is directly targeted by K5. We further demonstrate that BST2 reduces KSHV release upon inhibition of K5 expression by small interfering RNA (siRNA), suggesting that BST2 is part of the IFN-induced innate immune response to KSHV. Thus, in addition to contributing to viral evasion of cellular immune responses and remodeling EC function, K5 also counteracts the innate immune defense of the host cell.     

MicroRNA-21 orchestrates high glucose-induced signals to TOR complex 1, resulting in renal cell pathology in diabetes

Hyperglycemia induces a wide array of signaling pathways in the kidney that lead to hypertrophy and matrix expansion, eventually culminating in progressive kidney failure. High glucose-induced reduction of the tumor suppressor protein phosphatase and tensin homolog deleted in chromosome 10 (PTEN) contributes to renal cell hypertrophy and matrix expansion. We identified microRNA-21 (miR-21) as the molecular link between high glucose and PTEN suppression. Renal cortices from OVE26 type 1 diabetic mice showed significantly elevated levels of miR-21 associated with reduced PTEN and increased fibronectin content. In renal mesangial cells, high glucose increased the expression of miR-21, which targeted the 3'-UTR of PTEN mRNA to inhibit PTEN protein expression. Overexpression of miR-21 mimicked the action of high glucose, which included a reduction in PTEN expression and a concomitant increase in Akt phosphorylation. In contrast, expression of miR-21 Sponge, to inhibit endogenous miR-21, prevented down-regulation of PTEN and phosphorylation of Akt induced by high glucose. Interestingly, high glucose-stimulated miR-21 inactivated PRAS40, a negative regulator of TORC1. Finally, miR-21 enhanced high glucose-induced TORC1 activity, resulting in renal cell hypertrophy and fibronectin expression. Thus, our results identify a previously unrecognized function of miR-21 that is the reciprocal regulation of PTEN levels and Akt/TORC1 activity that mediate critical pathologic features of diabetic kidney disease.