Dynamic compression inhibits fibronectin fragment induced iNOS and COX-2 expression in chondrocyte/agarose constructs

Mechanical loading and the fibronectin fragments (FN-fs) are known to stimulate the anabolic and catabolic processes in articular cartilage, possible through pathways mediated by ^·NO. This study examined the combined effects of dynamic compression and the NH₂-hep I or COOH-hep II FN-fs on the expression levels of iNOS and COX-2 and production of ^·NO and PGE₂ release. Both types of fragments induced iNOS and COX-2 expression and stimulated the production of ^·NO release. This response was inhibited by dynamic compression. Inhibitor experiments indicated that both dynamic compression and the iNOS inhibitor were important in restoring cell proliferation and proteoglycan synthesis in the presence of the FN-fs. This is the first study which demonstrates a downregulation of the FN-f-induced iNOS and COX-2 expression by dynamic compression. The combination of mechanical and pharmacological interventions makes this study a powerful tool to examine further the interactions of biomechanics and cell signalling in osteoarthritis.     

The Herpes Simplex Virus Type 1 UL20 Protein and the Amino Terminus of Glycoprotein K (gK) Physically Interact with gB

Herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) and the UL20 protein (UL20p) are strictly required for virus-induced cell fusion, and mutations within either the gK or UL20 gene cause extensive cell fusion (syncytium formation). We have shown that gK forms a functional protein complex with UL20p, which is required for all gK and UL20p-associated functions in the HSV-1 life cycle. Recently, we showed that the amino-terminal 82 amino acids (aa) of gK (gKa) were required for the expression of the syncytial phenotype of the mutant virus gBΔ28 lacking the carboxyl-terminal 28 amino acids of gB (V. N. Chouljenko, A. V. Iyer, S. Chowdhury, D. V. Chouljenko, and K. G. Kousoulas, J. Virol. 83:12301-12313, 2009). This work suggested that the amino terminus of gK may directly or indirectly interact with gB and/or other viral glycoproteins. Two-way coimmunoprecipitation experiments revealed that UL20p interacted with gB in infected cells. Furthermore, the gKa peptide was coimmunoprecipitated with gB but not gD. Three recombinant baculoviruses were constructed, expressing the amino-terminal 82 aa of gKa together with either the extracellular portion of gB (30 to 748 aa), gD (1 to 340 aa), or gH (1 to 792 aa), respectively. Coimmunoprecipitation experiments revealed that gKa physically interacted with the extracellular portions of gB and gH but not gD. Three additional recombinant baculoviruses expressing gKa and truncated gBs encompassing aa 30 to 154, 30 to 364, and 30 to 500 were constructed. Coimmunoprecipitation experiments showed that gKa physically interacted with all three truncated gBs. Computer-assisted prediction of possible gKa binding sites on gB suggested that gKa may interact predominantly with gB domain I (E. E. Heldwein, H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. Harrison, Science 313:217-220, 2006). These results imply that the gK/UL20p protein complex modulates the fusogenic properties of gB and gH via direct physical interactions.Herpes simplex virus type 1 (HSV-1) can enter into cells via the fusion of its viral envelope with cellular membranes. Also, the virus can spread from infected to uninfected cells by causing virus-induced cell fusion, allowing virions to enter into uninfected cells without being exposed to extracellular spaces. These membrane fusion phenomena are known to be mediated by viral glycoproteins and other viral proteins (reviewed in reference 36). Although wild-type viruses cause a limited amount of virus-induced cell fusion, certain mutations cause extensive virus-induced cell-to-cell fusion (syncytial, or syn, mutations). These syncytial mutations are located predominantly within the UL20 gene (5, 27, 28); the UL24 gene (25, 38); the UL27 gene, encoding glycoprotein gB (7, 15, 18, 32); and the UL53 gene, coding for gK (6, 11, 24, 34, 35, 37).The presence of syncytial mutations within different viral genes, as well as other accumulating evidence, suggests that virus-induced cell fusion is mediated by the concerted action and interactions of the viral glycoproteins gD, gB, and gH/gL as well as gK and the membrane protein UL20p. Specifically, recent studies have shown that gD interacts with both gB and gH/gL (1, 2, 21). However, gB and gH/gL can also interact with each other even in the absence of gD (3). In this membrane fusion model, the binding of gD to its cognate receptors, including nectin-1, herpesvirus entry mediator (HVEM), and other receptors (8, 19, 30, 39-42), is thought to trigger sequential conformational changes in gH/gL and gB causing the fusion of the viral envelope with cellular membranes during virus entry as well as fusion among cellular membranes (22, 23). The transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (31, 43), suggesting that these four viral glycoproteins are necessary and sufficient for membrane fusion. However, this transient fusion system does not accurately depict virus-induced cell fusion. Specifically, viral glycoprotein K (gK) and the UL20 membrane protein (UL20p) have been shown to be strictly required for virus-induced cell fusion (10, 27, 29). Moreover, syncytial mutations within gK (6, 11, 24, 34, 35, 37) or UL20 (5, 27, 28) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than the wild-type virus into susceptible cells (17). In contrast, the transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while the coexpression of wild-type gK with gB, gD, and gH/gL was reported previously to inhibit cell fusion in certain cell lines (4). To date, there is no direct evidence that either gK or UL20p interacts with gB, gD, gH, or gL.The X-ray structure of the ectodomain of HSV-1 gB has been determined and was predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB (23). Single-amino-acid changes within the carboxyl terminus of gB located intracellularly as well as the deletion of the terminal 28 amino acids (aa) of gB cause extensive virus-induced cell fusion, presumably because they alter the extracellular conformation of gB (15, 31, 43). We have previously shown that HSV-1 gK and UL20p functionally and physically interact and that these interactions are absolutely necessary for their coordinate intracellular transport, cell surface expression, and functions in the HSV-1 life cycle (13, 16). In contrast to gB, syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (12), while syncytial mutations of UL20 are located within the amino terminus of UL20p shown to be located intracellularly (27).Recently, we showed that the a peptide composed of the amino-terminal 82 amino acids of gK (gKa) can complement in trans for gB-mediated cell fusion caused by the deletion of the carboxyl-terminal 28 amino acids of gB, suggesting that the gKa peptide interacted with gB or other viral glycoproteins involved in virus-induced cell fusion (10). In this work, we demonstrate that UL20p and the amino terminus of gKa physically interact with gB in infected cells, while the gKa peptide is also capable of binding to the extracellular portion of gH, suggesting that gK/UL20p modulates virus-induced cell fusion via direct interactions with gB and gH.     

FRAP analysis of membrane-associated proteins: lateral diffusion and membrane-cytoplasmic exchange

Obtaining quantitative kinetic parameters from fluorescence recovery after photobleaching (FRAP) experiments generally requires a theoretical analysis of protein mobility and appropriate solutions for FRAP recovery derived for a given geometry. Here we provide a treatment of FRAP recovery for a molecule undergoing a combined process of reversible membrane association and lateral diffusion on the plasma membrane for two commonly used bleach geometries: stripes, and boxes. Such analysis is complicated by the fact that diffusion of a molecule during photobleaching can lead to broadening of the bleach area, resulting in significant deviations of the actual bleach shape from the desired bleach geometry, which creates difficulty in accurately measuring kinetic parameters. Here we overcome the problem of deviations between actual and idealized bleach geometries by parameterizing, more accurately, the initial postbleach state. This allows for reconstruction of an accurate and analytically tractable approximation of the actual fluorescence distribution. Through simulated FRAP experiments, we demonstrate that this method can be used to accurately measure a broad range of combinations of diffusion constants and exchange rates. Use of this method to analyze the plextrin homology domain of PLC-δ1 in Caenorhabditis elegans results in quantitative agreement with prior analysis of this domain in other cells using other methods. Because of the flexibility, relative ease of implementation, and its use of standard, easily obtainable bleach geometries, this method should be broadly applicable to investigation of protein dynamics at the plasma membrane.     

C4' sugar oxidation of deoxyribonucleotide triphosphates by chromium(V) complexes

The Cr(V) complexes, bis(2-ethyl-2-hydroxybutyrato)oxochromate(V) ([OCr(V)(ehba)(2)](-)) and (2,2-bis(hydroxymethyl)-2-(bis(2-hydroxyethyl)amino)ethanolato)oxochromate(V) ([OCr(V)(BT)](2-)), were reacted with a series of deoxyribonucleotide triphosphates. Oxidation of deoxyribose at C4' was observed by measuring the amount of thiobarbituric acid reactive species (TBARS) produced in these reactions. For both compounds, the TBARS obtained with purine nucleotides was between 2.25 and 3.5 times greater than what was observed with pyrimidine nucleotide. This result suggests that the identity of the nucleic acid base can influence the hydrogen atom abstraction at C4'. Overall, the amount of product obtained with [OCr(V)(BT)](2-) was significantly less than what was observed with [OCr(V)(ehba)(2)](-), indicating that these two Cr(V) model complexes may oxidize DNA differently.     

Cutting Edge: Stimulation of dopamine D4 receptors induce T cell quiescence by up-regulating Kruppel-like factor-2 expression through inhibition of ERK1/ERK2 phosphorylation

The neurotransmitter dopamine (DA) is an important regulator of human T cell functions. Although it has been observed that DA, by acting through the D1/D5, D2, and D3 receptors, can activate resting T cells by stimulating the release of cytokines and the expression of surface integrins and also inhibit the proliferation of activated T cells by down-regulating nonreceptor tyrosine kinases, there is not yet a report indicating the functional significance of the D4 DA receptors present in these cells. The present work, for the first time, demonstrates that the stimulation of D4 DA receptors in human T cells induces T cell quiescence by up-regulating lung Krüppel-like factor-2 expression through the inhibition of ERK1/ERK2 phosphorylation. These results reveal a new link between the nervous system and T cell quiescence and indicate that D4 DA receptor agonists may have a therapeutic value in diseases with uncontrolled T cell proliferation.     

Molecular structure and linkage isomerization of isothiocyanato(3-thiapentane-1,5-dithiolato)oxorhenium(V) complex

The properties of isothiocyanato(3-thiapentane-1,5-dithiolato)oxorhenium(V) [ReO(SSS)NCS, (1a), (3+1) type], where isothiocyanato occupies the fifth position, have been studied. Two linkage isomers, i.e., ReO(SSS)NCS (1a) and ReO(SSS)SCN (1b), were found to be formed during syntheses. The sufficient quantities of 1a were isolated in the solid state, and characterized by X-ray crystallography and IR spectroscopy. From ¹H and ¹⁵N NMR measurements, it was found that 1a is in equilibrium with 1b in liquid state. In the solvents with low dielectric constant such as CH₂Cl₂, only 1a isomer was detected, while in the solvents with high such as CH₃CN, both 1a and 1b isomers were observed. We have obtained the equilibrium constant (K_iso) for the linkage isomerization reaction in CD₃CN by measuring ¹⁵N NMR spectra at various temperatures. The values of K_iso at 25 °C, the standard enthalpy (ΔH^°), and the standard entropy (ΔS^°) for the isomerization equilibrium were evaluated as 0.409, 14.4 kJ mol⁻¹, and 40.9 J K⁻¹ mol⁻¹, respectively.     

Immune Subversion by Mycobacterium tuberculosis through CCR5 Mediated Signaling: Involvement of IL-10

Tuberculosis is characterized by severe immunosuppression of the host macrophages, resulting in the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates C-C Chemokine Receptor 5 (CCR5) to enhance IL-10 production, indicating the possible involvement of CCR5 in regulation of the host immune response. Here, we found that Mycobacterium infection significantly increased CCR5 expression in macrophages there by facilitating the activation of its downstream signaling. These events culminated in up-regulation of the immunosuppressive cytokine IL-10 production, which was further associated with the down-regulation of macrophage MHC-II expression along with the up-regulation of CCR5 expression via engagement of STAT-3 in a positive feedback loop. Treatment of macrophages with CCR5 specific siRNA abrogated the IL-10 production and restored MHCII expression. While, in vivo CCR5 silencing was also effective for the restoration of host immune responses against tuberculosis. This study demonstrated that CCR5 played a very critical role for the immune subversion mechanism employed by the pathogen.