Improvement of Monoclonal Antibody Production in Hybridoma Cells by Dimethyl Sulfoxide

Biotechnology Progress. Jan-Feb, 2003  |  Pubmed ID: 12573019

Hybridoma cultures are routinely used as a source for monoclonal antibody (mAb) production necessary for preclinical evaluation. However, these cultures typically have low volumetric and specific productivities. In this article, we examined the use and the timing of addition of dimethyl sulfoxide (DMSO) as a medium additive to improve mAb production in our hybridoma clone 19 (c19) cultures. From shake flask studies, we defined the optimal DMSO concentration and time of addition for improved productivity. This timing coordinated with high cell viability and density. Hybridoma cultures treated with DMSO up to 0.3% (v/v) possessed cell densities and viabilities comparable to untreated control. We demonstrated that 0.2% (v/v) DMSO added to shake flask cultures at their maximal viable cell densities resulted in a 2-fold increase in specific mAb production. This procedure was scaleable up to 20 L Cellbags (Wave Bioreactors) with similar titer improvement. Moreover, DMSO treatment did not affect the bioactivity or glycosylation profiles of the mAb.

Altered Cutaneous Immune Parameters in Transgenic Mice Overexpressing Viral IL-10 in the Epidermis

The Journal of Clinical Investigation. Jun, 2003  |  Pubmed ID: 12813028

IL-10 is a pleiotropic cytokine that inhibits several immune parameters, including Th1 cell-mediated immune responses, antigen presentation, and antigen-specific T cell proliferation. Recent data implicate IL-10 as a mediator of suppression of cell-mediated immunity induced by exposure to UVB radiation (280-320 nm). To investigate the effects of IL-10 on the cutaneous immune system, we engineered transgenic mice that overexpress viral IL-10 (vIL-10) in the epidermis. vIL-10 transgenic mice demonstrated a reduced number of I-A(+) epidermal and dermal cells and fewer I-A(+) hapten-bearing cells in regional lymph nodes after hapten painting of the skin. Reduced CD80 and CD86 expression by I-A(+) epidermal cells was also observed. vIL-10 transgenic mice demonstrated a smaller delayed-type hypersensitivity response to allogeneic cells upon challenge but had normal contact hypersensitivity to an epicutaneously applied hapten. Fresh epidermal cells from vIL-10 transgenic mice showed a decreased ability to stimulate allogeneic T cell proliferation, as did splenocytes. Additionally, chronic exposure of mice to UVB radiation led to the development of fewer skin tumors in vIL-10 mice than in WT controls, and vIL-10 transgenic mice had increased splenic NK cell activity against YAC-1targets. These findings support the concept that IL-10 is an important regulator of cutaneous immune function.

Thalidomide Inhibits Tumor Necrosis Factor-alpha Production and Antigen Presentation by Langerhans Cells

The Journal of Investigative Dermatology. Nov, 2003  |  Pubmed ID: 14708607

Thalidomide is an effective treatment for several inflammatory and autoimmune disorders including erythema nodosum leprosum, Behcet's syndrome, discoid lupus erythematosus, and Crohn's disease. Thalidomide is believed to exert its anti-inflammatory effects, at least in part, by inhibiting tumor necrosis factor-alpha (TNF-alpha) production by monocytes. We studied the effects of thalidomide on epidermal Langerhans cells (LC). LCs are epidermal antigen-presenting dendritic cells that play important roles in skin immune responses. Using the murine epidermis-derived dendritic cell lines, XS106A from A/J mice and XS52 from BALB/c mice as surrogates for LC, we found that thalidomide inhibited TNF-alpha production in a concentration-dependent manner. Northern blot analysis revealed that thalidomide significantly decreased the peak-induced mRNA level of TNF-alpha in XS106A cells and XS52 cells. We then examined the effect of thalidomide on fresh LC enriched to approximately 98% using positive selection of Ia+ cells with antibodies conjugated to magnetic microspheres. TNF-alpha production was reduced by 67.7% at a thalidomide concentration of 200 microg per mL. Thalidomide also had a profound inhibitory effect on the ability of LC to present antigen to a responsive TH1 clone. Thalidomide inhibits TNF-alpha production and the antigen-presenting ability of epidermal LCs. These mechanisms may contribute to the therapeutic effects observed with this agent.

Detection and Quantification of the Human IgG4 Half-molecule, HL, from Unpurified Cell-culture Supernatants

Biotechnology and Applied Biochemistry. Dec, 2004  |  Pubmed ID: 14979869

The presence and absence of inter-heavy-chain disulphide linkages contribute to the existence of the tetrameric (H(2)L(2)) and half (HL) human IgG molecules, respectively. Reduced effector response in the human IgG4 subclass presents an alternative therapeutic platform in a monoclonal-antibody (mAb) development program. During the initial cell-selection stage, titres of the recombinant human antibody present in crude cell-culture supernatants are determined by ELISA, a technique requiring nanogram quantities of mAb. In the case of an IgG4 antibody, this material is represented mainly by the combination of the tetrameric (H(2)L(2)) and dimeric (HL) forms of the antibody. The determination of concentrations or ratios of tetramer and dimer usually requires at least one chromatographic purification step, and thus frequently this is evaluated later in the mAb development process when the number of potential clones has been reduced. In the present paper we describe a Western-blot-based method that detects and quantifies IgG4 half-molecules, HL, from crude cell-culture supernatants without purification so that H(2)L(2)/HL ratios can be included as a part of early clonal evaluation along with the screening of mAb titres. This method was demonstrated (1) to have a linear HL detection range of 0.5-10 ng, (2) to require microlitre volumes of culture and (3) to react specifically with human IgG4 produced from hybridoma and Chinese-hamster ovary cell cultures. Moreover, this protocol is applicable to evaluate and monitor potential H(2)L(2)/HL variations as a result of changes during the process-development stage of a mAb development program.

[Study on Quality Evaluation Methods of Pretreatment for Polystyrene-type Macroporous Absorbing Resins]

Zhongguo Zhong Yao Za Zhi = Zhongguo Zhongyao Zazhi = China Journal of Chinese Materia Medica. Nov, 2004  |  Pubmed ID: 15656131

To establish a GC method using wide bore open tubular columns f or controlling pretreatment for polystyrene-type macroporous absorbing resins and its eligible standard.

Zirconocene-mediated Intermolecular Coupling of Si-tethered Diynes with Alkynes, Ketones, Aldehydes, and Isocyanates by Means of Novel Skeletal Rearrangement of Zirconacyclobutene-silacyclobutene and Zirconacyclohexadiene-silacyclobutene Fused-ring Intermediates

Chemistry (Weinheim an Der Bergstrasse, Germany). Mar, 2005  |  Pubmed ID: 15674981

Bis(alkynyl)silanes react with low valent zirconocene species to afford zirconacyclobutene intermediates. These in situ generated reactive organometallic intermediates can react with alkynes, ketones, aldehydes, and isocyanates by means of a novel skeletal rearrangement. When a zirconacyclobutene intermediate was treated with an alkyne, an alpha-alkynylsilyl zirconacyclopentadiene was formed. Addition of dimethyl acetylenedicarboxylate (DMAD) and CuCl resulted in one-pot formation of an alkynylsilyl-benzene derivative from three different alkynes. At a higher temperature, the alpha-alkynylsilylzirconacyclopentadiene was transformed by means of an intramolecular skeletal rearrangement to a zirconacyclohexadiene-silacyclobutene fused-ring compound, which reacted with DMAD in the presence of CuCl affording the same alkynylsilyl-benzene derivative. When treated with a ketone, an aldehdye, or an isocyanate, the zirconocyclobutene intermediate also underwent the above-mentioned skeletal rearrangement, generating zirconocene-mediated cross-coupling products.

Metal Carbene-promoted Sequential Transformations for the Enantioselective Synthesis of Highly Functionalized Cycloheptadienes

Journal of the American Chemical Society. Feb, 2005  |  Pubmed ID: 15686344

A two-step, three-component coupling of an alkyne, enol ether, and vinyl diazoester was accomplished by use of successive metal carbene-catalyzed transformations. This efficient approach to cycloheptadienes is both diastereo- and enantioselective. Kinetic resolution was accomplished on dienol ethers bearing a racemic chiral center at the propargylic position. A model is offered which explains the observed selectivity and accounts for the reactivity difference between trans- and cis-dienol ethers.

Synthesis, Reactivity, and Structural Characterization of a 14-vertex Carborane

Angewandte Chemie (International Ed. in English). Mar, 2005  |  Pubmed ID: 15726559

Synthesis, Structure, and Reactivity of a Zirconocene-carboryne Precursor

Journal of the American Chemical Society. Oct, 2005  |  Pubmed ID: 16201782

Transition metal-benzyne complexes have found many applications in organic synthesis, mechanistic studies, and the synthesis of functional materials. In sharp contrast, the reaction chemistry of transition metal-carboryne complexes is virtually unknown although the theoretical calculations indicated that the formation of carboryne (1,2-C2B10H10) and benzyne is very energetically comparable. This communication reports a novel zirconocene-carboranyl complex Cp2Zr(mu-Cl)(mu-C2B10H10)Li(OEt2)2 (1), an efficient precursor of the zirconocene-carboryne species, prepared from the reaction of Cp2ZrCl2 with 1 equiv of Li2C2B10H10 in Et2O. The reactivity studies indicated that 1 resembles zirconocene-benzyne in reactions with polar unsaturated organic molecules. On the other hand, it shows no reactivity toward alkynes and alkenes, a reactivity pattern which is quite different from that of zirconocene-benzyne. This work also furnishes a novel method for the preparation of functional o-carboranes and their metal complexes which cannot be synthesized by other methods presently known.

Synthesis, Structure, and Reactivity of 13-vertex Carboranes and 14-vertex Metallacarboranes

Journal of the American Chemical Society. Apr, 2006  |  Pubmed ID: 16608358

Syntheses, properties, and synthetic applications of 13-vertex closo- and nido-carboranes are reported. Reactions of the nido-carborane salt [(CH2)3C2B10H10]Na2 with dihaloborane reagents afforded 13-vertex closo-carboranes 1,2-(CH2)3-3-R-1,2-C2B11H10 (R = H (2), Ph (3), Z-EtCH=C(Et) (4), E-(t)BuCH=CH (5)). Treatment of the arachno-carborane salt [(CH2)3C2B10H10]Li4 with HBBr2.SMe2 gave both the 13-vertex carborane 2 and a 14-vertex closo-carborane (CH2)3C2B12H12 (8). On the other hand, the reaction of [C6H4(CH2)2C2B10H10]Li4 with HBBr2.SMe2 generated only a 13-vertex closo-carborane 1,2-C6H4(CH2)2-1,2-C2B11H11 (9). Electrophilic substitution reactions of 2 with excess MeI, Br2, or I2 in the presence of a catalytic amount of AlCl3 produced the hexa-substituted 13-vertex carboranes 8,9,10,11,12,13-X6-1,2-(CH2)3-1,2-C2B11H5 (X = Me (10), Br (11), I (12)). The halogenated products 11 and 12 displayed unexpected instability toward moisture. The 13-vertex closo-carboranes were readily reduced by groups 1 and 2 metals. Accordingly, several 13-vertex nido-carborane dianionic salts [nido-1,2-(CH2)3-1,2-C2B11H11][Li2(DME)2(THF)2] (13), [[nido-1,2-(CH2)3-1,2-C2B11H11][Na2(THF)4]]n (13a), [[nido-1,2-(CH2)3-3-Ph-1,2-C2B11H10][Na2(THF)4]]n (14), [[nido-1,2-C6H4(CH2)2-1,2-C2B11H11][Na2(THF)4]]n (15), and [nido-1,2-(CH2)3-1,2-C2B11H11][M(THF)5] (M = Mg (16), Ca (17)) were prepared in good yields. These carbon-atom-adjacent nido-carboranes were not further reduced to the corresponding arachno species by lithium metal. On the other hand, like other nido-carborane dianions, they were useful synthons for the production of super-carboranes and supra-icosahedral metallacarboranes. Interactions of 13a with HBBr2.SMe2, (dppe)NiCl2, and (dppen)NiCl2 gave the 14-vertex carborane 8 and nickelacarboranes [eta5-(CH2)3C2B11H11]Ni(dppe) (18) and [eta5-(CH2)3C2B11H11]Ni(dppen) (19), respectively. All complexes were fully characterized by various spectroscopic techniques and elemental analyses. Some were further confirmed by single-crystal X-ray diffraction studies.

Synthesis and Structure of 14- and 15-vertex Ruthenacarboranes

Angewandte Chemie (International Ed. in English). Jun, 2006  |  Pubmed ID: 16729344

Nickel-mediated Regioselective [2 + 2 + 2] Cycloaddition of Carboryne with Alkynes

Journal of the American Chemical Society. Jun, 2006  |  Pubmed ID: 16771473

Transition metal-benzyne complexes have found many applications in organic synthesis, mechanistic studies, and the synthesis of functional materials. In sharp contrast, the chemistry of transition metal-carboryne complexes, especially late transition metal complexes, is virtually unknown. This communication reports a novel nickel-mediated regioselective [2 + 2 + 2] cycloaddition reaction of carboryne with alkynes via the Ni-carboryne intermediate (eta2-C2B10H10)Ni(PPh3)2. Because of the bulkiness of the carborane moiety, a high regioselectivity was achieved in the reactions involving unsymmetrical alkynes. This work furnishes a novel method for the preparation of highly substituted benzocarboranes which are difficult to obtain by other methods.

Vaccinia Virus Infection Attenuates Innate Immune Responses and Antigen Presentation by Epidermal Dendritic Cells

Journal of Virology. Oct, 2006  |  Pubmed ID: 17005676

Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-alpha), and IFN-gamma, it actively inhibited the production of proinflammatory cytokines TNF-alpha and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-alpha secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the DeltaE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IkappaBalpha. These results suggest a novel role for the E3L protein as an antagonist of the NF-kappaB signaling pathway. DeltaE3L-infected XS52 cells secreted higher levels of TNF-alpha and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. DeltaK1L-infected cells secreted higher levels of TNF-alpha and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.

Role of C,c'-linkage in the Formation and Stabilization of Supercarboranes. Synthesis and Structure of Carbon-atoms-apart 13-vertex Carborane and 14-vertex Metallacarborane

Journal of the American Chemical Society. Jan, 2007  |  Pubmed ID: 17199267

Synthesis, Structure, Reactivity, and Thermal Isomerization of Boron-substituted 13-vertex Cobaltacarboranes (eta5-Cp)Co(eta6-R2C2B10Me8H2) (R = H, Et)

Inorganic Chemistry. Apr, 2007  |  Pubmed ID: 17343377

Reduction of boron-substituted carboranes o-R2C2B10Me8H2 (R = H, Et), thermal isomerization, and nucleophilic reaction of the resultant 13-vertex cobaltacarboranes were studied. Reaction of o-C2B10Me8H4 (1) with excess potassium metal in tetrahydrofuran (THF) gave, after recrystallization from a THF solution of 18-crown-6 ether, [[K(18-crown-6)(THF)2][K(18-crown-6)]][[4-(18-crown-6)-2,3,5,8,9,11,12,13-Me8-4,1,6-KC2B10H4]2] (2) in 78% yield. Interaction of 1 with excess sodium or potassium metal in THF, followed by treatment with CoCl2/CpNa and then aerobatic oxidation, afforded two boron-substituted 13-vertex cobaltacarboranes, 4-Cp-2,3,5,8,9,11,12,13-Me8-4,1,6-CoC2B10Me8H4 (3) and 4-Cp-2,3,5,9,10,11,12,13-Me8-4,1,6-CoC2B10Me8H4 (4), in 15% and 8% yield, respectively. Subsequently, thermal isomerization of 3 and 4 yielded another two new isomers, 4-Cp-2,3,5,6,8,11,12,13-Me8-4,1,9-CoC2B10Me8H4 (5) and 4-Cp-2,3,5,6,7,11,12,13-Me8-4,1,9-CoC2B10Me8H4 (6). Treatment of 3 or 4 with strong bases such as nBuLi and MeLi generated unexpected nucleophilic substitution products 4-nBuCp-2,3,5,8,9,11,12,13-Me8-4,1,6-CoC2B10Me8H4 (7), 4-nBuCp-2,3,5,9,10,11,12,13-Me8-4,1,6-CoC2B10Me8H4 (8a), and 4-MeCp-2,3,5,9,10,11,12,13-Me8-4,1,6-CoC2B10Me8H4 (8b) in good yields. Under the same reaction conditions, however, only one 13-vertex cobaltacarborane, 4-Cp-1,9-Et2-2,5,6,7,8,11,12,13-Me8-4,1,9-CoC2B10Me8H4 (10), was isolated when o-Et2C2B10Me8H2 (9) was used as the starting material. Complex 10 is a thermodynamically stable product and has a substitution pattern different from that of 3-6. These results show that the substituents on either the cage carbon or boron atoms have an important effect on the formation and thermal stability of the 13-vertex metallacarboranes. The formation of these complexes can be rationalized by the diamond-square-diamond mechanism.

Dermoscopic and Reflectance Confocal Microscope Findings of Trichoepithelioma

Dermatology (Basel, Switzerland). 2007  |  Pubmed ID: 17911996

Trichoepitheliomas (TE) are benign neoplasms of follicular differentiation. Solitary lesions are often confused with basal cell carcinoma (BCC). Reflectance confocal microscopy (RCM) and dermoscopy are imaging tools for in vivo, noninvasive evaluation of skin lesions. To date, there has been no description of their findings in the evaluation of TE.

Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly

Journal of Virology. Dec, 2007  |  Pubmed ID: 17928345

The bioterror threat of a smallpox outbreak in an unvaccinated population has mobilized efforts to develop new antipoxviral agents. By screening a library of known drugs, we identified 13 compounds that inhibited vaccinia virus replication at noncytotoxic doses. The anticancer drug mitoxantrone is unique among the inhibitors identified in that it has no apparent impact on viral gene expression. Rather, it blocks processing of viral structural proteins and assembly of mature progeny virions. The isolation of mitoxantrone-resistant vaccinia strains underscores that a viral protein is the likely target of the drug. Whole-genome sequencing of mitoxantrone-resistant viruses pinpointed missense mutations in the N-terminal domain of vaccinia DNA ligase. Despite its favorable activity in cell culture, mitoxantrone administered intraperitoneally at the maximum tolerated dose failed to protect mice against a lethal intranasal infection with vaccinia virus.

Multiple Erythematous Eroded Patches and Papules on the Scalp

Archives of Dermatology. Jan, 2008  |  Pubmed ID: 18209179

Stabilization of Fully Reduced Iron-sulfur Clusters by Carbene Ligation: the [FenSn]0 Oxidation Levels (n = 4, 8)

Journal of the American Chemical Society. Jul, 2008  |  Pubmed ID: 18593124

The all-ferrous [Fe4S4](0) state has been demonstrated in the fully reduced Fe protein of the Azotobacter vinelandii nitrogenase complex. We seek synthetic analogues of this state more tractable than the recently prepared but highly unstable cluster [Fe4S4(CN)4](4-) (Scott, Berlinguette, Holm, and Zhou, Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9741). The N-heterocyclic carbene 1,3-diisopropyl-4,5-dimethylimidazol-2-ylidene (Pr(i)2NHCMe2) has been found to stabilize the fully reduced clusters [Fe8S8(Pr(i)2NHCMe2)6] (4) and [Fe4S4(Pr(i)2NHCMe2)4] (5), which are prepared by cluster assembly or phosphine substitution of FenSn (n = 8, 16) clusters. Cluster 4 is also obtained by reaction of the carbene with all-ferrous [Fe7S6(PEt3)5Cl2] (3) and cluster 5 by carbene cleavage of 4. Detailed structures of 3 (monocapped prismatic), 4, and 5 are described; the latter two are the first iron-sulfur clusters with Fe-C sigma bonds. Cluster 4 possesses the [Fe8(mu3-S) 6(mu4-S)2] edge-bridged double cubane structure and 5 the cubane-type [Fe4(mu3-S)4] stereochemistry. The all-ferrous formulations of the clusters are confirmed by X-ray structure parameters and (57)Fe isomer shifts. Both clusters are stable under conventional aprotic anaerobic conditions, enabling further study of reactivity. The collective properties of 5 indicate that it is a meaningful synthetic analogue of the core of the fully reduced protein-bound cluster.

Vaccinia Virus Subverts a Mitochondrial Antiviral Signaling Protein-dependent Innate Immune Response in Keratinocytes Through Its Double-stranded RNA Binding Protein, E3

Journal of Virology. Nov, 2008  |  Pubmed ID: 18715932

Skin keratinocytes provide a first line of defense against invading microorganisms in two ways: (i) by acting as a physical barrier to pathogen entry and (ii) by initiating a vigorous innate immune response upon sensing danger signals. How keratinocytes detect virus infections and generate antiviral immune responses is not well understood. Orthopoxviruses are dermatotropic DNA viruses that cause lethal disease in humans. Virulence in animal models depends on the virus-encoded bifunctional Z-DNA/double-stranded RNA (dsRNA)-binding protein E3. Here, we report that infection of mouse primary keratinocytes with a vaccinia DeltaE3L mutant virus triggers the production of beta interferon (IFN-beta), interleukin-6 (IL-6), CCL4, and CCL5. None of these immune mediators is produced by keratinocytes infected with wild-type vaccinia virus. The dsRNA-binding domain of E3 suffices to prevent activation of the innate immune response. DeltaE3L induction of IFN-beta, IL-6, CCL4, and CCL5 secretion requires mitochondrial antiviral signaling protein (MAVS; an adaptor for the cytoplasmic viral RNA sensors RIG-I and MDA5) and the transcription factor IRF3. IRF3 phosphorylation is induced in keratinocytes infected with DeltaE3L, an event that depends on MAVS. The response of keratinocytes to DeltaE3L is unaffected by genetic ablation of Toll-like receptor 3 (TLR3), TRIF, TLR9, and MyD88.

Mössbauer, Electron Paramagnetic Resonance, and Theoretical Studies of a Carbene-based All-ferrous Fe4S4 Cluster: Electronic Origin and Structural Identification of the Unique Spectroscopic Site

Inorganic Chemistry. Apr, 2009  |  Pubmed ID: 19326927

It is well established that the cysteinate-coordinated [Fe(4)S(4)] cluster of the iron protein of nitrogenase from Azotobacter vinelandii (Av2) can attain the all-ferrous core oxidation state. Mössbauer and electron paramagnetic resonance (EPR) studies have shown that the all-ferrous cluster has a ground-state spin S = 4 and an effective 3:1 site symmetry in the spin structure and (57)Fe quadrupole interactions. Recently, Deng and Holm reported the synthesis of [Fe(4)S(4)(Pr(i)(2)NHCMe(2))(4)],(2) (1; Pr(i)(2)NHCMe(2) = 1,3-diisopropyl-4,5-dimethylimidazol-2-ylidene) and showed that the all-ferrous carbene-coordinated cluster is amenable to physicochemical studies. Mössbauer and EPR studies of 1, reported here, reveal that the electronic structure of this complex is strikingly similar to that of the protein-bound cluster, suggesting that the ground-state spin and the 3:1 site ratio are consequences of spontaneous distortions of the cluster core. To gain insight into the origin of the peculiar ground state of the all-ferrous clusters in 1 and Av2, we have studied a theoretical model that is based on a Heisenberg-Dirac-van Vleck Hamiltonian whose exchange-coupling constants are a function of the Fe-Fe distances. By combining the exchange energies with the elastic deformation energies in the harmonic approximation, we obtain for a T(2) distortion a minimum with spin S = 4 and a C(3v) core structure in which one iron is unique and three irons are equivalent. This minimum has all of the spectroscopic and structural characteristics of the all-ferrous clusters of 1 and Av2. Our analysis maps the unique spectroscopic iron site to a specific site in the X-ray structure of the [Fe(4)S(4)](0) core both in complex 1 and in Av2.

Cleavage of Ni-(mu(2)-S)-Ni Bridges in Dinuclear Nickel(II) Dithiolate Pincer Complexes and Related Reactions

Inorganic Chemistry. Jul, 2009  |  Pubmed ID: 19459662

Pyridine-2,6-dimethanethiolate and pyridine-2,6-dithiocarboxylate form sparingly soluble Ni(II) pincer complexes formulated as [Ni(pdmt)](2) and [Ni(pdtc)](2), respectively, with two Ni-(mu(2)-S)-Ni bridges. In acetonitrile reaction systems, the latter undergoes the facile bridge cleavage reactions [Ni(pdtc)](2) + 2L(0,-) --> 2[Ni(pdtc)L](0,-) with an extensive set of nucleophiles to afford planar mononuclear products with L(-) = halide, CN, Me(3)SiO(-), RS(-) and L(0) = Et(3)P and a N-heterocyclic carbene. [Ni(pdmt)](2) is considerably less reactive toward bridge disruption. Cleavage products support several reactions of interest leading to other mononuclear species and to di- and trinuclear complexes. [Ni(pdtc)(OSiMe(3))](1-) deprotonates acetonitrile and acetone to form [Ni(pdtc)(CH(2)R)](1-) (R = CN, COMe). Reaction of [Ni(pdtc)SEt](1-) with Fe(II) yields the thiolate-bridged dimer {[Ni(pdtc)](2)(SEt)}(1-). Refluxing an acetonitrile solution of [Ni(pdtc)SH](1-) in air results in formation of trinuclear [Ni(pdtc)](3)S](2-) containing the rare unsupported Ni(3)(mu(3)-S) bridge core. Reaction of [Ni(pdtc)CN](1-) with [Fe(Me(6)tren)(OTf)](1+) forms the complex [Ni(pdtc)CNFe(Me(6)tren)](1+), the only example of a single Ni-C[triple bond]N-Fe bridge within a molecule. Structures of the various types of reaction products are presented. This work demonstrates the potential utility of bridge cleavage of polynuclear Ni(II) thiolates, an extensive family of compounds, to produce mononuclear products.

Zirconocene-mediated Ligand-switched Selective Cleavage of Active and Inert Carbon-carbon Bonds in Allylcyclopropanes

Chemical Communications (Cambridge, England). Aug, 2009  |  Pubmed ID: 19597610

Selective cleavage of both active and inert C-C bonds in substituted allylcyclopropanes was realized via a novel ligand-controlled approach using zirconocene(II)-1-butene or zirconocene(II)-benzyne complexes.

Cubane-type Co4S4 Clusters: Synthesis, Redox Series, and Magnetic Ground States

Journal of the American Chemical Society. Aug, 2009  |  Pubmed ID: 19722678

The recent demonstration that the carbene cluster [Fe(4)S(4)(Pr(i)(2)NHCMe(2))(4)] (9) is an accurate structural and electronic analogue of the fully reduced cluster of the iron protein of Azotobacter vinelandii nitrogenase, including a common S = 4 ground state, raises the issue of the existence and magnetism of other [M(4)S(4)L(4)](z) clusters, none of which are known with transition metals other than iron. The system CoCl(2)/Pr(i)(3)P/(Me(3)Si)(2)S/THF assembles [Co(4)S(4)(PPr(i)(3))(4)] (3), which is converted to [Co(4)S(4)(Pr(i)(2)NHCMe(2))(4)] (5) upon reaction with carbene. The clusters support the redox series [3](1-/0/1+) and [5](0/1+/2+); monocations (4, 6) have been isolated by chemical oxidation. Redox potentials and substitution reactions indicate that the carbene is the more effective electron donor to tetrahedral Fe(II) and Co(II) sites. Clusters 3-6 have the same overall cubane-type geometry as 9. Neutral clusters 3 and 5 have an S = 3 ground state. As with the S = 4 state of 9 with local spins S(Fe) = 2, the septet spin state can be described in terms of the coupling of three parallel and one antiparallel spins S(Co) = 3/2. The octanuclear clusters [Co(8)S(8)(PPr(i)(3))(6)](0,1+) were isolated as minor byproducts of the formation and chemical oxidation of 3. The clusters exhibit a rhomb-bridged noncubane (RBNC) structure, whereas clusters with the Fe(8)S(8) core possess edge-bridged double-cubane (EBDC) stereochemistry. There are two structural solutions for the M(8)S(8) core in the form of topological isomers whose stability may depend on valence electron count. A conceptual model for the RBNC <--> EBDC interconversion is presented. (Pr(i)(2)NHCMe(2) = C(11)H(20)N(2) = 1,3-diisopropyl-4,5-dimethylimidazol-2-ylidene).

The Modular Nature of All-ferrous Edge-bridged Double Cubanes

Inorganic Chemistry. Feb, 2010  |  Pubmed ID: 20073485

Two all-ferrous, edge-bridged 8Fe-8S clusters, one capped with carbenes (2) and the other with phosphenes (3), have been characterized by (57)Fe Mossbauer spectroscopy. The clusters have diamagnetic ground states that yield spectra consisting of one quadrupole doublet with a large splitting (25% of absorption) and one (3) or two (2) doublets with much smaller splittings (75% of absorption). These patterns closely resemble those observed for all-ferrous 4Fe-4S clusters. Structurally, the 4Fe-4S fragments of 2 and 3 are remarkably similar to all-ferrous 4Fe-4S clusters, sharing with them the characteristic 3:1 pattern of the iron sites, a differentiation that has been shown previously to reflect spontaneous distortions of the cluster core. These spectroscopic and geometric similarities suggest that the diamagnetic ground state of the 8Fe-8S cluster results from antiferromagnetic exchange coupling of two identical 4Fe-4S modules, each carrying spin S(4Fe) = 4. The iron atoms with the largest quadrupole splittings are located at the opposite ends of the body diagonal containing the bridging sulfides.

MRNA Stability and Antibody Production in CHO Cells: Improvement Through Gene Optimization

Biotechnology Journal. Apr, 2010  |  Pubmed ID: 20222103

The productivity of stably transfected cell lines is of critical importance for the manufacturing of therapeutic proteins. Various methods have been successfully implemented to increase the production output of mammalian cell cultures. Increasing evidence suggests that optimization of the gene coding sequences of an expression vector can improve specific cell line yield of the recombinant protein. Here we demonstrate that gene optimization substantially enhances antibody production in Chinese hamster ovary cells. When gene optimization was applied to the heavy and light chain genes of a therapeutic antibody, we observed increased antibody production in transient transfection. Elevated heavy chain mRNA level was associated with the increase of antibody production. Further analysis suggested that the increased antibody expression is attributable to enhanced mRNA stability resulting from gene optimization. Gene optimization also led to increased antibody production in stable clones.

[Preliminary Study on Mechanism of Therapeutic Effect of Huganjiexian Decoction on Hepatic Fibrosis]

Zhonghua Gan Zang Bing Za Zhi = Zhonghua Ganzangbing Zazhi = Chinese Journal of Hepatology. Mar, 2010  |  Pubmed ID: 20380795

To observe the effects of Huganjiexian decoction on rat hepatic fibrosis and the creation of cytokines.

Stabilization of 3:1 Site-differentiated Cubane-type Clusters in the [Fe(4)S(4)](1+) Core Oxidation State by Tertiary Phosphine Ligation: Synthesis, Core Structural Diversity, and S = 1/2 Ground States

Inorganic Chemistry. Dec, 2010  |  Pubmed ID: 21038882

An extensive series of 3:1 site-differentiated cubane-type clusters [Fe(4)S(4)(PPr(i)(3))(3)L] (L = Cl(-), Br(-), I(-), RO(-), RS(-), RSe(-)) has been prepared in 40-80% yield by two methods: ligand substitution of [Fe(4)S(4)(PPr(i)(3))(4)](1+) in tetrahydrofuran (THF)/acetonitrile by reaction with monoanions, and reductive cleavage of ligand substrates (RSSR, RSeSeR, I(2)) by the all-ferrous clusters [Fe(8)S(8)(PPr(i)(3))(6)]/[Fe(16)S(16)(PPr(i)(3))(8)] in THF. These neutral clusters are stable and do not undergo ligand redistribution reactions involving charged species in benzene and THF solutions. X-ray structural studies confirm the cubane stereochemistry but with substantial and variable distortions of the [Fe(4)S(4)](1+) core from idealized cubic core geometry. Based on Fe-S bond lengths, seven clusters were found to have compressed tetragonal distortions (4 short and 8 long bonds), and the remaining seven display other types of distortions with different combinations of long, short, and intermediate bond lengths. These results further emphasize the facile deformabililty of this core oxidation state previously observed in [Fe(4)S(4)(SR)(4)](3-) clusters. The Fe(2.25+) mean oxidation state was demonstrated from (57)Fe isomer shifts, and the appearance of two quadrupole doublets arises from the spin-coupled |9/2,4,1/2> state. The S = 1/2 ground state was further supported by electron paramagnetic resonance spectra and magnetic susceptibility data.

Effects of P-CREB-1 on Transforming Growth Factor-β3 Auto-regulation in Hepatic Stellate Cells

Journal of Cellular Biochemistry. Apr, 2011  |  Pubmed ID: 21308733

Previous studies have demonstrated that transforming growth factor-β3 (TGF-β3) protected liver against fibrosis in vivo and vitro, but its regulation is poorly understood. In addition, the cAMP-responsive element (CRE) in TGF-β3 promoter is recognized as an important regulatory site for TGF-β3 auto-regulation. Thus, we hypothesize that transcription factor CRE-binding protein-1 (CREB-1) regulates the auto-induction of TGF-β3 in hepatic stellate cells (HSCs). We used exogenous TGF-β3 to activate the signal pathway of TGF-β3 auto-regulation in HSCs, results indicated that exogenous TGF-β3 could up-regulate the protein and mRNA expressions of TGF-β3, and provoke the phosphorylation of CREB-1 on Ser-133, besides, it could induce the DNA binding activity of p-CREB-1 and activate TGF-β3 promoter as well. Additionally, we used pGenesil-1.1-shRNA-CREB-1 and pRSV-CREB-1 expression vector to silence and up-regulate CREB-1 gene expression respectively, and the results indicated that inhibition of CREB-1 suppressed exogenous TGF-β3 stimulation of TGF-β3 mRNA and protein expressions in HSCs, whereas up-regulation of CREB-1 induced this stimulation. Our results indicate that exogenous TGF-β3 up-regulates the activity of TGF-β3 promoter by activating CREB-1, then induces the mRNA and protein expressions of TGF-β3. Especially, p-CREB-1 is a critical transcription factor in mediating TGF-β3 auto-induction.

Myxoma Virus Induces Type I Interferon Production in Murine Plasmacytoid Dendritic Cells Via a TLR9/MyD88-, IRF5/IRF7-, and IFNAR-dependent Pathway

Journal of Virology. Oct, 2011  |  Pubmed ID: 21835795

Poxviruses are large DNA viruses that replicate in the cytoplasm of infected cells. Myxoma virus is a rabbit poxvirus that belongs to the Leporipoxvirus genus. It causes a lethal disease called myxomatosis in European rabbits but cannot sustain any detectable infection in nonlagomorphs. Vaccinia virus is a prototypal orthopoxvirus that was used as a vaccine to eradicate smallpox. Myxoma virus is nonpathogenic in mice, whereas systemic infection with vaccinia virus can be lethal even in immunocompetent mice. Plasmacytoid dendritic cells (pDCs) are potent type I interferon (IFN)-producing cells that play important roles in antiviral innate immunity. How poxviruses are sensed by pDCs to induce type I IFN production is not well understood. Here we report that infection of primary murine pDCs with myxoma virus, but not with vaccinia virus, induces IFN-α, IFN-β, tumor necrosis factor (TNF), and interleukin-12p70 (IL-12p70) production. Using pDCs derived from genetic knockout mice, we show that the myxoma virus-induced innate immune response requires the endosomal DNA sensor TLR9 and its adaptor MyD88, transcription factors IRF5 and IRF7, and the type I IFN positive-feedback loop mediated by IFNAR1. It is independent of the cytoplasmic RNA sensing pathway mediated by the mitochondrial adaptor molecule MAVS, the TLR3 adaptor TRIF, or the transcription factor IRF3. Using pharmacological inhibitors, we demonstrate that myxoma virus-induced type I IFN and IL-12p70 production in murine pDCs is also dependent on phosphatidylinositol 3-kinase (PI3K) and Akt. Furthermore, our results reveal that the N-terminal Z-DNA/RNA binding domain of vaccinia virulence factor E3, which is missing in the orthologous M029 protein expressed by myxoma virus, plays an inhibitory role in poxvirus sensing and innate cytokine production by murine pDCs.

[Components of Myrsinane-type Diterpenes from Euphorbia Prolifera]

Zhejiang Da Xue Xue Bao. Yi Xue Ban = Journal of Zhejiang University. Medical Sciences. Jul, 2011  |  Pubmed ID: 21845750

To extract and isolate the component from myrsinane-type diterpenes of Euphorbia prolifera.

[Effects of Exogenous Transforming Growth Factor-β3 on the Activities of Its Promoter and CAMP-responsive Element Binding Protein-1 in Rat Hepatic Stellate Cell]

Zhonghua Yi Xue Za Zhi. Sep, 2011  |  Pubmed ID: 22321755

To explore the effects of exogenous transforming growth factor-β3 (TGF-β3) on the activities of its promoter and cAMP-responsive element binding protein-1 (CREB-1) in rat hepatic stellate cell (HSC-T6).