A functional model for pMMO (particulate methane monooxygenase): Hydroxylation of alkanes with H2O2 catalyzed by beta-diketiminatocopper(II) complexes

The reaction of copper(II) complexes supported by a series of beta-diketiminate ligands ((R1,R2)L, [(Dipp)N-C(R(2))-C(R(1))-C(R(2))-N(Dipp)](-), Dipp=2,6-diisopropylphenyl; see ) and H(2)O(2) has been examined spectroscopically at a low temperature. The beta-diketiminatocopper(II) complexes with R(2)=H (no substituent on the beta-carbon) provided a copper-oxygen intermediate that exhibited the same spectroscopic features as those of the bis(mu-oxo)dicopper(III) complex generated by the reaction of corresponding beta-diketiminatocopper(I) complex and O(2). On the other hand, the beta-diketiminatocopper(II) complexes with methyl substituent on the beta-carbon (R(2)=Me) did not produce such an intermediate in the same reaction. The beta-diketiminatocopper(II) complexes carrying an electron-withdrawing substituent on the alpha-carbon (R(1)=NO(2) or CN) but no beta-substituent (R(2)=H) exhibited a high catalytic activity in the oxygenation reaction of alkanes with H(2)O(2). Mechanism of the catalytic oxygenation reaction as well as the substituent effects of the ligands on the copper(II)-H(2)O(2) reactivity is discussed.     

Cysteine Residues in the Major Capsid Protein,Vp1, of the JC Virus Are Important for Protein Stability and Oligomer Formation

The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.     

Common and Distinct Clinical Features in Adult Patients with Anti-Aminoacyl-tRNA Synthetase Antibodies: Heterogeneity within the Syndrome

Objective

To identify similarities and differences in the clinical features of adult Japanese patients with individual anti-aminoacyl-tRNA synthetase antibodies (anti-ARS Abs).

Methods

This was a retrospective analysis of 166 adult Japanese patients with anti-ARS Abs detected by immunoprecipitation assays. These patients had visited Kanazawa University Hospital or collaborating medical centers from 2003 to 2009.

Results

Anti-ARS Ab specificity included anti-Jo-1 (36%), anti-EJ (23%), anti-PL-7 (18%), anti-PL-12 (11%), anti-KS (8%), and anti-OJ (5%). These anti-ARS Abs were mutually exclusive, except for one serum Ab that had both anti-PL-7 and PL-12 reactivity. Myositis was closely associated with anti-Jo-1, anti-EJ, and anti-PL-7, while interstitial lung disease (ILD) was correlated with all 6 anti-ARS Abs. Dermatomyositis (DM)-specific skin manifestations (heliotrope rash and Gottron’s sign) were frequently observed in patients with anti-Jo-1, anti-EJ, anti-PL-7, and anti-PL-12. Therefore, most clinical diagnoses were polymyositis or DM for anti-Jo-1, anti-EJ, and anti-PL-7; clinically amyopathic DM or ILD for anti-PL-12; and ILD for anti-KS and anti-OJ. Patients with anti-Jo-1, anti-EJ, and anti-PL-7 developed myositis later if they had ILD alone at the time of disease onset, and most patients with anti-ARS Abs eventually developed ILD if they did not have ILD at disease onset.

Conclusion

Patients with anti-ARS Abs are relatively homogeneous. However, the distribution and timing of myositis, ILD, and rashes differ among patients with individual anti-ARS Abs. Thus, identification of individual anti-ARS Abs is beneficial to define this rather homogeneous subset and to predict clinical outcomes within the “anti-synthetase syndrome.”     

Long-term adaptation of the Bombyx mori BmN4 cell line to grow in serum-free culture

Bombyx mori ovary-derived BmN4 cells have been successfully adapted to a commercial serum-free medium (SFM; SF900-II) by gradually reducing the serum-containing TC-100 medium content from 100 to 0% (v/v). The BmN4 cells adapted to the SFM (BmN-SFM) adhered strongly to the culture flask and showed altered cell morphology. The BmN-SFM was subcultured 200 times, and the population doubling time was 4.70 d. Infection studies showed that BmN-SFM cells were easily susceptible to B. mori nucleopolyhedrovirus (BmNPV), and both the multiplication of budded virus and the promoter activity of the polyhedrin gene in BmN-SFM cells were almost the same as those in BmN4 cells before adaptation. Additionally, mouse interleukin-3 expressed by a recombinant BmNPV was normally secreted and modified with N-linked glycans in BmN-SFM cells. These findings indicate that BmN-SFM is particularly useful for a BmNPV-based baculovirus expression vector system with serum-free conditions.     

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.     

Organ-specific pancreatic tumor growth properties and tumor immunity

We established a model of orthotopic injection of a syngeneic pancreatic tumor cell line in C57BL/6 mice and evaluated the effects of organ site on induction of immunity to a tumor-specific antigen, MUC1. Mice were challenged with a syngeneic pancreatic adenocarcinoma cell line that expressed MUC1 (Panc02-MUC1) by orthotopic injection into the pancreas, or by subcutaneous injection. Tumor cells injected into the pancreas grew much faster than those injected subcutaneously. Mice challenged subcutaneously with Panc02-MUC1 rejected tumors or developed slowly growing tumors that were negative for MUC1 expression. In contrast, mice challenged orthotopically into the pancreas developed progressive tumors that were positive for MUC1 expression. Sera from mice that rejected Panc02-MUC1 (tumor-immune mice) showed no detectable IgG1 and IgM titers against the MUC1 tandem-repeat peptide, whereas mice with progressive tumor growth had significant titers of IgG1 and IgM specific for MUC1. This suggests that the humoral immune response was ineffective in mediating tumor rejection. The results show that the growth properties and immunological rejection of pancreatic tumors is affected by the organ site at which the tumor grows. Received: 25 April 1998 / Accepted: 7 October 1998     

Heme-Cu complexes as oxygen-activating functional models for the active site of cytochrome c oxidase

Tri(2-pyridylmethyl)amineCu complex-linked iron meso-tetraphenylporphyine derivatives were prepared to model the active site of cytochrome c oxidase. Exposure to oxygen converted the reduced forms of the complexes to the corresponding stable mu-peroxo species in spite of the presence of three coordination sites, two on the heme and one on the Cu. The oxy forms were characterized spectroscopically. Kinetic analyses of the oxygenation reactions of the reduced forms suggests that preferential O2 binding occurs at the Cu site over the heme. This mechanism is also supported by examination of the redox potentials of the two metal ions. Since the peroxy complexes of the models exhibit a structure similar to that of the previously reported fully-oxidized form, the relevance of the model chemistry to the enzyme reaction is discussed.     

Molecular requirements for CD8-mediated rejection of a MUC1-expressing pancreatic carcinoma: implications for tumor vaccines

Previous studies have indicated that different effector cells are required to eliminate MUC1-expressing tumors derived from different organ sites and that different vaccine strategies may be necessary to generate these two different MUC1-specific immune responses. In this study, we characterized molecular components that are required to produce immune responses that eliminate Panc02.MUC1 tumors in vivo by utilizing mice genetically deficient in molecules related to immunity. A parallel study has been reported for a B16.MUC1 tumor model. We confirmed that a CD8(+) effector cell was required to eliminate MUC1-expressing Panc02 tumors, and demonstrated that T cells expressing TCR-alpha/beta and co-stimulation through CD28 and CD40:CD40L interactions played critical roles during the initiation of the anti-Panc02.MUC1 immune response. TCR-alpha/beta(+) cells were required to eliminate Panc02.MUC1 tumors, while TCR-gamma/delta(+) cells played a suppressive non-MUC1-specific role in anti-Panc02 tumor immunity. Type 1 cytokine interferon-gamma (IFN-gamma), but not interleukin-12 (IL-12), was essential for eliminating MUC1-expressing tumors, while neither IL-4 nor IL-10 (type 2 cytokines) were required for tumor rejection. In vitro studies demonstrated that IFN-gamma upregulated MHC class I, but not MHC class II, on Panc02.MUC1 tumor cells. Surprisingly, both perforin and FasL played unique roles during the effector phase of immunity to Panc02.MUC1, while lymphotoxin-alpha, but not TNFR-1, was required for immunity against Panc02.MUC1 tumors. The findings presented here and in parallel studies of B16.MUC1 immunity clearly demonstrate that different effector cells and cytolytic mechanisms are required to eliminate MUC1-expressing tumors derived from different organ sites, and provide insight into the immune components required to eliminate tumors expressing the same antigen but derived from different tissues.     

Phosphorylation of Fanconi anemia protein, FANCA, is regulated by Akt kinase

Phosphorylation of the Fanconi anemia complementation group A (FANCA) protein is thought to be important for the function of the FA pathway. However, the kinase for FANCA (so-called FANCA-PK) remains to be identified. FANCA has a consensus sequence for Akt kinase near serine 1149 (Ser1149), suggesting that Akt can phosphorylate FANCA. We performed in vitro kinase assays using as substrate either a GST-fusion wild-type (WT) FANCA fragment or a GST-fusion FANCA fragment containing a mutation from serine to alanine at 1149 (FANCA-S1149A). These experiments confirmed that FANCA is phosphorylated at Ser 1149, in vitro. However, (32)P-orthophosphate labeling experiments revealed that FANCA-S1149A was more efficiently phosphorylated than WT-FANCA. Furthermore, phosphorylation of wild-type FANCA was blocked by coexpression of a constitutively active (CA)-Akt and enhanced by a dominant-negative (DN) Akt. Our results suggest that Akt is a negative regulator of FANCA phosphorylation.