Crystal structure of an aromatic ring opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions

AbstractBackground: Sphingomonas paucimobilis SYK-6 utilizes an extradiol-type catecholic dioxygenase, the LigAB enzyme (a protocatechuate 4,5-dioxygenase), to oxidize protocatechuate (or 3,4-dihydroxybenzoic acid, PCA). The enzyme belongs to the family of class III extradiol-type catecholic dioxygenases catalyzing the ring-opening reaction of protocatechuate and related compounds. The primary structure of LigAB suggests that the enzyme has no evolutionary relationship with the family of class II extradiol-type catecholic dioxygenases. Both the class II and class III enzymes utilize a non-heme ferrous center for adding dioxygen to the substrate. By elucidating the structure of LigAB, we aimed to provide a structural basis for discussing the function of class III enzymes.Results: The crystal structure of substrate-free LigAB was solved at 2.2 Å resolution. The molecule is an α2β2 tetramer. The active site contains a non-heme iron coordinated by His12, His61, Glu242, and a water molecule located in a deep cleft of the β subunit, which is covered by the α subunit. Because of the apparent oxidation of the Fe ion into the nonphysiological Fe(III) state, we could also solve the structure of LigAB complexed with a substrate, PCA. The iron coordination sphere in this complex is a distorted tetragonal bipyramid with one ligand missing, which is presumed to be the O2-binding site.Conclusions: The structure of LigAB is completely different from those of the class II extradiol-type dioxygenases exemplified by the BphC enzyme, a 2,3-dihydroxybiphenyl 1,2-dioxygenase from a Pseudomonas species. Thus, as already implicated by the primary structures, no evolutionary relationship exists between the class II and III enzymes. However, the two classes of enzymes share many geometrical characteristics with respect to the nature of the iron coordination sphere and the position of a putative catalytic base, strongly suggesting a common catalytic mechanism

High-dimensional covariance matrix estimation under the SSE model (New Developments in Statistical Model)

In this paper, we consider the estimation for the inverse matrix of a high-dimensional covariance matrix under the strongly spiked eigenvalue model. One of the well-known estimation methods is the principal orthogonal complement thresholding (POET) given by Fan et al. [5]. We show that the POET has consistency properties only under several severe conditions in high-dimensional settings. In order to overcome the difficulty, we consider applying the noise-reduction (NR) method given by Yata and Aoshima [8, 9] to the POET. We propose a new estimation of the inverse covariance matrix called the NR-POET. We compare the performance of the NR-POET with the POET by several simulations

Manipulating histone acetylation leads to antitumor effects in hemangiosarcoma cells

Canine hemangiosarcoma (HSA) is a malignant tumour derived from endothelial cells. No effective treatment has yet been developed because of the lack of understanding of its pathogenesis. Histone acetylation, an epigenetic modification, is highly associated with cancer pathogenesis. Manipulating histone acetylation by histone deacetylase inhibitors (HDACi) or bromodomain and extraterminal domain inhibitors (BETi) is one approach to treat various cancers. However, the role of histone acetylation in HSA remains unknown. This study aimed to investigate how histone acetylation functions in HSA pathogenesis using two HDACi, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), and one BETi, JQ1, in vitro and in vivo. Histone acetylation levels were high in cell lines and heterogeneous in clinical cases. SAHA and JQ1 induced apoptosis in HSA cell lines. HSA cell lines treated with SAHA and VPA upregulated inflammatory-related genes and attracted macrophage cell line RAW264 cells, which suggests that SAHA and VPA can affect immune responses. JQ1 stimulated autophagy and inhibited the cell cycle in HSA cell lines. Finally, we demonstrated that JQ1 suppressed HSA tumour cell proliferation in vivo although SAHA and VPA did not affect tumour growth. These results suggest that BETi can be alternative drugs for HSA treatment. Although further research is required, our study indicated that dysregulation of histone acetylation is likely to be involved in HSA malignancy

Exogenous Expression of Equine MHC Class I Molecules in Mice Increases Susceptibility to Equine Herpesvirus 1 Pulmonary Infection

Equine herpesvirus 1 (EHV-1) uses equine major histocompatibility complex class I (MHC class I) as an entry receptor. Exogenous expression of equine MHC class I genes in murine cell lines confers susceptibility to EHV-1 infection. To examine the in vivo role of equine MHC class I as an entry receptor for EHV-1, we generated transgenic (Tg) mice expressing equine MHC class I under the control of the CAG promoter. Equine MHC class I protein was expressed in the liver, spleen, lung, and brain of Tg mice, which was confirmed by Western blot. However, equine MHC class I antigen was only detected in bronchiolar epithelium and not in other tissues, using the immunofluorescence method employed in this study. Both Tg and wild-type (WT) mice developed pneumonia 3 days after intranasal infection with EHV-1. The bronchiolar epithelial cells of Tg mice showed more severe necrosis, compared with those in WT mice. In addition, the number of virus antigen-positive cells in the lungs was higher in Tg mice than in WT mice. These results suggest that exogenous expression of equine MHC class I renders mice more susceptible to EHV-1 infection

Establishment of alternative culture method for spermatogonial stem cells using knockout serum replacement.

Since spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to daughter cells for subsequent spermatogenesis, the development of an efficient in vitro culture system is essential for studies related to spermatogenesis. Although the currently available system is serum-free and contains only chemically-defined components, it highly relies upon bovine serum albumin (BSA), a component with batch-to-batch quality variations similar to those of fetal bovine serum. Thus, we searched for an alternative BSA-free culture system that preserved the properties of SSCs. In this study, we utilized Knockout Serum Replacement (KSR) in the SSC culture medium, as a substitute for BSA. The results demonstrated that KSR supported the continuous growth of SSCs in vitro and the SSC activity in vivo without BSA, in a feeder-cell combination with mouse embryonic fibroblasts. The addition of BSA to KSR further facilitated cell cycle progression, whereas a transplantation assay revealed that the addition of BSA did not affect the number of SSCs in vivo. The combination of KSR with BSA also allowed the elimination of GFRA1 and FGF2, and the reduction of the GDNF concentration from 20 ng/ml to 5 ng/ml, while maintaining the growth rate and the expression of SSC markers. Furthermore, KSR was also useful with SSCs from non-DBA/2 strains, such as C57BL/6 and ICR. These results suggested that KSR is an effective substitute for BSA for long-term in vitro cultures of SSCs. Therefore, this method is practical for various studies related to SSCs, including spermatogenesis and germ stem cell biology

Potential for transmission of sporadic Creutzfeldt-Jakob disease through peripheral routes

Five sporadic Creutzfeldt-Jakob disease (CJD) strains have been identified to date, based on differences in clinicopathological features of the patients, the biochemical properties of abnormal prion proteins, and transmission properties. Recent advances in our knowledge about iatrogenic transmission of sporadic CJD have raised the possibility that the infectivity of sporadic CJD strains through peripheral routes is different from that of intracranial infection. To test this possibility, here we assessed systematically the infectivity of sporadic CJD strains through the peripheral route for the first time using a mouse model expressing human prion protein. Although the infectivity of the V2 and M1 sporadic CJD strains is almost the same in intracerebral transmission studies, the V2 strain infected more efficiently than the M1 strain through the peripheral route. The other sporadic CJD strains examined lacked infectivity. Of note, both the V2 and M1 strains showed preference for mice with the valine homozygosity at the PRNP polymorphic codon. These results indicate that the V2 strain is the most infectious sporadic CJD strain for infection through peripheral routes. In addition, these findings raise the possibility that individuals with the valine homozygosity at the PRNP polymorphic codon might have higher risks of infection through peripheral routes compared with the methionine homozygotes. Thus, preventive measures against the transmission of the V2 sporadic CJD strain will be important for the eradication of iatrogenic CJD transmission through peripheral routes. The authors sought to determine whether the infectivity of sporadic Creutzfeldt-Jakob disease prion strains through peripheral routes is different from that from intracranial infection. They found that among the strains, V2 is the most infectious through peripheral routes. Thus, preventive measures against transmission of this strain will be important for the eradication of iatrogenic transmission of Creutzfeldt-Jakob disease

Minocycline prevents primary duck neurons from duck Tembusu virus-induced death

Duck Tembusu virus (DTMUV), a neurotropic flavivirus, is a causative agent of severe neurological diseases in different birds. No approved vaccines or antiviral therapeutic treatments are available to date. The poultry industry experiences significant economic losses due to DTMUV infections. Minocycline is a second- generation semi-synthetic tetracycline analogue that is commonly used as an antimicrobial treatment. Experimental studies have indicated the successful protective effects of minocycline against neuronal cell death from neurodegenerative diseases and viral encephalitis. The aim of this study was to investigate the effects of minocycline on DTMUV infection in neurons. Primary duck neurons were treated with minocycline, which exhibited neuroprotective effects via anti-apoptotic function rather than through viral replication inhibition. Minocycline might serve as a potential effective drug in DTMUV infection