Jab1 mediates cytoplasmic localization and degradation of West Nile virus capsid protein

The clinical manifestations of West Nile virus (WNV), a member of the Flavivirus family, include febrile illness, sporadic encephalitis, and paralysis. The capsid (Cp) of WNV is thought to participate in these processes by inducing apoptosis through mitochondrial dysfunction and activation of caspase-9 and caspase-3. To further identify the molecular mechanism of the WNV capsid protein (WNVCp), yeast two-hybrid assays were employed using WNV-Cp as bait. Jab1, the fifth subunit of the COP9 signalosome, was subsequently identified as a molecule that interacts with WNVCp. Immunoprecipitation and glutathione S-transferase pulldown assays confirmed that direct interaction could occur between WNVCp and Jab1. Immunofluorescence microscopy demonstrated that the overexpressed WNVCp, which localized to the nucleolus, was translocated to the cytoplasm upon its co-expression with Jab1. When treated with leptomycin B, Jab1-facilitated nuclear exclusion of WNVCp was prevented, which indicated that the CRM1 complex is required for Jab1-facilitated nuclear export of WNVCp. Moreover, Jab1 promoted the degradation of WNVCp in a proteasome-dependent way. Consistent with this, WNVCp-mediated cell cycle arrest at the G(2) phase in H1299 was prevented by exogenous Jab1. Finally, an analysis of WNVCp deletion mutants indicated that the first 15 amino acids were required for interaction with Jab1. Furthermore, the double-point mutant of the WNVCp, P5A/P8A, was incapable of binding to Jab1. These results indicate that Jab1 has a potential protective effect against pathogenic WNVCp and might provide a novel target site for the treatment of disease caused by WNV.     

Intrinsic resistance of neural stem cells to toxic metabolites may make them well suited for cell non-autonomous disorders: evidence from a mouse model of Krabbe leukodystrophy

While transplanted neural stem cells (NSCs) have been shown to hold promise for cell replacement in models of a number of neurological disorders, these examples have typically been under conditions where the host cells become dysfunctional due to a cell autonomous etiology, i.e. a 'sick' cell within a relatively supportive environment. It has long been held that cell replacement in a toxic milieu would not likely be possible; donor cells would succumb in much the same way as endogenous cells had. Many metabolic diseases are characterized by this situation, suggesting that they would be poor targets for cell replacement therapies. On the other hand, models of such diseases could prove ideal for testing the capacity for cell replacement under such challenging conditions. In the twitcher (twi ) mouse -- as in patients with Krabbe or globoid cell leukodystrophy (GLD), for which it serves as an authentic model -- loss of galactocerebrosidase (GalC) activity results in the accumulation of psychosine, a toxic glycolipid. Twi mice, like children with GLD, exhibit inexorable neurological deterioration presumably as a result of dysfunctional and ultimately degenerated oligodendrocytes with loss of myelin. It is believed that GLD pathophysiology is related to a psychosine-filled environment that kills not only host oligodendrocytes but theoretically any new cells placed into that milieu. Through the implantation of NSCs into the brains of both neonatal and juvenile/young adult twi mice, we have determined that widespread oligodendrocyte replacement and remyelination is feasible. NSCs appear to be intrinsically resistant to psychosine -- more so in their undifferentiated state than when directed ex vivo to become oligodendrocytes. This resistance can be enhanced by engineering the NSCs to over-express GalC. Some twi mice grafted with such engineered NSCs had thicker white tracts and lived 2-3 times longer than expected. While their brains had detectable levels of GalC, it was probably more significant that their psychosine levels were lower than in twi mice that died at a younger age. This concept of resistance based on differentiation state extended to human NSCs which could similarly survive within the twi brain. Taken together, these results suggest a number of points regarding cellular therapies against degenerative diseases with a prominent cell non-autonomous component: Cell replacement is possible if cells resistant to the toxic environment are employed. Furthermore, an important aspect of successful treatment will likely be not only cell replacement but also cross-correction of host cells to provide them with enzyme activity and hence resistance. While oligodendrocyte replacement alone was not a sufficient treatment for GLD (even when extensive), the replacement of both cells and molecules -- e.g. with NSCs that could both become oligodendrocytes and 'pumps' for GalC -- emerges as a promising basis for a multidisciplinary strategy. Most neurological disease are complex in this way and will likely require multifaceted approaches, perhaps with NSCs serving as the 'glue'.     

Genome sequence of Lactobacillus johnsonii PF01, isolated from piglet feces

Lactobacillus johnsonii PF01, an autochthonous bacterium of the gastrointestinal tract, was isolated from a fecal sample from a piglet. The strain adhered specifically to the duodenal and jejunal epithelial cells of the piglet and had high bile resistance activity. Here we report the genomic sequence of L. johnsonii PF01.     

Subclinical inflammation on MRI of hand and foot of anticitrullinated peptide antibody–negative arthralgia patients at risk for rheumatoid arthritis

Introduction

It is known that anticitrullinated peptide antibody (ACPA)–positive rheumatoid arthritis (RA) has a preclinical phase. Whether this phase is also present in ACPA-negative RA is unknown. To determine this, we studied ACPA-negative arthralgia patients who were considered prone to progress to RA for local subclinical inflammation observed on hand and foot magnetic resonance imaging (MRI) scans.

Methods

We studied a total of 64 ACPA-negative patients without clinically detectable arthritis and with arthralgia of the small joints within the previous 1 year. Because of the character of the patients’ symptoms, the rheumatologists considered these patients to be prone to progress to RA. For comparisons, we evaluated 19 healthy, symptom-free controls and 20 ACPA-negative RA patients, who were identified according to the 1987 American Rheumatism Association criteria. All participants underwent MRI of unilateral wrist, metacarpophalangeal and metatarsophalangeal joints. Synovitis and bone marrow oedema (BME) were scored according to the OMERACT rheumatoid arthritis magnetic resonance imaging scoring system, and the scores were summed to yield the ‘MRI inflammation score’. Scores were compared between groups. Among the ACPA-negative arthralgia patients, MRI inflammation scores were related to C-reactive protein (CRP) levels and the tenderness of scanned joints.

Results

MRI inflammation scores increased progressively among the groups of controls and ACPA-negative arthralgia and RA patients (median scores = 0, 1 and 10, respectively; P < 0.001). The MRI inflammation scores of ACPA-negative arthralgia patients were significantly higher than those of controls (P = 0.018). In particular, the synovitis scores were higher in ACPA-negative arthralgia patients (P = 0.046). Among the ACPA-negative arthralgia patients, inflammation was observed predominantly in the wrist (53%). The synovitis scores were associated with CRP levels (P = 0.007) and joint tenderness (P = 0.026). Despite the limited follow-up duration, five patients developed clinically detectable arthritis. These five patients had higher scores for MRI inflammation (P = 0.001), synovitis (P = 0.002) and BME (P = 0.003) compared to the other patients.

Conclusion

Subclinical synovitis was observed in the small joints of ACPA-negative arthralgia patients, and especially in patients whose conditions progressed to clinically detectable arthritis. This finding suggests the presence of a preclinical phase in ACPA-negative RA. Further longitudinal studies of these lesions and patients are required to confirm this hypothesis.     

Partial Purification and Properties of a Cysteine Protease from Citrus Red Mite Panonychus citri

Several studies have reported that the citrus red mites Panonychus citri were an important allergen of citrus-cultivating farmers in Jeju Island. The aim of the present study was to purify and assess properties of a cysteine protease from the mites acting as a potentially pathogenic factor to citrus-cultivating farmers. A cysteine protease was purified using column chromatography of Mono Q anion exchanger and Superdex 200 HR gel filtration. It was estimated to be 46 kDa by gel filtration column chromatography and consisted of 2 polypeptides, at least. Cysteine protease inhibitors, such as trans poxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) and iodoacetic acid (IAA) totally inhibited the enzyme activities, whereas serine or metalloprotease inhibitors did not affect the activities. In addition, the purified enzyme degraded human IgG, collagen, and fibronectin, but not egg albumin. From these results, the cysteine protease of the mites might be involved in the pathogenesis such as tissue destruction and penetration instead of nutrient digestion.     

Age-Associated Lipidome Changes in Metaphase II Mouse Oocytes

The quality of mammalian oocytes declines with age, which negatively affects fertilization and developmental potential. The aging process often accompanies damages to macromolecules such as proteins, DNA, and lipids. To investigate if aged oocytes display an altered lipidome compared to young oocytes, we performed a global lipidomic analysis between oocytes from 4-week-old and 42 to 50-week-old mice. Increased oxidative stress is often considered as one of the main causes of cellular aging. Thus, we set up a group of 4-week-old oocytes treated with hydrogen peroxide (H₂O₂), a commonly used oxidative stressor, to compare if similar lipid species are altered between aged and oxidative-stressed oocytes. Between young and aged oocytes, we identified 26 decreased and 6 increased lipids in aged oocytes; and between young and H₂O₂-treated oocytes, we identified 35 decreased and 26 increased lipids in H₂O₂-treated oocytes. The decreased lipid species in these two comparisons were overlapped, whereas the increased lipid species were distinct. Multiple phospholipid classes, phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), and lysophosphatidylserine (LPS) significantly decreased both in H₂O₂-treated and aged oocytes, suggesting that the integrity of plasma membrane is similarly affected under these conditions. In contrast, a dramatic increase in diacylglycerol (DG) was only noted in H₂O₂-treated oocytes, indicating that the acute effect of H₂O₂-caused oxidative stress is distinct from aging-associated lipidome alteration. In H₂O₂-treated oocytes, the expression of lysophosphatidylcholine acyltransferase 1 increased along with increases in phosphatidylcholine. Overall, our data reveal that several classes of phospholipids are affected in aged oocytes, suggesting that the integrity of plasma membrane is associated with maintaining fertilization and developmental potential of mouse oocytes.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

CD8 T-cell activation in mice injected with a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean Plasmodium vivax

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8(+) T-cells and CD4(+) T-cells. However, in mice immunized using a gene gun, significantly higher (P<0.05) proportions of CD8(+) cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique.     

Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration

We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies.