IL-22/STAT3-Induced Increases in SLURP1 Expression within Psoriatic Lesions Exerts Antimicrobial Effects against Staphylococcus aureus

Background

SLURP1 is the causal gene for Mal de Meleda (MDM), an autosomal recessive skin disorder characterized by diffuse palmoplantar keratoderma and transgressive keratosis. Moreover, although SLURP1 likely serves as an important proliferation/differentiation factor in keratinocytes, the possible relation between SLURP1 and other skin diseases, such as psoriasis and atopic dermatitis, has not been studied, and the pathophysiological control of SLURP1 expression in keratinocytes is largely unknown.

Objectives

Our aim was to examine the involvement of SLURP1 in the pathophysiology of psoriasis using an imiquimod (IMQ)-induced psoriasis model mice and normal human epidermal keratinocytes (NHEKs).

Results

SLURP1 expression was up-regulated in the skin of IMQ-induced psoriasis model mice. In NHEKs stimulated with the inflammatory cytokines IL-17, IL-22 and TNF-α, which are reportedly expressed in psoriatic lesions, SLURP1 mRNA expression was significantly up-regulated by IL-22 but not the other two cytokines. The stimulatory effect of IL-22 was completely suppressed in NHEKs treated with a STAT3 inhibitor or transfected with siRNA targeting STAT3. Because IL-22 induces production of antimicrobial proteins in epithelial cells, the antibacterial activity of SLURP1 was assessed against Staphylococcus aureus (S. aureus), which is known to be associated with disease severity in psoriasis. SLURP1 significantly suppressed the growth of S. aureus.

Conclusions

These results indicate SLURP1 participates in pathophysiology of psoriasis by regulating keratinocyte proliferation and differentiation, and by suppressing the growth of S. aureus.     

The Relationship between Processing Speed and Regional White Matter Volume in Healthy Young People

Processing speed is considered a key cognitive resource and it has a crucial role in all types of cognitive performance. Some researchers have hypothesised the importance of white matter integrity in the brain for processing speed; however, the relationship at the whole-brain level between white matter volume (WMV) and processing speed relevant to the modality or problem used in the task has never been clearly evaluated in healthy people. In this study, we used various tests of processing speed and Voxel-Based Morphometry (VBM) analyses, it is involves a voxel-wise comparison of the local volume of gray and white, to assess the relationship between processing speed and regional WMV (rWMV). We examined the association between processing speed and WMV in 887 healthy young adults (504 men and 383 women; mean age, 20.7 years, SD, 1.85). We performed three different multiple regression analyses: we evaluated rWMV associated with individual differences in the simple processing speed task, word–colour and colour–word tasks (processing speed tasks with words) and the simple arithmetic task, after adjusting for age and sex. The results showed a positive relationship at the whole-brain level between rWMV and processing speed performance. In contrast, the processing speed performance did not correlate with rWMV in any of the regions examined. Our results support the idea that WMV is associated globally with processing speed performance regardless of the type of processing speed task.     

Artificial ground shelters for overwintering phytoseiid mites in orchards

Biological control in orchards strongly depends on winter survival of natural enemies, especially in temperate regions. Predacious phytoseiid mites overwinter on trees or on the ground depending on the characteristics of the species. However, the overwintering ecology of phytoseiid mites on the ground is less well known than that of those on trees. We investigated the usefulness of artificial overwintering shelters as a tool for studying the overwintering ecology of phytoseiid mites on the ground. Four kinds of artificial shelter (shading net, felt, cardboard, and urethane foam) were placed on the ground in an apple orchard in Korea. Two dominant phytoseiid species, Neoseiulus makuwa (Ehara) and N. womersleyi (Schicha) (Acari: Phytoseiidae), overwintered in the artificial ground shelters, and numbers were highest in the urethane foam among the four kinds of shelter, and next highest in the shading net. On the other hand, the numbers of phytoseiid mites collected in the ground vegetation plus soil samples under the ground shelters were not significantly different among the five shelter treatments, including the no-shelter control. Our results suggest that artificial overwintering shelters are efficient tools for investigating overwintering ecology of phytoseiid mites on the ground, as well as on trees, in orchards. Furthermore, the artificial shelters would be good sampling units because they are easily formed into identical sizes and can be used almost anywhere in the field with less laborious work. We also discuss some implications about the effects of sheltered structures on the ground on the populations of phytoseiid mites during winter.     

Age-related changes of the stretch reflex excitability in human ankle muscles

The purpose of this study was to characterize the effects of aging on the stretch reflex in the ankle muscles, and in particular to compare the effects on the ankle dorsi-flexor (tibialis anterior: TA) and the plantar-flexor (soleus: SOL). Stretch reflex responses were elicited in the TA and SOL at rest and during weak voluntary contractions in 20 elderly and 23 young volunteers. The results indicated that, in the TA muscle, the elderly group had a remarkably larger long-latency reflex (LLR), whereas no aging effect was found in the short latency reflex (SLR). These results were very different from those in the SOL muscle, which showed significant aging effects in the SLR and medium latency reflex (MLR), but not in the LLR. Given the fact that the LLR of the TA stretch reflex includes the cortical pathway, it is probable that the effects of aging on the TA stretch reflex involve alterations not only at the spinal level but also at the cortical level. The present results indicate that the stretch reflexes of each of the ankle antagonistic muscles are affected differently by aging, which might have relevance to the neural properties of each muscle.     

Changes of Gap and Tight Junctions during Differentiation of Human Nasal Epithelial Cells Using Primary Human Nasal Epithelial Cells and Primary Human Nasal Fibroblast Cells in a Noncontact Coculture System

The epithelium of upper respiratory tissues such as nasal mucosa forms a continuous barrier to a wide variety of exogenous antigens. The epithelial barrier function is regulated in large part by the intercellular junctions, referred to as gap and tight junctions. However, changes of gap and tight junctions during differentiation of human nasal epithelial (HNE) cells are still unclear. In the present study, to investigate changes of gap and tight junctions during differentiation of HNE cells in vitro, we used primary human HNE cells cocultured with primary human nasal fibroblast (HNF) cells in a noncontact system. In HNE cells cocultured with HNF cells for 2 weeks, numerous elongated cilia-like structures were observed compared to those without HNF cells. In the coculture, downregulation of Cx26 and upregulation of Cx30.3 and Cx31 were observed together with extensive gap junctional intercellular communication. Furthermore, expression of the tight junction proteins claudin-1, claudin-4, occludin and ZO-2 was increased. These results suggest that switching in expression of connexins and induction of tight junction proteins may be closely associated with differentiation of HNE cells in vitro and that differentiation of HNE cells requires unknown soluble factors secreted from HNF cells.     

A structured viroid RNA serves as a substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RNA-induced silencing complex-mediated degradation

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.     

Structural and functional studies of the HAMP domain of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli

The HAMP domain plays an essential role in signal transduction not only in histidine kinase but also in a number of other signal-transducing receptor proteins. Here we expressed the EnvZ HAMP domain (Arg(180)-Thr(235)) with the R218K mutation (termed L(RK)) or with L(RK) connected with domain A (Arg(180)-Arg(289)) (termed LA(RK)) of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli, by fusing it with protein S. The L(RK) and LA(RK) proteins were purified after removing protein S. The CD analysis of the isolated L protein revealed that it consists of a random structure or is unstructured. This suggests that the EnvZ HAMP domain by itself is unable to form a stable structure and that this structural fragility may be important for its role in signal transduction. Interestingly the substitution of Ala(193) in the EnvZ HAMP domain with valine or leucine in Tez1A1, a chimeric protein of Tar and EnvZ, caused a constitutive OmpC phenotype. The CD analysis of LA(RK)(A193L) revealed that this mutated HAMP domain possesses considerable secondary structures and that the thermostability of this entire LA(RK)(A193L) became substantially lower than that of LA(RK) or just domain A, indicating that the structure of the HAMP domain with the A193L mutation affects the stability of downstream domain A. This results in cooperative thermodenaturation of domain A with the mutated HAMP domain. These results are discussed in light of the recently solved NMR structure of the HAMP domain from a thermophilic bacterium (Hulko, M., Berndt, F., Gruber, M., Linder, J. U., Truffault, V., Schultz, A., Martin, J., Schultz, J. E., Lupas, A. N., and Coles, M. (2006) Cell 126, 929-940).     

Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

Human β-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of α- and β-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using α-1,6-mannosyltransferase-deficient (och1Δ) yeast as the host. Genes encoding the α- and β-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (αα) and HexB (ββ). A total of 57 mg of β-hexosaminidase isozymes, of which 13 mg was HexA (αβ), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the β-subunit. The purified HexA was treated with α-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 ± 0.1 and 1.7 ± 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the β-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.     

Sensitive detection of FGFR3 mutations in bladder cancer and urine sediments by peptide nucleic acid-mediated real-time PCR clamping

Somatic mutations of the fibroblast growth factor receptor 3 (FGFR3) gene were detected by peptide nucleic acid (PNA)-mediated real-time PCR clamping. Mutation was detected in negative control containing only wild-type DNA due to a misincorporation of dNTPs to PNA binding sites when the amount of template DNA was decreased to 1 ng. Thus, the amount of template DNA was critical determinant of the assay sensitivity in PNA-mediated PCR clamping. Assay conditions were optimized to detect FGFR3 mutations in exons 7, 10, and 15, at a concentration of more than 1% mutated DNA using 50 ng of genomic DNA as the template. Mutations were detected in 12 of 13 (92.3%) tumor tissues and 11 of 13 (84.6%) urine samples from patients with superficial bladder cancer, while no mutations were detected in tissues and/or urine samples from patients with muscle-invasive bladder cancer or chronic cystitis.     

IRS-1 transgenic mice show increased epididymal fat mass and insulin resistance

Insulin receptor substrate-1 (IRS-1) is the major substrate of both the insulin receptor and the IGF-1 receptor. In this study, we created IRS-1 transgenic (IRS-1-Tg) mice which express human IRS-1 cDNA under control of the mouse IRS-1 gene promoter. In the IRS-1-Tg mice, IRS-1 mRNA expression was significantly increased in almost all tissues, but its protein expression was increased in very limited tissues (epididymal fat and skeletal muscle). IRS-1-Tg mice showed glucose intolerance and significantly enlarged epididymal fat mass, as well as elevated serum TNF-α concentrations. Importantly insulin signaling was significantly attenuated in the liver of IRS-1-Tg mice, which may contribute to the glucose intolerance. Our results suggest that excess IRS-1 expression may not provide a beneficial impact on glucose homeostasis in vivo.