Regulation of inflammatory arthritis by the upstream kinase mitogen activated protein kinase kinase 7 in the c-Jun N-Terminal kinase pathway

Introduction

The c-Jun N-terminal kinase (JNK) is a key regulator of matrix metalloproteinase (MMP) and cytokine production in rheumatoid arthritis (RA) and JNK deficiency markedly protects mice in animal models of arthritis. Cytokine-induced JNK activation is strictly dependent on the mitogen-activated protein kinase kinase 7 (MKK7) in fibroblast-like synoviocytes (FLS). Therefore, we evaluated whether targeting MKK7 using anti-sense oligonucleotides (ASO) would decrease JNK activation and severity in K/BxN serum transfer arthritis.

Methods

Three 2''-O-methoxyethyl chimeric ASOs for MKK7 and control ASO were injected intravenously in normal C57BL/6 mice. PBS, control ASO or MKK7 ASO was injected from Day -8 to Day 10 in the passive K/BxN model. Ankle histology was evaluated using a semi-quantitative scoring system. Expression of MKK7 and JNK pathways was evaluated by quantitative PCR and Western blot analysis.

Results

MKK7 ASO decreased MKK7 mRNA and protein levels in ankles by about 40% in normal mice within three days. There was no effect of control ASO on MKK7 expression and MKK7 ASO did not affect MKK3, MKK4 or MKK6. Mice injected with MKK7 ASO had significantly less severe arthritis compared with control ASO (P < 0.01). Histologic evidence of synovial inflammation, bone erosion and cartilage damage was reduced in MKK7 ASO-treated mice (P < 0.01). MKK7 deficiency decreased phospho-JNK and phospho-c-Jun in ankle extracts (P < 0.05), but not phospho-MKK4. Interleukin-1beta (IL-1β), MMP3 and MMP13 gene expression in ankle joints were decreased by MKK7 ASO (P < 0.01).

Conclusions

MKK7 plays a critical regulatory role in the JNK pathway in a murine model of arthritis. Targeting MKK7 rather than JNK could provide site and event specificity when treating synovitis.     

Selection of phosphate-solubilizing diazotrophic Herbaspirillum and Burkholderia strains and their effect on rice crop yield and nutrient uptake

Background and Aims

Plant growth-promoting bacteria, mainly diazotrophs and phosphate solubilizers, can reduce the use of chemical fertilizers for rice crops. Here, diazotrophic bacteria isolated from rice were screened for their ability to solubilize inorganic P (P_i) in vitro and in association with rice plants cultivated in pots.

Methods

Forty-nine isolates were tested for the ability to solubilize P_i on NBRIP and GL agar plate media and seven selected strains were further evaluated in NBRIP liquid medium. Three of these strains were inoculated in rice plants grown in soil pots containing ¹⁵N-labeled fertilizer and two sources of P: tricalcium phosphate (TCP) or simple superphosphate (SSP). The dry matter, yield, N, P, and the ¹⁵N content accumulated in plant tissues were measured at 135 days after planting.

Results

Seven strains belonging to the genera Herbaspirillum and Burkholderia formed a halo of solubilized P_i on agar plates. The Burkholderia strains showed peak soluble P (around 200 mg P L^?1) on the fifth day when grown in NBRIP liquid medium for 14 days. Inoculation of Herbaspirillum strains (H18, ZA15) and a Burkholderia vietaminensis strain (AR114) increased rice grain yield from 33 to 47 % with TCP and 18 to 44 % with TSS, respectively. The bacterial inoculation led to enhanced N-use efficiency of the ¹⁵N-labeled fertilizer.

Conclusion

These results suggest that the selection and use of P-solubilizing diazotrophic bacteria are a good strategy to promote P solubilization and/or N use efficiency in rice plants.     

Sequence variation in nuclear ribosomal small subunit,internal transcribed spacer and large subunit regions of Rhizophagus irregularis and Gigaspora margarita is high and isolate‐dependent

Arbuscular mycorrhizal (AM) fungi are known to exhibit high intra‐organism genetic variation. However, information about intra‐ vs. interspecific variation among the genes commonly used in diversity surveys is limited. Here, the nuclear small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and large subunit (LSU) rRNA gene portions were sequenced from 3 to 5 individual spores from each of two isolates of Rhizophagus irregularis and Gigaspora margarita. A total of 1482 Sanger sequences (0.5 Mb) from 239 clones were obtained, spanning ~4370 bp of the ribosomal operon when concatenated. Intrasporal and intra‐isolate sequence variation was high for all three regions even though variant numbers were not exhausted by sequencing 12–40 clones per isolate. Intra‐isolate nucleotide variation levels followed the expected order of ITS > LSU > SSU, but the values were strongly dependent on isolate identity. Single nucleotide polymorphism (SNP) densities over 4 SNP/kb in the ribosomal operon were detected in all four isolates. Automated operational taxonomic unit picking within the sequence set of known identity overestimated species richness with almost all cut‐off levels, markers and isolates. Average intraspecific sequence similarity values were 99%, 96% and 94% for amplicons in SSU, LSU and ITS, respectively. The suitability of the central part of the SSU as a marker for AM fungal community surveys was further supported by its level of nucleotide variation, which is similar to that of the ITS region; its alignability across the entire phylum; its appropriate length for next‐generation sequencing; and its ease of amplification in single‐step PCR.     

Neural progenitor genes. Germinal zone expression and analysis of genetic overlap in stem cell populations

The identification of the genes regulating neural progenitor cell (NPC) functions is of great importance to developmental neuroscience and neural repair. Previously, we combined genetic subtraction and microarray analysis to identify genes enriched in neural progenitor cultures. Here, we apply a strategy to further stratify the neural progenitor genes. In situ hybridization demonstrates expression in the central nervous system germinal zones of 54 clones so identified, making them highly relevant for study in brain and neural progenitor development. Using microarray analysis we find 73 genes enriched in three neural stem cell (NSC)-containing populations generated under different conditions. We use the custom microarray to identify 38 "stemness" genes, with enriched expression in the three NSC conditions and present in both embryonic stem cells and hematopoietic stem cells. However, comparison of expression profiles from these stem cell populations indicates that while there is shared gene expression, the amount of genetic overlap is no more than what would be expected by chance, indicating that different stem cells have largely different gene expression patterns. Taken together, these studies identify many genes not previously associated with neural progenitor cell biology and also provide a rational scheme for stratification of microarray data for functional analysis.     

A Novel Family of Serine/Threonine Kinases Participating in Spermiogenesis

The molecular mechanisms regulating the spectacular cytodifferentiation observed during spermiogenesis are poorly understood. We have recently identified a murine testis-specific serine kinase (tssk) 1, constituting a novel subfamily of serine/threonine kinases. Using low stringency screening we have isolated and molecularly characterized a second closely related family member, tssk 2, which is probably the orthologue of the human DGS-G gene. Expression of tssk 1 and tssk 2 was limited to the testis of sexually mature males. Immunohistochemical staining localized both kinases to the cytoplasm of late spermatids and to structures resembling residual bodies. tssk 1 and tssk 2 were absent in released sperms in the lumen of the seminiferous tubules and the epididymis, demonstrating a tight window of expression restricted to the last stages of spermatid maturation. In vitro kinase assays of immunoprecipitates containing either tssk 1 or tssk 2 revealed no autophosphorylation of the kinases, however, they led to serine phosphorylation of a coprecipitating protein of ~65 kD. A search for interacting proteins using the yeast two-hybrid system with tssk 1 and tssk 2 cDNA as baits and a prey cDNA library from mouse testis, led to the isolation of a novel cDNA, interacting specifically with both tssk 1 and tssk 2, and encoding the coprecipitated 65-kD protein phosphorylated by both kinases. Interestingly, expression of the interacting clone was also testis specific and paralleled the developmental expression observed for the kinases themselves. These results represent the first demonstration of the involvement of a distinct kinase family, the tssk serine/threonine kinases, together with a substrate in the cytodifferentiation of late spermatids to sperms.     

Reduced C(beta) statistical potentials can outperform all-atom potentials in decoy identification

We developed a series of statistical potentials to recognize the native protein from decoys, particularly when using only a reduced representation in which each side chain is treated as a single C(beta) atom. Beginning with a highly successful all-atom statistical potential, the Discrete Optimized Protein Energy function (DOPE), we considered the implications of including additional information in the all-atom statistical potential and subsequently reducing to the C(beta) representation. One of the potentials includes interaction energies conditional on backbone geometries. A second potential separates sequence local from sequence nonlocal interactions and introduces a novel reference state for the sequence local interactions. The resultant potentials perform better than the original DOPE statistical potential in decoy identification. Moreover, even upon passing to a reduced C(beta) representation, these statistical potentials outscore the original (all-atom) DOPE potential in identifying native states for sets of decoys. Interestingly, the backbone-dependent statistical potential is shown to retain nearly all of the information content of the all-atom representation in the C(beta) representation. In addition, these new statistical potentials are combined with existing potentials to model hydrogen bonding, torsion energies, and solvation energies to produce even better performing potentials. The ability of the C(beta) statistical potentials to accurately represent protein interactions bodes well for computational efficiency in protein folding calculations using reduced backbone representations, while the extensions to DOPE illustrate general principles for improving knowledge-based potentials.     

Heterogeneous Nature and Distribution of Interruptions in Dinucleotides May Indicate the Existence of Biased Substitutions Underlying Microsatellite Evolution

Some aspects of microsatellite evolution, such as the role of base substitutions, are far from being fully understood. To examine the significance of base substitutions underlying the evolution of microsatellites we explored the nature and the distribution of interruptions in dinucleotide repeats from the human genome. The frequencies that we inferred in the repetitive sequences were statistically different from the frequencies observed in other noncoding sequences. Additionally, we detected that the interruptions tended to be towards the ends of the microsatellites and 5'-3' asymmetry. In all the estimates nucleotides forming the same repetitive motif seem to be affected by different base substitution rates in AC and AG. This tendency itself could generate patterning and similarity in flanking sequences and reconcile these phenomena with the high mutation rate found in flanking sequences without invoking convergent evolution. Nevertheless, our data suggest that there is a regional bias in the substitution pattern of microsatellites. The accumulation of random substitutions alone cannot explain the heterogeneity and the asymmetry of interruptions found in this study or the relative frequency of different compound microsatellites in the human genome. Therefore, we cannot rule out the possibility of a mutational bias leading to convergent or parallel evolution in flanking sequences.