Tissue inhibitor of metalloproteinase-2 (TIMP-2) regulates neuromuscular junction development via a beta1 integrin-mediated mechanism

Extracellular matrix (ECM) molecules play critical roles in muscle function by participating in neuromuscular junction (NMJ) development and the establishment of stable, cytoskeleton-associated adhesions required for muscle contraction. Matrix metalloproteinases (MMPs) are neutral endopeptidases that degrade all ECM components. While the role of MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), has been investigated in many tissues, little is known about their role in muscle development and mature function. TIMP-2 -/- mice display signs of muscle weakness. Here, we report that TIMP-2 is expressed at the NMJ and its expression is greater in fast-twitch (extensor digitorum longus, EDL) than slow-twitch (soleus) muscle. EDL muscle mass is reduced in TIMP-2-/- mice without a concomitant change in fiber diameter or number. The TIMP-2-/- phenotype is not likely due to increased ECM proteolysis because net MMP activity is actually reduced in TIMP-2-/- muscle. Most strikingly, TIMP-2 colocalizes with beta1 integrin at costameres in the wild-type EDL and beta1 integrin expression is significantly reduced in TIMP-2-/- EDL. We propose that reduced beta1 integrin in fast-twitch muscle may be associated with destabilized ECM-cytoskeletal interactions required for muscle contraction in TIMP-2-/- muscle; thus, explaining the muscle weakness. Given that fast-twitch fibers are lost in muscular dystrophies and age-related sarcopenia, if TIMP-2 regulates mechanotransduction in an MMP-independent manner it opens new potential therapeutic avenues.     

Critical role of the major histocompatibility complex and IL-10 in matrilin-1-induced relapsing polychondritis in mice

Relapsing polychondritis (RP) is an autoimmune disease that affects extra-articular cartilage. Matrilin-1-induced relapsing polychondritis (MIRP) is a model for RP and is useful for studies of the pathogenic mechanisms in this disease. There are indications that the major histocompatibility complex (MHC) class II plays a major role in RP, since DR4⁺ patients are more commonly affected than controls. We have now addressed the role of the MHC region, as well as the non-MHC contribution, using congenic mouse strains. Of the MHC congenic strains, B10.Q (H2 ^q ) was the most susceptible, the B10.P (H2 ^p ) and B10.R (H2 ^r ) strains developed mild disease, while B10 strains carrying the v, b, f, or u H2 haplotypes were resistant. A slight variation of susceptibility of H2 ^q strains (B10.Q> C3H.Q> DBA/1) was observed and the (B10.Q × DBA/1)F₁ was the most susceptible of all strains. Furthermore, macrophages and CD4⁺ T cells were the most prominent cell types in inflammatory infiltrates of the tracheal cartilage. Macrophages are the major source of many cytokines, such as interleukin-10 (IL-10), which is currently being tested as a therapeutic agent in several autoimmune diseases. We therefore investigated B10.Q mice devoid of IL-10 through gene deletion and found that they developed a significantly more severe disease, with an earlier onset, than their heterozygous littermates. In conclusion, MHC genes, as well as non-MHC genes, are important for MIRP induction, and IL-10 plays a major suppressive role in cartilage inflammation of the respiratory tract.     

The planetary biology of cytochrome P450 aromatases

Background  

Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system.     

A method to optimize selection on multiple identified quantitative trait loci

A mathematical approach was developed to model and optimize selection on multiple known quantitative trait loci (QTL) and polygenic estimated breeding values in order to maximize a weighted sum of responses to selection over multiple generations. The model allows for linkage between QTL with multiple alleles and arbitrary genetic effects, including dominance, epistasis, and gametic imprinting. Gametic phase disequilibrium between the QTL and between the QTL and polygenes is modeled but polygenic variance is assumed constant. Breeding programs with discrete generations, differential selection of males and females and random mating of selected parents are modeled. Polygenic EBV obtained from best linear unbiased prediction models can be accommodated. The problem was formulated as a multiple-stage optimal control problem and an iterative approach was developed for its solution. The method can be used to develop and evaluate optimal strategies for selection on multiple QTL for a wide range of situations and genetic models.     

RNAi‐suppression of barley caffeic acid O‐methyltransferase modifies lignin despite redundancy in the gene family

Caffeic acid O‐methyltransferase (COMT), the lignin biosynthesis gene modified in many brown‐midrib high‐digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl‐to‐guaiacyl ratio was reduced by ~50%, the 5‐hydroxyguaiacyl (5‐OH‐G) unit incorporated into lignin at 10‐–15‐fold higher levels than normal, and the amount of p‐coumaric acid ester‐linked to cell walls was reduced by ~50%. No brown‐midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown‐midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.     

Isolation and characterization of monoclonal antibodies to proline dehydrogenase from Escherichia coli K-12

The PutA protein of Escherichia coli K-12 serves as both proline dehydrogenase and the repressor controlling the expression of genes putP and putA. Thirty-eight hybridoma cell lines were isolated using mice immunized with proline dehydrogenase purified from a bacterial membrane extract. The monoclonal antibodies secreted by those cells showed varying affinities for proline dehydrogenase by enzyme-linked immunosorbent assay (ELISA). Nine antibodies labelled the PutA protein in Western blots after sodium dodecyl sulfate--polyacrylamide gel electrophoresis and two of the five tested also labelled the undenatured PutA protein. Three antibodies bound proteins present in a peripheral membrane protein fraction from both putA+ bacteria and a putA::Tn5 mutant strain. Urea denaturation eliminated the proline:2,6-dichloroindophenol (DCIP) oxidoreductase activity, but did not alter the immunoreactivity of the PutA protein. Tween 20, which caused 1.8-fold increases in Km (proline) and Vmax for proline:DCIP oxidoreductase, increased the avidity of the antibody from hybridoma line 2.1C10.3 fivefold. The antibodies from hybridoma lines 2.1C10.2, 1.2C10.3, and 1.1B07.1 were shown by electron microscopy of immunogold-labelled preparations or by ELISA to bind the membrane-associated PutA protein, whereas those from hybridoma lines 2.1A08.2 and 1.4C09.1 failed to recognize that antigen form. These antibodies will serve as probes of the relationships among protein domain, conformation, and function for the PutA protein.     

Groundwater input affecting plant distribution by controlling ammonium and iron availability

Question: How does groundwater input affect plant distribution in Alnus glutinosa (black alder) carrs? Location: Alder carrs along the river Meuse, SE Netherlands. Methods: Three types of site, characterized by groundwater flow, were sampled in 17 A. glutinosa carrs. Vegetation and abiotic data (soil and water chemistry) were collected and analysed using a Canonical Correspondence Analysis. Based on the results, a laboratory experiment tested the effect of groundwater input (Ca²⁺) on pore water chemistry (NH₄⁺ availability). Results: Environmental factors indicating groundwater input (Ca²⁺ and Fe²⁺), correlating with the NH⁺₄ concentration in the pore water, best explained the variation in plant distribution. NH₄⁺ availability was determined by Ca²⁺ input via the groundwater and subsequent competition for exchange sites in the sediment. As a result, nutrient‐poor seepage locations fully fed by groundwater were dominated by small iron resistant plants such as Caltha palustris and Equisetum fluviatile. More nutrient‐rich locations, fed by a combination of groundwater and surface water, allowed the growth of taller iron resistant plant species such as Carex paniculata. Nutrient‐rich locations with stagnating surface water were hardly fed by groundwater, allowing the occurrence of fast growing and less iron tolerant wetland grasses such as Glyceria fluitans and G. maxima. Conclusion: Groundwater input affects plant composition in A. glutinosa carrs along the river Meuse by determining nutrient availability (ammonium) and concentrations of toxic iron.     

Intestinal inflammatory cytokine response in relation to tumorigenesis in the Apc(Min/+) mouse

The etiology of colon cancer is a complex phenomenon that involves both genetic and environmental factors. However, only about 20% have a familial basis with the largest fraction being attributed to environmental causes that can lead to chronic inflammation. While the link between inflammation and colon cancer is well established, the temporal sequence of the inflammatory response in relation to tumorigenesis has not been characterized. We examined the timing and magnitude of the intestinal inflammatory cytokine response in relation to tumorigenesis in the Apc^Min/+ mouse. Apc^Min/+ mice and wildtype mice were sacrificed at one of 4 time-points: 8, 12, 16, and 20 weeks of age. Intestinal tissue was analyzed for polyp burden (sections 1, 4 and 5) and mRNA expression and protein concentration of MCP-1, IL-1β, IL-6 and TNF-α (sections 2 and 3). The results show that polyp burden was increased at 12, 16 and 20 weeks compared to 8 weeks (P < 0.05). Gene expression (mRNA) of MCP-1, IL-1β, IL-6 and TNF-α was increased in sections 2 and 3 starting at week 12 (P < 0.05), with further increases in MCP-1, IL-1β and IL-6 at 16 weeks (P < 0.05). Protein concentration for these cytokines followed a similar pattern in section 3. Similarly, circulating MCP-1 was increased at 12 weeks (P < 0.05) and then again at 20 weeks (P < 0.05). In general, overall polyp number and abundance of large polyps were significantly correlated with the inflammatory cytokine response providing further support for a relationship between polyp progression and these markers. These data confirm the association between intestinal cytokines and tumorigenesis in the Apc^Min/+ mouse and provide new information on the timing and magnitude of this response in relation to polyp development. These findings may lead to the development of inflammatory mediators as important biomarkers for colon cancer progression. Further, these data may be relevant in the design of future investigations of therapeutic interventions to effectively target inflammatory processes in rodent models.