Constitutive expression of IL-22 in the human intervertebral disc and its reduction by exposure to pro-inflammatory cytokines in vitro

The importance of cytokines in disc degeneration is well recognized. Little is known about IL-22 expression in the human intervertebral disc. We investigated IL-22 immuno-localization in disc tissue, and molecular expression and production of IL-22 by annulus cells cultured in three-dimensional (3D) culture. We examined human disc tissue using immunohistochemistry and we cultured isolated annulus cells in 3D to analyze IL-22 expression and production, and its receptor, IL-22R, in conditioned media. Ingenuity pathway analysis (IPA) also was used to identify significant gene expression networks within the molecular data. IL-22 and IL-22R were immunolocalized in many cells in the human outer and inner annulus; fewer cells exhibited localization in the nucleus. Three-dimensional culture of annulus cells demonstrated production of IL-22 in conditioned media; exposure to IL-1ß or TNF-α significantly reduced IL-22 levels. Significant decreases also were identified in conditioned media assayed for IL-22R in TNF-α treated cells. IPA analysis showed that IL-22 ranked among the top canonical pathways. We found constitutive expression and production of IL-22 and IL-22R in the disc, which expands our understanding of the effect of pro-inflammatory cytokines on IL-22 expression and production. Three-dimensional cultured annulus cells exposed to IL-1ß or TNF produced significantly lower levels of IL-22 into their conditioned media compared to levels produced by control cells. Our findings have clinical relevance because of the elevated pro-inflammatory milieu within the degenerating human disc.     

Trichoderma spp. and Bacillus spp. as growth promoters in maize (Zea mays L.)

Microbes that are beneficial to plants are used to enhance the crop growth, yield and are alternatives to chemical fertilizers. Trichoderma and Bacillus are the predominant plant growth-promoting fungi and bacteria. The objective of this study was select, characterize, and evaluate isolates of Trichoderma spp. and Bacillus spp. native from the northern region of Sinaloa, Mexico, and assess their effect on growth promotion in maize (Zea mays L.). In greenhouse conditions, four Trichoderma isolates and twenty Bacillus isolates, as well as two controls, were tested in a completely randomized design with three replicates. We selected the two best strains of Trichoderma and Bacillus: TB = Trichoderma asperellum, TF = Trichoderma virens, B14 = Bacillus cereus sensu lato and B17 = Bacillus cereus, which were evaluated in the field in a completely randomized blocks in factorial arrangement design with three replicates applying different rates of nitrogen fertilizer (0, 150 kg N/ha, and 300 kg N/ha). Treatments 5 (B17 = B. cereus) and 11 (TF = T. virens) both fertilized with 150 kg N/ha showed similar yields and they did not reveal significant differences from the treatments fertilized with 300 kg N/ha. This indicated that treatment 5 (B17= B. cereus with 150 kg N/ha) and treatment 11 (TF= T. virens with 150 kg N/ha) were efficient as growth promoters, by not showing significant differences in root volume and dry weight of foliage. The results indicated a reduction of 50% in the rate of nitrogen to fertilizer required for maize (Zea mays L.) crops. These microorganisms Trichoderma and Bacillus could be an alternative to reduce the use of chemical fertilizers in maize.     

Phylogenetic analysis of carbamoylphosphate synthetase genes: complex evolutionary history includes an internal duplication within a gene which can root the tree of life

Carbamoylphosphate synthetase (CPS) catalyzes the first committed step in pyrimidine biosynthesis, arginine biosynthesis, or the urea cycle. Organisms may contain either one generalized or two specific CPS enzymes, and these enzymes may be heterodimeric (encoded by linked or unlinked genes), monomeric, or part of a multifunctional protein. In order to help elucidate the evolution of CPS, we have performed a comprehensive phylogenetic analysis using the 21 available complete CPS sequences, including a sequence from Sulfolobus solfataricus P2 which we report in this paper. This is the first report of a complete CPS gene sequence from an archaeon, and sequence analysis suggests that it encodes an enzyme similar to heterodimeric CPSII. We confirm that internal similarity within the synthetase domain of CPS is the result of an ancient gene duplication that preceded the divergence of the Bacteria, Archaea, and Eukarya, and use this internal duplication in phylogenetic tree construction to root the tree of life. Our analysis indicates with high confidence that this archaeal sequence is more closely related to those of Eukarya than to those of Bacteria. In addition to this ancient duplication which created the synthetase domain, our phylogenetic analysis reveals a complex history of further gene duplications, fusions, and other events which have played an integral part in the evolution of CPS.      

A rate-independent technique for analysis of nucleic acid sequences: evolutionary parsimony

The method of evolutionary parsimony--or operator invariants--is a technique of nucleic acid sequence analysis related to parsimony analysis and explicitly designed for determining evolutionary relationships among four distantly related taxa. The method is independent of substitution rates because it is derived from consideration of the group properties of substitution operators rather than from an analysis of the probabilities of substitution in branches of a tree. In both parsimony and evolutionary parsimony, three patterns of nucleotide substitution are associated one-to-one with the three topologically linked trees for four taxa. In evolutionary parsimony, the three quantities are operator invariants. These invariants are the remnants of substitutions that have occurred in the interior branch of the tree and are analogous to the substitutions assigned to the central branch by parsimony. The two invariants associated with the incorrect trees must equal zero (statistically), whereas only the correct tree can have a nonzero invariant. The chi 2-test is used to ascertain the nonzero invariant and the statistically favored tree. Examples, obtained using data calculated with evolutionary rates and branchings designed to camouflage the true tree, show that the method accurately predicts the tree, even when substitution rates differ greatly in neighboring peripheral branches (conditions under which parsimony will consistently fail). As the number of substitutions in peripheral branches becomes fewer, the parsimony and the evolutionary-parsimony solutions converge. The method is robust and easy to use.      

Increase in levels of microbiota recoverable from male and larval Myrmecia gulosa (Fabricius) (Hymenoptera: Formicidae) following segregation from worker ants

The segregation of laboratory maintained male and larval Myrmecia gulosa from workers resulted in increased levels of culturable microbiota. After 29 days, microbial levels recovered from segregated males and larvae were 27 and 126 times greater than from males and larvae not segregated from workers. These findings are consistent with the hypothesis that metapleural gland secretions, absent in larvae and males, are transferred from workers to larvae, and males contribute to the inhibition of cuticular microbiota.     

Hypercholesterolemia boosts joint destruction in chronic arthritis. An experimental model aggravated by foam macrophage infiltration

Objective

The aim of this study was to determine whether hypercholesterolemia increases articular damage in a rabbit model of chronic arthritis.

Methods

Hypercholesterolemia was induced in 18 rabbits by administrating a high-fat diet (HFD). Fifteen rabbits were fed normal chow as controls. Chronic antigen-induced arthritis (AIA) was induced in half of the HFD and control rabbits, previously immunized, by intra-articular injections of ovalbumin. After sacrifice, lipid and systemic inflammation markers were analyzed in blood serum. Synovium was analyzed by Krenn score, multinucleated cell counting, immunohistochemistry of RAM11 and CD31, and TNF-α and macrophage chemoattractant protein-1 (MCP-1) gene expression. Active bone resorption was assessed by protein expression of receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) and quantification of cathepsin K, contact surface and the invasive area of pannus into bone.

Results

Rabbits receiving the HFD showed higher total serum cholesterol, HDL, triglycerides and CRP levels than rabbits fed a normal diet. Synovitis score was increased in HFD, and particularly in AIA and AIA + HFD groups. AIA + HFD synovium was characterized by a massive infiltration of RAM11+ cells, higher presence of multinucleated foam cells and bigger vascularization than AIA. Cathepsin K+ osteoclasts and the contact surface of bone resorbing pannus were also increased in rabbits with AIA + HFD compared with AIA alone. Synovial TNF-α and MCP-1 gene expression was increased in AIA and HFD rabbits compared with healthy animals. RANKL protein expression in AIA and AIA + HFD groups was higher compared with either HFD or normal groups.

Conclusions

This experimental model demonstrates that hypercholesterolemia increments joint tissue damage in chronic arthritis, with foam macrophages being key players in this process.     

Does progressive nuclear staining with hemalum (alum hematoxylin) involve DNA,and what is the nature of the dye-chromatin complex?

Previous investigators have disagreed about whether hemalum stains DNA or its associated nucleoproteins. I review here the literature and describe new experiments in an attempt to resolve the controversy. Hemalum solutions, which contain aluminum ions and hematein, are routinely used to stain nuclei. A solution containing 16 Al³⁺ ions for each hematein molecule, at pH 2.0–2.5, provides selective progressive staining of chromatin without cytoplasmic or extracellular “background color.” Such solutions contain a red cationic dye-metal complex and an excess of Al³⁺ ions. The red complex is converted to an insoluble blue compound, assumed to be polymeric, but of undetermined composition, when stained sections are blued in water at pH 5.5–8.5. Staining experiments with DNA, histone and DNA + histone mixtures support the theory that DNA, not histone, is progressively colored by hemalum. Extraction of nucleic acids, by either a strong acid or nucleases at near neutral pH, prevented chromatin staining by a simple cationic dye, thionine, pH 4, and by hemalum, with pH adjustments in the range, 2.0–3.5. Staining by hemalum at pH 2.0–3.5 was not inhibited by methylation, which completely prevented staining by thionine at pH 4. Staining by hemalum and other dye-metal complexes at pH ≤ 2 may be due to the high acidity of DNA-phosphodiester (pK_a ~ 1). This argument does not explain the requirement for a much higher pH to stain DNA with those dyes and fluorochromes not used as dye-metal complexes. Sequential treatment of sections with Al₂(SO₄)₃ followed by hematein provides nuclear staining that is weaker than that attainable with hemalum. Stronger staining is seen if the pH is raised to 3.0–3.5, but there is also coloration of cytoplasm and other materials. These observations do not support the theory that Al³⁺ forms bridges between chromatin and hematein. When staining with hematein is followed by an Al₂(SO₄)₃ solution, there is no significant staining. Taken together, the results of my study indicate that the red hemalum cation is electrostatically attracted to the phosphate anion of DNA. The bulky complex cation is too large to intercalate between base pairs of DNA and is unlikely to fit into the minor groove. The short range van der Waals forces that bind planar dye cations to DNA probably do not contribute to the stability of progressive hemalum staining. The red cation is precipitated in situ as a blue compound, insoluble in water, ethanol and water-ethanol mixtures, when a stained preparation is blued at pH > 5.5.     

Phylogenetic analysis of the South American electric fishes (order Gymnotiformes) and the evolution of their electrogenic system: a synthesis based on morphology, electrophysiology, and mitochondrial sequence data

The order Gymnotiformes (South American electric fishes) is a fascinating assemblage of freshwater fishes that share the unusual ability to produce and sense electric fields used for electrolocation and social communication. In the last few decades, the electrogenic and electrosensory systems (EES) of these fish have served as an excellent model to study motor and sensory physiology in vertebrates. In an attempt to the evolution of characters associated with the EES in the group, we applied maximum-parsimony (MP), minimum-evolution (ME), and maximum-likelihood (ML) methods to analyze 302 aligned bases of the mitochondrial 12S rRNA and 416 bases of the mitochondrial 16S rRNA of 19 gymnotiform genera representing all six recognized families. Six catfish genera (order Siluriformes) were also sequenced and used as outgroups. The phylogenetic hypothesis resultant from molecular data analysis differs in some respects from previous hypotheses based on morphological studies. Our results were most informative within the family level, as we were unable to elucidate the relationships among deeper branches in this order with sufficient confidence by using molecular data alone. The phylogenetic information of both mitochondrial DNA segments appears to be affected by functional constraints, and the resultant topologies were sensitive to different weighting schemes and the algorithm used. Nonetheless, we found unanimous support for the following phylogenetic relationships: (1) the family Sternopygidae is an unnatural group, and Sternopygus is the sole representative of a unique lineage within the order; (2) the family Hypopomidae is not monophyletic; and (3) the order Gymnotiformes is composed of at least six natural clades: Sternopygus, family Apteronotidae, a new clade consisting of the remaining sternopygids, families Hypopomidae + Rhamphicthyidae, family Electrophoridae, and family Gymnotidae. By combining molecular, morphological, and physiological information, we propose a new hypothesis for the phylogeny of this group and suggest a new family Eigenmanniidae n. (order Gymnotiformes).