Inhibition of GSK-3β Rescues the Impairments in Bone Formation and Mechanical Properties Associated with Fracture Healing in Osteoblast Selective Connexin 43 Deficient Mice

Connexin 43 (Cx43) is the most abundant gap junction protein in bone and is required for osteoblastic differentiation and bone homeostasis. During fracture healing, Cx43 is abundantly expressed in osteoblasts and osteocytes, while Cx43 deficiency impairs bone formation and healing. In the present study we selectively deleted Cx43 in the osteoblastic lineage from immature osteoblasts through osteocytes and tested the hypothesis that Cx43 deficiency results in delayed osteoblastic differentiation and impaired restoration of biomechanical properties due to attenuated β-catenin expression relative to wild type littermates. Here we show that Cx43 deficiency results in alterations in the mineralization and remodeling phases of healing. In Cx43 deficient fractures the mineralization phase is marked by delayed expression of osteogenic genes. Additionally, the decrease in the RankL/ Opg ratio, osteoclast number and osteoclast size suggest decreased osteoclast bone resorption and remodeling. These changes in healing result in functional deficits as shown by a decrease in ultimate torque at failure. Consistent with these impairments in healing, β-catenin expression is attenuated in Cx43 deficient fractures at 14 and 21 days, while Sclerostin (Sost) expression, a negative regulator of bone formation is increased in Cx43cKO fractures at 21 days, as is GSK-3β, a key component of the β-catenin proteasomal degradation complex. Furthermore, we show that alterations in healing in Cx43 deficient fractures can be rescued by inhibiting GSK-3β activity using Lithium Chloride (LiCl). Treatment of Cx43 deficient mice with LiCl restores both normal bone formation and mechanical properties relative to LiCl treated WT fractures. This study suggests that Cx43 is a potential therapeutic target to enhance fracture healing and identifies a previously unknown role for Cx43 in regulating β-catenin expression and thus bone formation during fracture repair.     

Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Impact on energy metabolism of quantitative and functional cyclosporine-induced damage of kidney mitochondria

In this study we have measured, under experimental conditions which maintained efficient coupling, respiratory intensity, respiratory control, oxidative phosphorylation capacity and protonmotive force. Succinate cytochrome-c reductase and cytochrome-c oxidase activities were also studied. These investigations were carried out using kidney mitochondria from cyclosporine-treated rats (in vivo studies) and from untreated rats in the presence of cyclosporine (in vitro studies). Inhibition of respiratory intensity by cyclosporine did not exceed 21.1% in vitro and 15.9% in vivo. Since there was no in vitro inhibition of succinate cytochrome-c reductase and cytochrome-c oxidase activities, the slowing of electron flow observed can be interpreted as a consequence of an effect produced by cyclosporine between cytochromes b and c₁. Cyclosporine had no effect on respiratory control either in vitro or in vivo. Statistically significant inhibition of the oxidative phosphorylation was observed both in vitro (6.6%) and in vivo (12.1%). Moreover, cyclosporine did not induce any change of membrane potential either in vivo or in vitro. Our findings show that cyclosporine is neither a protonophore, nor a potassium ionophore. In cyclosporine-treated rats we noticed a decrease of protein in subcellular fraction, including the mitochondrial fraction. The role of the inhibition respiratory characteristics by cyclosporine in nephrotoxicity in vivo must take account of these two parameters: inhibition of the respiratory characteristics measured in vitro and diminution of mitochondrial protein in cyclosporine-treated rats.     

A pertussis toxin-sensitive G-protein mediates some aspects of insulin action in BC3H-1 murine myocytes.

The involvement of G-proteins in the insulin signal transduction system has been studied in detail using the murine BC3H-1 myocyte system. Pertussis toxin (PT) treatment, previously shown to attenuate some of the metabolic effects of insulin in this cell line (Luttrell, L.M., Hewlett, E.L., Romero, G., and Rogol, A.D. (1988) J. Biol. Chem. 263, 6134-6141), abolished insulin-induced generation of diacylglycerol and inositolglycan mediators with no effects on either the autophosphorylation of the insulin receptor or the phosphorylation of the major endogenous substrates for insulin-stimulated tyrosine kinase activity (pp185 and pp42-45). In vitro ADP-ribosylation and immunoblotting studies suggest that the major PT substrate is a 40-kDa protein of the G alpha family. This protein band did not exhibit detectable tyrosine phosphorylation upon stimulation of either intact cells or cell membranes with insulin. In the presence of low concentrations of GTP, insulin treatment of isolated myocyte plasma membranes resulted in a small (30-40%) but significant stimulation of GTP hydrolysis. This effect was best observed in the presence of small concentrations of sodium dodecyl sulfate. The rate of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to BC3H-1 membranes was also significantly increased in the presence of insulin. The effects of insulin on GTP hydrolysis and GTP gamma S binding were found to be dependent on the concentration of insulin. These effects were not detected in plasma membranes prepared from PT-pretreated BC3H-1 myocytes. In contrast, pretreatment with the B (inactive) subunit of PT did not alter the response of myocyte membranes to insulin. High affinity binding of [125I]iodoinsulin to myocyte plasma membranes was reduced by 60-70% in the presence of guanine nucleotides. Similar effects on insulin binding were produced by PT pretreatment of the cells. In contrast, adenine nucleotides had no effect on insulin binding. Scatchard analysis of the binding data showed that the observed effects of guanine nucleotides and PT on insulin binding resulted either from a reduction in the number of high affinity insulin binding sites or from a significant reduction of the affinity of insulin for its receptor. Low affinity binding sites did not appear to be affected by either guanine nucleotides nor PT pretreatment. These results provide substantial evidence suggestive of a noncovalent interaction between the insulin receptor and a regulatory G-protein system during the process of insulin signaling.     

NINJA-OPS: Fast Accurate Marker Gene Alignment Using Concatenated Ribosomes

The explosion of bioinformatics technologies in the form of next generation sequencing (NGS) has facilitated a massive influx of genomics data in the form of short reads. Short read mapping is therefore a fundamental component of next generation sequencing pipelines which routinely match these short reads against reference genomes for contig assembly. However, such techniques have seldom been applied to microbial marker gene sequencing studies, which have mostly relied on novel heuristic approaches. We propose NINJA Is Not Just Another OTU-Picking Solution (NINJA-OPS, or NINJA for short), a fast and highly accurate novel method enabling reference-based marker gene matching (picking Operational Taxonomic Units, or OTUs). NINJA takes advantage of the Burrows-Wheeler (BW) alignment using an artificial reference chromosome composed of concatenated reference sequences, the “concatesome,” as the BW input. Other features include automatic support for paired-end reads with arbitrary insert sizes. NINJA is also free and open source and implements several pre-filtering methods that elicit substantial speedup when coupled with existing tools. We applied NINJA to several published microbiome studies, obtaining accuracy similar to or better than previous reference-based OTU-picking methods while achieving an order of magnitude or more speedup and using a fraction of the memory footprint. NINJA is a complete pipeline that takes a FASTA-formatted input file and outputs a QIIME-formatted taxonomy-annotated BIOM file for an entire MiSeq run of human gut microbiome 16S genes in under 10 minutes on a dual-core laptop.     

Regulated nitrate transport in the cyanobacterium Anacystis nidulans.

Intracellular accumulation of nitrate, indicative of the operation of an active nitrate transport system, has been measured in intact cells of the cyanobacterium Anacystis nidulans. The ability of the cells to accumulate nitrate was effectively hindered by either ammonium addition or selective inhibition of CO2 fixation by DL-glyceraldehyde, with the effect of either compound being prevented by previously blocking ammonium assimilation. The results support the contention that nitrate utilization in cyanobacteria is regulated at the level of nitrate transport through the concerted action of ammonium assimilation and CO2 fixation.     

Glutathione content and GSH S-transferase activity in midgut gland of Procambarus clarkii. Sex differences, the effect of fasting, and their implications in cadmium toxicity

1. Glutathione content and GSH S-transferase activity in the midgut gland of Procambarus clarkii (P. c.) of different sex and body weight are presented. 2. Procambarus clarkii females' GSH concentration in the midgut gland decreases to a higher extent upon fasting, compared with males. 3. Procambarus clarkii females, both in control and fasting conditions, have a slightly higher GSH S-transferase activity than males. 4. Cadmium present in water only affects GSH content and GSH S-transferase activity (after 96 hr) in midgut gland, with cadmium chloride concentrations higher than 100 micrograms/l.     

Interleukin-1 stimulates prostaglandin biosynthesis by human amnion

The purpose of these studies was to determine if Interleukin-1 (IL-1) alters the rate of prostaglandin biosynthesis by human amnion. Primary monolayer cultures of amnion cells were established from women undergoing elective cesarean section before the onset of labor. Natural purified and recombinant human IL-1 alpha and IL-1 beta were incubated with amnion cells in culture, and prostaglandin E2 (PGE2) biosynthesis was measured by radioimmunoassay in cell-free media. A concentration-dependent increase in PGE2 production by amnion cells occurred in response to natural purified and recombinant IL-1 preparations. No differences in the parameters of the dose-response curves between the two IL-1 gene products could be determined (p greater than 0.05). Indomethacin blocked the effect of IL-1 in prostaglandin biosynthesis by human amnion. Interleukin-1, a fever mediator, could serve as a signal for the initiation of labor in cases of intrauterine or systemic infection.