The kinase activity of IL-1 receptor-associated kinase 4 is required for interleukin-1 receptor/toll-like receptor-induced TAK1-dependent NFkappaB activation

Two parallel interleukin-1 (IL-1)-mediated signaling pathways have been uncovered for IL-1R-TLR-mediated NFkappaB activation: TAK1-dependent and MEKK3-dependent pathways, respectively. The TAK1-dependent pathway leads to IKKalpha/beta phosphorylation and IKKbeta activation, resulting in classic NFkappaB activation through IkappaBalpha phosphorylation and degradation. The TAK1-independent MEKK3-dependent pathway involves IKKgamma phosphorylation and IKKalpha activation, resulting in NFkappaB activation through dissociation of phosphorylated IkappaBalpha from NFkappaB without IkappaBalpha degradation. IL-1 receptor-associated kinase 4 (IRAK4) belongs to the IRAK family of proteins and plays a critical role in IL-1R/TLR-mediated signaling. IRAK4 kinase-inactive mutant failed to mediate the IL-1R-TLR-induced TAK1-dependent NFkappaB activation pathway, but mediated IL-1-induced TAK1-independent NFkappaB activation and retained the ability to activate substantial gene expression, indicating a structural role of IRAK4 in mediating this alternative NFkappaB activation pathway. Deletion analysis of IRAK4 indicates the essential structural role of the IRAK4 death domain in receptor proximal signaling for mediating IL-1R-TLR-induced NFkappaB activation.     

Quantitative associations between osteocyte density and biomechanics, microcrack and microstructure in OVX rats vertebral trabeculae

Osteocytes actively regulate bone modeling and remodeling, direct skeletal mineralization, and regulate calcium/phosphate homeostasis and extracellular matrix metabolism; yet the specific role of osteocytes in maintaining bone structural integrity and strength is unknown. Studies have shown that the density of osteocytes decreases with age and estrogen deficiency, as seen in postmenopausal women. Here, we examined the relationships between osteocyte density and the related variables, including biomechanics, bone mineral density, microcrack and microstructure of vertebral trabeculae, in ovariectomized rats. We found that osteocyte density correlated with some of the parameters that determine the biomechanical quality of bone. Our findings suggest that osteocytes could play a crucial role in maintaining the mechanical quality of bone, and osteocyte density could be considered as an alternative index in assessing bone quality.     

Activated macrophages inhibit enterocyte gap junctions via the release of nitric oxide

Enterocytes exist in close association with tissue macrophages, whose activation during inflammatory processes leads to the release of nitric oxide (NO). Repair from mucosal injury requires the migration of enterocytes into the mucosal defect, a process that requires connexin43 (Cx43)-mediated gap junction communication between adjacent enterocytes. Enterocyte migration is inhibited during inflammatory conditions including necrotizing enterocolitis, in part, through impaired gap junction communication. We now hypothesize that activated macrophages inhibit gap junctions of adjacent enterocytes and seek to determine whether NO release from macrophages was involved. Using a coculture system of enterocytes and macrophages, we now demonstrate that "activation" of macrophages with lipopolysaccharide and interferon reduces the phosphorylation of Cx43 in adjacent enterocytes, an event known to inhibit gap junction communication. The effects of macrophages on enterocyte gap junctions could be reversed by treatment of macrophages with the inducible nitric oxide synthase (iNOS) inhibitor l-Lysine omega-acetamidine hydrochloride (l-NIL) and by incubation with macrophages from iNOS(-/-) mice, implicating NO in the process. Activated macrophages also caused a NO-dependent redistribution of connexin43 in adjacent enterocytes from the cell surface to an intracellular location, further suggesting NO release may inhibit gap junction function. Treatment of enterocytes with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) markedly inhibited gap junction communication as determined using single cell microinjection of the gap junction tracer Lucifer yellow. Strikingly, activated macrophages inhibited enterocyte migration into a scraped wound, which was reversed by l-NIL pretreatment. These results implicate enterocyte gap junctions as a target of the NO-mediated effects of macrophages during intestinal inflammation, particularly where enterocyte migration is impaired.     

Stromal-derived factor-1 (SDF-1) expression during early chick development

Cell migration plays a fundamental role in a wide variety of biological processes including development, tissue repair and disease. These processes depend on directed cell migration along and through cell layers. Chemokines are small secretory proteins that exert their effects by activating a family of G-protein coupled receptors and have been shown to play numerous fundamental roles in the control of physiological and pathological processes during development and in adult tissues, respectively. Stromal-derived factor-1 (SDF-1/CXCL12), a ligand of the chemokine receptor, CXCR4, is involved in providing cells with directional cues as well as in controlling their proliferation and differentiation. Here we studied the expression pattern of SDF-1 in the developing chick embryo. We could detect a specific expression of SDF-1 in the ectoderm, the sclerotome, the intersomitic spaces and the developing limbs. The expression domains of SDF-1 reflect its role in somitic precursor migration and vessel formation in the limbs.     

Analysis of metabolic flux in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing human-like collagen in fed-batch culture

Metabolic flux distributions of recombinant Escherichia coli BL21 expressing human-like collagen were determined by means of a stoichiometric network and metabolic balancing. At the batch growth stage, the fluxes of the pentose phosphate pathway were higher than the fluxes of the fed-batch growth phase and the production stage. After the temperature was increased, there was a substantially elevated energy demand for synthesizing human-like collagen and heat-shock proteins, which resulted in changes in metabolic fluxes. The activities of the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle were significantly enhanced, leading to a reduction in the fluxes of the pentose phosphate pathway and other anabolic pathways. The temperature upshift also caused an increase in NADPH production by isocitrate dehydrogenase in the tricarboxylic acid cycle. The metabolic model predicted the involvement of a transhydrogenase that generates additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. These data indicated that the maintenance energy for cellular activity increased with the increase in biomass in fed-batch culture, and that cell growth and synthesis of human-like collagen could clearly represent the changes in metabolic fluxes. At the production stage, more NADPH was used to synthesize human-like collagen than for maintaining cellular activity, cell growth, and cell propagation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Microcalorimetric Investigation on Metabolic Activity and Effects of La (III) in Mitochondria Isolated from Indica Rice 9311

Thermogenic metabolic curves were determined by the ampoule method at 303 K using a TAM air isothermal microcalorimeter in mitochondria isolated from rice 9311 (Oryza sativa L). From the thermogenic curves the activity recovery rate constant k and the maximum heat power P _m were obtained. Both were positively correlated to the protein content of rice mitochondria. The corresponding correlation coefficients were 0.9959 and 0.9950, respectively, indicating that the in vitro metabolic activity of mitochondria can be reliably expressed by these parameters. Addition of La (III) ions in concentrations ranging from 0 to 130 μg/mL resulted in significantly higher k and P _m values. Concentrations from 140 to 180 μg/mL had the opposite effect. These results are consistent with previous reports on the effects of rare earth elements on plant growth. We propose that the lanthanum-induced change of mitochondrial metabolic activity is a possible mechanism by which La (III) ions influence indica rice 9311 growth.     

Transient receptor potential vanilloid 1-mediated expression and secretion of endothelial cell-derived calcitonin gene-related peptide

Calcitonin gene-related peptide (CGRP), the principal transmitter in sensory nerves, could also be expressed in vascular endothelium. Transient receptor potential vanilloid 1(TRPV1), which modulates the synthesis and release of CGRP in sensory nerves, is also present in endothelial cells. The present study tested whether TRPV1 modulates the release and synthesis of CGRP in endothelial cells, and evaluated the protective effect of endothelial cell-derived CGRP. Human umbilical vein endothelial cells (HUVECs) were treated with capsaicin or hyperthermia. The level of CGRP mRNA was detected by RT-PCR, and protein level was measured by radioimmunoassay. Endothelial cell injury was induced by lysophosphatidylcholine, and evaluated by cell viability and lactate dehydrogenase activity. HUVECs expressed CGRP, both alpha- and beta-subtype. Capsaicin increased the level of CGRP in the culture medium, and up-regulated the expression of CGRP in endothelial cells. Hyperthermia also increased the level of CGRP mRNA. These effects were abolished by capsazepine, a competitive antagonist of TRPV1. Capsaicin significantly attenuated the endothelial cell damage induced by LPC, which was prevented and aggravated by capsazepine or CGRP(8-37,) antagonist of CGRP receptor. These results indicate that TRPV1 also regulates the expression and secretion of endothelial cell-derived CGRP, which affords protective effects on endothelial cells.     

A basidiomycetous yeast, Pseudozyma crassa, produces novel diastereomers of conventional mannosylerythritol lipids as glycolipid biosurfactants

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by the yeast strains of the genus Pseudozyma. These compounds show not only excellent surface-active properties, but also versatile biochemical actions. During a survey of new MEL producers, we found that a basidiomycetous yeast, Pseudozyma crassa, extracellularly produces three glycolipids. When glucose and oleic acid were used as the carbon source, the total amount of glycolipids reached approximately 4.6 g/L in the culture medium. The structures of these glycolipids were similar to those of well-known MEL-A, -B, and -C, respectively. Very interestingly, in all the present glycolipids, the configuration of the erythritol moiety was entirely opposite to that of conventional MELs. The present glycolipids were identified to have the carbohydrate structure of 4-O-β-d-mannopyranosyl-(2R,3S)-erythritol, stereochemically different from 4-O-β-d-mannopyranosyl-(2S,3R)-erythritol of conventional MELs. Furthermore, these new glycolipids possessed both short-chain acids (C₂ or C₄) and long-chain acids (C₁₄, C₁₆, or C₁₈) on the mannose moiety. The major component of the present glycolipids clearly showed different interfacial and biological properties, compared to conventional MELs comprising two medium-chain acids on the mannose moiety. Accordingly, the novel MEL diastereomers produced by P. crassa should provide us with different glycolipid functions, and facilitate a broad range of applications of MELs.     

Anticancer drugs are synergistic with freezing in induction of apoptosis in HCC cells

Cryotherapy has been shown to be an important therapeutic alternative to surgery in the treatment of hepatocellular carcinoma (HCC). Here, the influence of cryo-chemotherapy on HCC was examined in vitro using the human HCC cell line Bel-7402, a drug-resistant HCC cell line originating from Bel-7402 cells (Bel-7402/R), as well as two control cell lines, the HCC cell line SMMC-7721 and a colorectal tumor cell line HIC-251. Cells were treated with either exposure to different freezing temperatures (ranging from −15 to −80 °C for 20 min), exposure to sub-lethal concentrations of anticancer chemotherapy drugs or a combination of cryotherapy and chemotherapy. Cell viability and apoptosis under each condition were investigated. We found that the combined treatment resulted in increases in both cell death and apoptosis compared to either treatment alone. The increased level of apoptosis observed in Bel-7402 cells after cryo-chemotherapy was inhibited in the presence of caspase inhibitors. Furthermore, Bax expression was increased 2- to 3-fold in cells exposed to the combination treatment compared with cells treated by freezing or drugs alone. In contrast, Bcl-2 levels remained constant. Although Bel-7402/R cells originated from the Bel-7402 cell line, they were more sensitive to the freezing procedure than the parental cell line. The level of Bax expression in Bel-7402/R cells was also higher than that observed in the parental cell line. In addition, we found that Bel-7402/R cells had lower levels of survivin mRNA than the parental Bel-7402 cells, in both untreated and treated cells. In conclusion, our data show that in HCC cells, apoptosis induced by cryotherapy can be synergistically enhanced using anticancer drugs.