Determination of acetylsalicylic acid and salicylic acid in skin and plasma by high-performance liquid chromatography

This study describes a HPLC method to determine the concentrations of acetylsalicylic acid (ASA) and salicylic acid (SA) in human stratum corneum and in plasma. The stratum corneum layers for ASA/SA analysis were removed from three patients with postherpetic hyperalgesia treated with topical and oral aspirin. Blood samples were also collected from the same patients. Tape strippings were placed in acetonitrile and sonicated for 15 min. After centrifuging, aliquots of the supernatant were injected into the chromatograph. ASA and SA from plasma samples were extracted on Isolute C₈ columns. Due to interfering peaks in the tape samples, HPLC conditions were slightly different for tape and plasma samples. ASA and SA were separated on a LiChrospher 100 RP-18 column at 1 ml/min using a water–phosphate buffer (pH 2.5)–acetonitrile mobile phase (35:40:25, v/v/v). A linear response to quantities of ASA from 0.1 to 100 μg/cm² and of SA from 0.1 to 5 μg/cm² in tape and to quantities of ASA 0.1 to 2 μg/ml and 1 to 50 μg/ml was obtained and the recovery from tape and plasma samples was over 98%. The method is sensitive (0.1 μg/cm²) and specific enough to allow the determination of the drugs in the skin not only after topical but also after oral administration. A good sensitivity was also obtained in plasma (0.1 μg/ml) allowing study of the kinetics of ASA and SA in plasma after oral administration. Concentrations of ASA after topical administration were 100–200 times higher than after oral administration. Plasma levels of ASA and SA after oral administration were similar to those previously found. No ASA or SA were detected in plasma after topical ASA administration.     

Loss of RNA binding protein HuD facilitates the production of the senescence-associated secretory phenotype

HuD, an RNA binding protein, plays a role in the regulation of gene expression in certain types of cells, including neuronal cells and pancreatic β-cells, via RNA metabolism. Its aberrant expression is associated with the pathogenesis of several human diseases. To explore HuD-mediated gene regulation, stable cells expressing short hairpin RNA against HuD were established using mouse neuroblastoma Neuro2a (N2a) cells, which displayed enhanced phenotypic characteristics of cellular senescence. Two approaches, RNA immunoprecipitation (RNA IP)-NanoString profiling and cytokine array, were used to subsequently identify a subset of putative HuD targets that act as senescence-associated secretory phenotype (SASP), including C-C motif ligand 2 (CCL2), CCL20, C-X-C motif chemokine ligand 2 (CXCL2), and interleukin-6 (IL-6). Here, we further demonstrated that HuD regulates the expression of CCL2, a SASP candidate upregulated in cells following HuD knockdown, by binding to the 3′-untranslated region (UTR) of Ccl2 mRNA. Downregulation of HuD increased the level of CCL2 in N2a cells and the brain tissues of HuD knockout (KO) mice. Exposure to γ-irradiation induced cellular senescence in N2a cells and HuD knockdown facilitated stress-induced cellular senescence. Our results reveal that HuD acts as a novel regulator of CCL2 expression, and its aberrant expression may contribute to cellular senescence by regulating SASP production.Subject terms: Senescence, RNA quality control     

Induction of human choriogonadotropin in HeLa-cell cultures by aliphatic monocarboxylates and inhibitors of deoxyribonucleic acid synthesis

The ectopic production of the glycopeptide hormone human placental choriogonadotropin by HeLa₆₅ cells was measured by radioimmunoassay with antiserum against the β-subunit of choriogonadotropin and with the ¹²⁵I-labelled β-subunit as a tracer antigen. Choriogonadotropin synthesis was markedly (500-fold) stimulated by sodium butyrate. Kinetic studies and the use of an inhibitor of protein synthesis, cycloheximide, indicated that protein synthesis was required for this induction. Investigation of the efficiency of 22 aliphatic short-chain fatty acids and derivatives in causing increased choriogonadotropin synthesis by HeLa cells showed stringent structural requirements. Induction of choriogonadotropin synthesis in HeLa cells was not restricted to butyrate. Other aliphatic acids (propionate, isobutyrate, valerate and hexanoate) were also capable of inducing choriogonadotropin synthesis at 10–50% of the efficiency of butyrate. Hydroxy derivatives of monocarboxylate inducers, related mono- and di-carboxylic acids, alcohols, amines, ketones, esters and sulphoxide were ineffective in increasing choriogonadotropin production by HeLa cells. A saturated C₄ straight-chain acid without substituent hydroxyl groups but with a methyl group at one end and a carboxyl moiety at the other appeared to be most efficient in activating choriogonadotropin production. A second clonal line of HeLa cells, HeLa₇₁, showed a higher constitutive synthesis of choriogonadotropin than HeLa₆₅ cells, which was also markedly increased by butyrate. Butyrate and other aliphatic monocarboxylate inducers of choriogonadotropin synthesis inhibited HeLa-cell growth and DNA synthesis. This inhibition of DNA replication may be related to the mechanism of choriogonadotropin synthesis, since two well-characterized anti-neoplastic inhibitors of DNA synthesis, hydroxyurea and 1-β-d-arabinofuranosylcytosine, also stimulated a 300-fold increase in choriogonadotropin synthesis in HeLa cells and were synergistic with butyrate in promoting choriogonadotropin synthesis. Thus activation in tumour cells of genes normally expressed by foetal tissue and the consequent ectopic synthesis of polypeptide hormones may require neither cell division nor DNA synthesis.     

IL-6 Amplifies TLR Mediated Cytokine and Chemokine Production: Implications for the Pathogenesis of Rheumatic Inflammatory Diseases

The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.     

On the antibiotic activity of oxazolomycin

Structural analysis of oxazolomycin and simpler fragments containing a common 3-hydroxy-2,2-dimethylpropanamide moiety has indicated that a U-shaped conformation is preferred, in some cases stabilised by hydrogen bonding between the N–H and O–H residues, as shown by a combination of molecular modelling, NMR spectroscopic and single crystal X-ray analysis. A direct synthesis of this unit has been established via the opening of β-lactones by a range of amines, and their antibacterial activity been shown to vary with the hydrophobic character of the substituents.     

Growth-Coupled Lipid Synthesis in Mortierella alpina LPM 301, a Producer of Arachidonic Acid

Mortierella alpina LPM 301, a producer of arachidonic acid (ARA), was found to possess a unique property of intense lipid synthesis in the period of active mycelium growth. Under batch cultivation of this strain in glucose-containing media with potassium nitrate or urea, the bulk of lipids (28–35% of dry biomass) was produced at the end of the exponential growth phase and remained almost unaltered in the stationary phase. The ARA content of lipids comprised 42–50% at the beginning of the stationary phase and increased continuously after glucose depletion in the medium due to the turnover of intracellular fatty acids; by the end of fermentation (189–210 h), the amount of ARA reached 46–60% of the total fatty acids (16–19% of dry mycelium). Plausible regulatory mechanisms of the growth-coupled lipid synthesis in microorganisms are discussed.     

Clonidine induces calcitonin gene-related peptide expression via nitric oxide pathway in endothelial cells

The present study was to determine whether clonidine could induce calcitonin gene-related peptide (CGRP) production and the underlying mechanisms. Human umbilical vein endothelial cells were treated with clonidine and the dose–effect or time–effect relationship of clonidine on CGRP production was examined. Youhimbine (a α₂-adrenoceptor blocker) and l-NAME (an antagonist of nitric oxide synthase, NOS) were chosen to explore the role of α₂-adrenoceptor and nitric oxide pathway in the effect of clonidine on endothelial cell-derived CGRP production. The level of CGRP mRNA or protein was detected by Real Time-PCR or radioimmunoassay. Nitric oxide content was measured by nitroreduction assay. The study showed that clonidine was able to induce CGRP mRNA (α- and β-isoforms) expression in a dose-dependent manner in endothelial cells. The effect of clonidine on endothelial cell-derived CGRP synthesis and secretion was attenuated in the presence of youhimbine. l-NAME treatment could also inhibit clonidine-induced CGRP synthesis and secretion concomitantly with the decreased NO content in culture medium. These results suggest that clonidine could stimulate CGRP synthesis and secretion in endothelial cells through the activation of α₂-adrenoceptor, which is related to the NO pathway.     

CCL20 and Beta-Defensin 2 Production by Human Lung Epithelial Cells and Macrophages in Response to Brucella abortus Infection

Both CCL20 and human β-defensin 2 (hBD2) interact with the same membrane receptor and display chemotactic and antimicrobial activities. They are produced by airway epithelia in response to infectious agents and proinflammatory cytokines. Whereas Brucella spp. can infect humans through inhalation, their ability to induce CCL20 and hBD2 in lung cells is unknown. Here we show that B. abortus induces CCL20 expression in human alveolar (A549) or bronchial (Calu-6) epithelial cell lines, primary alveolar epithelial cells, primary human monocytes, monocyte-derived macrophages and the monocytic cell line THP-1. CCL20 expression was mainly mediated by JNK1/2 and NF-kB in both Calu-6 and THP-1 cells. CCL20 secretion was markedly induced in A549, Calu-6 and THP-1 cells by heat-killed B. abortus or a model Brucella lipoprotein (L-Omp19) but not by the B. abortus lipopolysaccharide (LPS). Accordingly, CCL20 production by B. abortus-infected cells was strongly TLR2-dependent. Whereas hBD2 expression was not induced by B. abortus infection, it was significantly induced in A549 cells by conditioned media from B. abortus-infected THP-1 monocytes (CMB). A similar inducing effect was observed on CCL20 secretion. Experiments using blocking agents revealed that IL-1β, but not TNF-α, was involved in the induction of hBD2 and CCL20 secretion by CMB. In the in vitro antimicrobial assay, the lethal dose (LD) 50 of CCL20 for B. abortus (>50 μg/ml) was markedly higher than that against E. coli (1.5 μg/ml) or a B. abortus mutant lacking the O polysaccharide in its LPS (8.7 ug/ml). hBD2 did not kill any of the B. abortus strains at the tested concentrations. These results show that human lung epithelial cells secrete CCL20 and hBD2 in response to B. abortus and/or to cytokines produced by infected monocytes. Whereas these molecules do not seem to exert antimicrobial activity against this pathogen, they could recruit immune cells to the infection site.     

Redox Control of the Senescence Regulator Interleukin-1α and the Secretory Phenotype

Senescent cells accumulate in aged tissue and are causally linked to age-associated tissue degeneration. These non-dividing, metabolically active cells are highly secretory and alter tissue homeostasis, creating an environment conducive to metastatic disease progression. IL-1α is a key senescence-associated (SA) proinflammatory cytokine that acts as a critical upstream regulator of the SA secretory phenotype (SASP). We established that SA shifts in steady-state H₂O₂ and intracellular Ca²⁺ levels caused an increase in IL-1α expression and processing. The increase in intracellular Ca²⁺ promoted calpain activation and increased the proteolytic cleavage of IL-1α. Antioxidants and low oxygen tension prevented SA IL-1α expression and restricted expression of SASP components IL-6 and IL-8. Ca²⁺ chelation or calpain inhibition prevented SA processing of IL-1α and its ability to induce downstream cytokine expression. Conditioned medium from senescent cells treated with antioxidants or Ca²⁺ chelators or cultured in low oxygen markedly reduced the invasive capacity of proximal metastatic cancer cells. In this paracrine fashion, senescent cells promoted invasion by inducing an epithelial-mesenchymal transition, actin reorganization, and cellular polarization of neighboring cancer cells. Collectively, these findings demonstrate how SA alterations in the redox state and Ca²⁺ homeostasis modulate the inflammatory phenotype through the regulation of the SASP initiator IL-1α, creating a microenvironment permissive to tumor invasion.     

Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in -minimal essential medium containing either vehicle, genistein (10^–7–10^–5 M) or daidzein (10^–7–10^–5 M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [³H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein (10^–5 M) or daidzein (10^–5 M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10^–7 M) or cycloheximide (10^–6 M) in the absence or presence of isoflavones. Moreover, when genistein (10^–7–10^–5 M) or daidzein (10^–6 and 10^–5 M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10^–7 M). In addition, [³H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10^–6 and 10^–5 M) or daidzein (10^–5 M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.     

Synthesis and pharmacological evaluation of 1,1,3-substituted urea derivatives as potent TNF-α production inhibitors

A three substituted urea derivative, SA13353 (compound 1a), exhibited potent inhibitory activity against lipopolysaccharide (LPS)-induced TNF-α production. We focused on the 1,1-substituted moiety (R¹ and R²) of SA13353 and investigated substituent effects of this moiety on LPS-induced TNF-α production by oral administration in rats. The synthesis of the urea derivatives was performed rapidly in a one-pot manner using a manual synthesizer. Several compounds containing hydrophobic substituents at this moiety showed more potent inhibitory activities than SA13353.     

TWEAK Activates the Non-Canonical NFκB Pathway in Murine Renal Tubular Cells: Modulation of CCL21

TWEAK is a member of the TNF superfamily of cytokines that contribute to kidney tubulointerstitial injury. It has previously been reported that TWEAK induces transient nuclear translocation of RelA and expression of RelA-dependent cytokines in renal tubular cells. Additionally, TWEAK induced long-lasting NFκB activation suggestive of engagement of the non-canonical NFκB pathway. We now explore TWEAK-induced activation of NFκB2 and RelB, as well as expression of CCL21, a T-cell chemotactic factor, in cultured murine tubular epithelial cells and in healthy kidneys in vivo. In cultured tubular cells, TWEAK and TNFα activated different DNA-binding NFκB complexes. TWEAK-induced sustained NFκB activation was associated with NFκB2 p100 processing to p52 via proteasome and nuclear translocation and DNA-binding of p52 and RelB. TWEAK, but not TNFα used as control), induced a delayed increase in CCL21a mRNA (3.5±1.22-fold over control) and CCL21 protein (2.5±0.8-fold over control), which was prevented by inhibition of the proteasome, or siRNA targeting of NIK or RelB, but not by RelA inhibition with parthenolide. A second NFκB2-dependent chemokine, CCL19, was upregulates by TWEAK, but not by TNFα. However, both cytokines promoted chemokine RANTES expression (3-fold mRNA at 24 h). In vivo, TWEAK induced nuclear NFκB2 and RelB translocation and CCL21a mRNA (1.5±0.3-fold over control) and CCL21 protein (1.6±0.5-fold over control) expression in normal kidney. Increased tubular nuclear RelB and tubular CCL21 expression in acute kidney injury were decreased by neutralization (2±0.9 vs 1.3±0.6-fold over healthy control) or deficiency of TWEAK (2±0.9 vs 0.8±0.6-fold over healthy control). Moreover, anti-TWEAK treatment prevented the recruitment of T cells to the kidney in this model (4.1±1.4 vs 1.8±1-fold over healthy control). Our results thus identify TWEAK as a regulator of non-canonical NFκB activation and CCL21 expression in tubular cells thus promoting lymphocyte recruitment to the kidney during acute injury.     

Cortisol Biosynthesis in the Human Ocular Surface Innate Immune Response

Innate immune responses have a critical role in regulating sight-threatening ocular surface (OcS) inflammation. While glucocorticoids (GCs) are frequently used to limit tissue damage, the role of intracrine GC (cortisol) bioavailability via 11-beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in OcS defense, remains unresolved. We found that primary human corneal epithelial cells (PHCEC), fibroblasts (PHKF) and allogeneic macrophages (M1, GM-CSF; M2, M-CSF) were capable of generating cortisol (M1>PHKF>M2>PHCEC) but in corneal cells, this was independent of Toll-like receptor (TLR) activation. While PolyI∶C induced maximal cytokine and chemokine production from both PHCEC (IFNγ, CCL2, CCL3, and (CCL4), IL6, CXCL10, CCL5, TNFα) and PHKF (CCL2, IL-6, CXCL10, CCL5), only PHKF cytokines were inhibited by GCs. Both Poly I∶C and LPS challenged-corneal cells induced M1 chemotaxis (greatest LPS-PHKF (250%), but down-regulated M1 11β-HSD1 activity (30 and 40% respectively). These data were supported by clinical studies demonstrating reduced human tear film cortisol∶cortisone ratios (a biomarker of local 11β-HSD1 activity) in pseudomonas keratitis (1∶2.9) versus healthy controls (1∶1.3; p<0.05). This contrasted with putative TLR3-mediated OcS disease (Stevens-Johnson Syndrome, Mucous membrane pemphigoid) where an increase in cortisol∶cortisone ratio was observed (113.8∶1; p<0.05). In summary, cortisol biosynthesis in human corneal cells is independent of TLR activation and is likely to afford immunoprotection under physiological conditions. Contribution to ocular mucosal innate responses is dependent on the aetiology of immunological challenge.     

Enhanced production of pigment-free pullulan by a morphogenetically arrested Aureobasidium pullulans (ATCC 42023) in a two-stage fermentation with shift from soy bean oil to sucrose

A two-stage fermentation process was established for the production of pigment-free pullulan by the yeast-like fungus Aureobasidium pullulans (ATCC 42023). In the first stage, starting at pH 4.5 with soy bean oil as the carbon source and glutamate as the nitrogen source, a cell mass of about 15 g l^–1 dry cell weight was obtained, the population being restricted mainly to the yeast form of the microorganism (yeast form more than 90% of total cells) and the formation of pigment in the culture being prevented. Small amounts of pullulan (less than 2 g l^–1) are produced at this phase, and the viscosity remained low throughout the entire growth stage. When the oil and glutamate source were nearly exhausted (below 5% of initial amounts), the cells were shifted to a production stage with sucrose as the carbon source with continued nitrogen depletion. Production of pullulan started immediately with no lag period. During 50 h of the production phase more than 35 g l^–1 of pullulan was produced (productivity approx. 0.7 g l^–1), resulting in a large increase in the viscosity of the broth. The production yield of pollulan on the sugar was about 0.6 g g^–1. Morphogenesis from the yeast form of the microorganism to chlamydospores was still restrained and no pigment was formed in the culture during the production stage. A pigment-free polysaccharide, with a molecular mass in the range of 600–750 kDa, was recovered from the supernatant of the broth after solvent precipitation.     

Construction of the plasmid for expression of ETA-EGFP fusion protein under control of the cytomegalovirus promoter and its effects in HeLa cells

Tumor-targeted vectors encoding toxic protein genes are promising tools for treating malignant tumors. We used the pEGFP-N1 vector to construct a novel plasmid (pCMV-ETA-EGFP) for eukaryotic expression of a truncated Pseudomonas aeruginosa exotoxin A (ETA) that is known to inhibit protein synthesis, and subsequently induce cell death, by inactivation of elongation factor-2. ETA was linked to the enhanced green fluorescent protein (EGFP) gene, and ETA-EGFP gene expression was driven by the cytomegalovirus (CMV) promoter. The time-lapse effects of pCMV-ETA-EGFP expression were examined in transiently transfected HeLa cells. HeLa cells transfected with pCMV-ETA-EGFP or cotransfected with pCMV-ETA-EGFP and рЕGFP-N1 showed lower fluorescence intensity than cells transfected with pEGFP-N1 alone. Analysis of the number of dead cells further confirmed the highly toxic effect of the ETA-EGFP fusion protein on cells transfected with pCMV-ETA-EGFP or cotransfected with pCMV-ETA-EGFP and рЕGFP-N1. ETA-EGFP fusion protein induced apoptotic cell death through the caspase-3 activation. By using the antibody against a marker nucleolar antigen A3 [Grigoryev, A.A., Bulycheva, T.I., Sheval, E.V., Kalinina, I.A., Zatsepina, O.V., 2008. Cytological indicators of the overall suppression of protein synthesis revealed by staining with new monoclonal antibody. Cell Tissue Biol. 2, 191–199], the distribution of which changes when HeLa cells are treated with known translation inhibitors, we obtained evidence to support the idea that protein synthesis is inhibited in transfected cells in situ. ETA-EGFP fusion protein was identified in lysates of transfected cells using anti-GFP-BL antibodies. Collectively, our results indicate that HeLa cells transfected with pCMV-ETA-EGFP synthesize the ETA-EGFP fusion protein that efficiently inhibits protein synthesis, leading to massive cell death by an apoptosis-mediated pathway with a participation of caspase-3. The constructed vector can be used in suicidal gene therapy of cancer and may also be useful for investigating the general effects of translational downregulation in human cancer cells. We also suggest a novel approach for detecting the activity of new vectors in transfected cells, which is based on the redistribution of nucleolar proteins in transfected cells.     

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Introduction

The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.

Methods

Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.

Results

LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.

Conclusions

Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.     

The Pseudomonas aeruginosa rhlG Gene Encodes an NADPH-Dependent β-Ketoacyl Reductase Which Is Specifically Involved in Rhamnolipid Synthesis

A Pseudomonas aeruginosa gene homologous to the fabG gene, which encodes the NADPH-dependent β-ketoacyl-acyl carrier protein (ACP) reductase required for fatty acid synthesis, was identified. The insertional mutation of this fabG homolog (herein called rhlG) produced no apparent effect on the growth rate and total lipid content of P. aeruginosa cells, but the production of rhamnolipids was completely abrogated. These results suggest that the synthetic pathway for the fatty acid moiety of rhamnolipids is separate from the general fatty acid synthetic pathway, starting with a specific ketoacyl reduction step catalyzed by the RhlG protein. In addition, the synthesis of poly-β-hydroxyalkanoate (PHA) is delayed in this mutant, suggesting that RhlG participates in PHA synthesis, although it is not the only reductase involved in this pathway. Traits regulated by the quorum-sensing response, other than rhamnolipid production, including production of proteases, pyocyanine, and the autoinducer butanoyl-homoserine lactone (PAI-2), were not affected by the rhlG mutation. We conclude that the P. aeruginosa rhlG gene encodes an NADPH-dependent β-ketoacyl reductase absolutely required for the synthesis of the β-hydroxy acid moiety of rhamnolipids and that it has a minor role in PHA production. Expression of rhlG mRNA under different culture conditions is consistent with this conclusion.