Identification of an integral plasma membrane pool of protein kinase C in mammalian tissues and cells

Protein kinase C (PKC) is a family of serine/threonine protein kinases that are pivotal in cellular regulation. Since its discovery in 1977, PKCs have been known as cytosolic and peripheral membrane proteins. However, there are reports that PKC can insert into phospholipids vesicles in vitro. Given the intimate relationship between the plasma membrane and the activation of PKC, it is important to determine whether such "membrane-inserted" form of PKC exists in mammalian cells or tissues. Here, we report the identification of an integral plasma membrane pool for all the 10 PKC isozymes in vivo by their ability to partition into the detergent-rich phase in Triton X-114 phase partitioning, and by their resistance to extractions with 0.2 M sodium carbonate (pH 11.5), 2 M urea and 2 M sodium chloride. The endogenous integral membrane pool of PKC in mouse fibroblasts is found to be acutely regulated by phorbol ester or diacylglycerol, suggesting that this pool of PKC may participate in cellular processes known to be regulated by PKC. At least for PKC(alpha), the C2-V3 region at the regulatory domain of the kinase is responsible for membrane integration. Further exploration of the function of this novel integral plasma membrane pool of PKC will not only shed new light on molecular mechanisms underlying its cellular functions but also provide new strategies for pharmaceutical modulation of this important group of kinases.     

Cellulose Derivatives Enhanced Stability of Alginate-Based Beads Loaded with <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> LAB12 against Low pH,High Temperature and Prolonged Storage

The susceptibility of probiotics to low pH and high temperature has limited their use as nutraceuticals. In this study, enhanced protection of probiotics via microencapsulation was achieved. Lactobacillus plantarum LAB12 were immobilised within polymeric matrix comprised of alginate (Alg) with supplementation of cellulose derivatives (methylcellulose (MC), sodium carboxymethyl cellulose (NaCMC) or hydroxypropyl methylcellulose (HPMC)). L. plantarum LAB12 encapsulated in Alg-HPMC(1.0) and Alg-MC(1.0) elicited improved survivability (91%) in simulated gastric conditions and facilitated maximal release (～100%) in simulated intestinal condition. Alg-HPMC(1.0) and Alg-MC(1.0) significantly reduced (P < 0.05) the viability loss of LAB12 (viability loss <7%) when compared to Alg alone (viability loss <13%) under extreme temperatures (75 and 90 °C). Four-week storage of encapsulated LAB12 at 4 °C yielded viable counts >7 log CFU g^?1. Alg-MC and Alg-HPMC improved the survival of LAB12 against simulated gastric condition (9.24 and 9.55 log CFU g^?1, respectively), temperature up to 90 °C (9.54 and 9.86 log CFU g^?1, respectively) and 4-week of storage at 4 °C (8.61 and 9.23 log CFU g^?1, respectively) with sustained release of probiotic in intestinal condition (>9 log CFU g^?1). These findings strongly suggest the potential of cellulose derivatives supplemented Alg bead as protective micro-transport for probiotic strains. They can be safely incorporated into new functional food or nutraceutical products.     

Metabolipidomic profiling reveals an age‐related deficiency of skeletal muscle pro‐resolving mediators that contributes to maladaptive tissue remodeling

Specialized pro‐resolving mediators actively limit inflammation and support tissue regeneration, but their role in age‐related muscle dysfunction has not been explored. We profiled the mediator lipidome of aging muscle via liquid chromatography‐tandem mass spectrometry and tested whether treatment with the pro‐resolving mediator resolvin D1 (RvD1) could rejuvenate the regenerative ability of aged muscle. Aged mice displayed chronic muscle inflammation and this was associated with a basal deficiency of pro‐resolving mediators 8‐oxo‐RvD1, resolvin E3, and maresin 1, as well as many anti‐inflammatory cytochrome P450‐derived lipid epoxides. Following muscle injury, young and aged mice produced similar amounts of most pro‐inflammatory eicosanoid metabolites of cyclooxygenase (e.g., prostaglandin E₂) and 12‐lipoxygenase (e.g., 12‐hydroxy‐eicosatetraenoic acid), but aged mice produced fewer markers of pro‐resolving mediators including the lipoxins (15‐hydroxy‐eicosatetraenoic acid), D‐resolvins/protectins (17‐hydroxy‐docosahexaenoic acid), E‐resolvins (18‐hydroxy‐eicosapentaenoic acid), and maresins (14‐hydroxy‐docosahexaenoic acid). Similar absences of downstream pro‐resolving mediators including lipoxin A₄, resolvin D6, protectin D1/DX, and maresin 1 in aged muscle were associated with greater inflammation, impaired myofiber regeneration, and delayed recovery of strength. Daily intraperitoneal injection of RvD1 had minimal impact on intramuscular leukocyte infiltration and myofiber regeneration but suppressed inflammatory cytokine expression, limited fibrosis, and improved recovery of muscle function. We conclude that aging results in deficient local biosynthesis of specialized pro‐resolving mediators in muscle and that immunoresolvents may be attractive novel therapeutics for the treatment of muscular injuries and associated pain in the elderly, due to positive effects on recovery of muscle function without the negative side effects on tissue regeneration of non‐steroidal anti‐inflammatory drugs.     

Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR beta-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vbeta subsets (Vbeta2, Vbeta4, and Vbeta16 positive) with highly conserved TCRbeta complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR beta-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR alpha-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR alpha-chain vs TCR beta-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.     

Dimeric progestins from rhizomes of Ligusticum chuanxiong

Five dimeric phthalides were isolated from rhizomes of Ligusticum chuanxiong and their structures deduced based on spectral data. All compounds and their parent extracts were assessed for progesterone-like activity using a progesterone receptor driven reporter-gene bioassay. Among all the compounds, riligustilide, displayed weak progesterone-like activity (EC50 approximately 81 microM), whereas, (3Z')-(3a'R,6'R,3R,6R,7R)-3,8-dihydro-6.6',7.3a'-diligustilide (Mr: 382, EC50 approximately 90 nM), was found to be a potent and specific activator of the progesterone receptor. Levistolide A, although having a very similar plenary structure, was inactive indicating the importance of stereochemistry of chiral centers and flexibility of butylidene side chain for progestogenic activity. These bioactive phthalides and their parent extracts (EC50 approximately 8 microg/ml) may have utility for treatment of conditions requiring progesterone action.     

Antioxidant activity of phenylpropanoid esters isolated and identified from Platycodon grandiflorum A. DC

Platycodon grandiflorum A. DC (Campanulaceae) is used as a traditional oriental medicine and also as a food in Korea. Here we investigated its antioxidant activity, and isolated and identified its active compounds. Petroleum ether extracts from the whole root of P. grandiflorum were fractionated by silica gel column chromatography using a solvent gradient (petroleum ether:diethyl ether, v/v; 9:1-5:5). The 8:2 fraction showed a higher radical scavenging activity than the other fractions, and active compounds were purified from this fraction by reversed-phased HPLC. Two active compounds were identified as coniferyl alcohol esters of palmitic and oleic acids by FAB-MS, UV, IR and NMR spectroscopy. The antioxidant activities of these two compounds, which were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide and nitric oxide radical scavenging capacity, were found to be as high as those of BHT or BHA.     

Association between polymorphisms of matrix metalloproteinase 11 (MMP-11) and Kawasaki disease in the Korean population

AimsThis study was conducted to investigate the associations between single nucleotide polymorphisms (SNPs) of matrix metalloproteinases (MMPs) and Kawasaki disease (KD) in the Korean population.Main methodsA total 0f 101 KD patients and 306 healthy controls were examined. MMP7 (rs10502001, G/A, Arg77His), MMP11 (rs738792, T/C, Ala38Val), MMP12 (rs652438, A/G, Ile357Val) and MMP26 (rs2499953, A/G, Lys43Glu) genes were genotyped from the genomic DNA using direct sequencing. The results were then analyzed using logistic regression models, adjusting for gender as covariates.Key findingsThe four SNPs were in Hardy–Weinberg equilibrium. Only the MMP11 polymorphism (rs738792) was associated with KD. The SNP (rs738792) showed a statistically significant association with KD in the codominant (OR = 1.61, 95% CI = 1.11–2.34, P = 0.011) and dominant (OR = 1.92, 95% CI = 1.21–3.06, P = 0.006) models. However, there was no association between polymorphisms of other MMP genes and KD.SignificanceOverall, the results of this study indicate that MMP11 polymorphism may be associated with KD in the Korean population.     

Thickness sensing of hMSCs on collagen gel directs stem cell fate

Mechanically compliant substrate provides crucial biomechanical cues for multipotent stem cells to regulate cellular fates such as differentiation, proliferation and maintenance of their phenotype. Effective modulus of which cells sense is not only determined by intrinsic mechanical properties of the substrate, but also the thickness of substrate. From our study, it was found that interference from underlying rigid support at hundreds of microns away could induce significant cellular response. Human mesenchymal stem cells (hMSCs) were cultured on compliant biological gel, collagen type I, of different thickness but identical ECM composition and local stiffness. The cells sensed the thin gel (130 μm) as having a higher effective modulus than the thick gel (1440 μm) and this was reflected in their changes in morphology, actin fibers structure, proliferation and tissue specific gene expression. Commitment into neuronal lineage was observed on the thin gel only. Conversely, the thick gel (1440 μm) was found to act like a substrate with lower effective modulus that inhibited actin fiber polymerization. Stem cells on the thick substrate did not express tissue specific genes and remained at their quiescent state. This study highlighted the need to consider not only the local modulus but also the thickness of biopolymer gel coating during modulation of cellular responses.