Heparin inhibits melanosome uptake and inflammatory response coupled with phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways in human epidermal keratinocytes

To gain insight for the role of mast cell‐produced heparin in the regulation of epidermal homeostasis and skin pigmentation, we have investigated the effect of heparin on melanosome uptake and proinflammatory responses in normal human epidermal keratinocytes (NHEKs). We quantified phagocytic activity of NHEKs with uptake of melanosomes or fluorescent microspheres. Heparin exhibited the inhibitory effect on keratinocyte phagocytosis through blocking PI3k/Akt and MEK/ERK signaling pathways. In fact, the heparin‐treated NHEKs showed impaired activation of Akt and ERK during phagocytosis, whereas PI3k and MEK inhibitors significantly suppressed melanosome uptake by NHEKs. In addition, the inflammation marker cycloxygenase‐2 (COX‐2) expression and prostaglandin E₂ (PGE₂) production were induced during phagocytosis, while these effects were downregulated in the presence of heparin. Our observations suggest that heparin may play an antiphagocytic and anti‐inflammation role in epidermis of human skin.     

Genetic variations in humans associated with differences in the course of hepatitis C

The outcome of hepatitis C virus (HCV) infection varies among individuals, but the genetic factors involved remain unknown. We conducted a population-based association study in which 238 Japanese individuals positive for anti-HCV antibody were genotyped for 269 single nucleotide polymorphisms (SNPs) in 103 candidate genes that might influence the course of infection. Altogether, 50 SNPs in 32 genes were listed. Genetic polymorphisms in IL4, IL8RB, IL10RA, PRL, ADA, NFKB1, GRAP2, CABIN1, IFNAR2, IFI27, IFI41, TNFRSF1A, ALDOB, AP1B1, SULT2B1, EGF, EGFR, TGFB1, LTBP2, and CD4 were associated with persistent viremia (P < 0.05), whereas those in IL1B, IL1RL1, IL2RB, IL12RB1, IL18R1, STAT5A, GRAP2, CABIN1, IFNAR1, Mx1, BMP8, FGL1, LTBP2, CD34, and CD80 were associated with different serum alanine aminotransferase levels in HCV carriers (P < 0.05). The sorted genes allow us to draw novel hypotheses for future studies of HCV infection to ultimately identify bona fide genes and their variations.     

Polymorphisms Influencing Expression of Dermonecrotic Toxin in Bordetella bronchiseptica

Bordetella bronchiseptica is a pathogenic bacterium causing respiratory infections in a broad range of mammals. Recently, we determined the whole genome sequence of B. bronchiseptica S798 strain isolated from a pig infected with atrophic rhinitis and found four single-nucleotide polymorphisms (SNPs) at positions -129, -72, +22, and +38 in the region upstream of dnt encoding dermonecrotic toxin (DNT), when compared with a rabbit isolate, RB50. DNT is known to be involved in turbinate atrophy observed in atrophic rhinitis. Immunoblotting, quantitative real-time PCR, and β-galactosidase reporter assay revealed that these SNPs resulted in the increased promoter activity of dnt and conferred the increased ability to produce DNT on the bacteria. Similar or identical SNPs were also found in other pig isolates kept in our laboratory, all of which produce a larger amount of DNT than RB50. Our analysis revealed that substitution of at least two of the four bases, at positions -72 and +22, influenced the promoter activity for dnt. These results imply that these SNPs are involved in the pathogenicity of bordetellae specific to pig diseases.     

Cessation of annual apical growth and partial death of cambium in stem of Abies sachalinensis under intensive shading

Journal of Plant Research - This study evaluated variation in the height at which absent rings and internodes were detected along stem of Abies sachalinensis trees grown under shade for...     

Molecular cloning of a cDNA for alpha-subunit of rat liver electron transfer flavoprotein

Two cDNA clones for the alpha-subunit of rat liver electron transfer flavoprotein were isolated and their nucleotide sequences were determined. The longer cDNA contained a protein-coding region of 900 nucleotides and 3'-noncoding region of 335 nucleotides. The identity of the clone was confirmed by matching the amino acid sequence predicted from the cDNA with the sequence of one of the lysyl endopeptidase-digested peptides from the purified alpha-subunit. The molecular weight of the protein calculated from the protein-coding nucleotides was approx. 3,000 daltons smaller than that of the precursor, suggesting that the cDNA was not of full length. The derived amino acid composition fairly agreed with the chemically determined amino acid composition of the purified alpha-subunit, indicating that the protein-coding region contains most of the mature alpha-subunit.     

Effects of light on fruit-body formation in a basidiomycete, Coprinus macrorhizus

A basidiomycete, Coprinus macrorhizus produced only vegetativemycelia, when cultured under continuous darkness. Under continuouswhite light, visible tiny primordia formed on the 6th day afterinoculation, followed by normal development to fruit-bodies.Spores disseminated on the 11th day. However, when cultureswhere transferred to continuous darkness after the formationof primordia, rudimentary pilei with slender stalks formed.Abundant hairy hyphae were produced along the entire surfaceof the slender stalks. Thus, light was required-for both theinitiation and development of the fungal fruit-body. A fairy ring of primordia formed along the narrow region ofmycelia formed just prior to the exposure to light, provideda colony was pre-grown for more than 4 days in the dark, thenexposed to light for 24 hr or longer. Exposure to light for at least 48 hr during the period between24th and 96th hr after primordium initiation was required fornormal maturation of the fruit-body. This 48 hr light periodneeded for maturation of the fruit-body could be substitutedby two, 2 hr light periods given at the beginning and the endof the 48 hr period. We have tentatively concluded that lightis required at two definite stages for fruit-body developmentafter formation of the primordium. Effective wavelengths for both the initiation and developmentof fruit-bodies were in the near ultraviolet and blue regions. (Received July 24, 1972; )     

Improved Technique for Identification and Enumeration of Methanogenic Bacterial Colonies on Roll Tubes by Epifluorescence Microscopy

Methanogenic fluorescent colonies can be clearly identified on roll tubes by using an epifluorescence microscope equipped with a × 2 objective. Methanogenic and nonmethanogenic colonies could be counted in roll tubes prepared from methanogenic enrichment cultures. Late-developing colonies appearing after 25 days of incubation were mainly methanogenic.     

MARCKS dephosphorylation is involved in bradykinin-induced neurite outgrowth in neuroblastoma SH-SY5Y cells

Bradykinin (BK) plays a major role in producing peripheral sensitization in response to peripheral inflammation and in pain transmission in the central nerve system (CNS). Because BK activates protein kinase C (PKC) through phospholipase C (PLC)-β and myristoylated alanine-rich C kinase substrate (MARCKS) has been found to be a substrate of PKC, we explored the possibility that BK could induce MARCKS phosphorylation and regulate its function. BK stimulation induced transient MARCKS phosphorylation on Ser159 with a peak at 1 min in human neuroblastoma SH-SY5Y cells. By contrast, PKC activation by the phorbol ester phorbol 12,13-dibutyrate (PDBu) elicited MARCKS phosphorylation which lasted more than 10 min. Western blotting analyses and glutathione S-transferase (GST) pull-down analyses showed that the phosphorylation by BK was the result of activation of the PKC-dependent RhoA/Rho-associated coiled-coil kinase (ROCK) pathway. Protein phosphatase (PP) 2A inhibitors calyculin A and fostriecin inhibited the dephosphorylation of MARCKS after BK-induced phosphorylation. Moreover, immunoprecipitation analyses showed that PP2A interacts with MARCKS. These results indicated that PP2A is the dominant PP of MARCKS after BK stimulation. We established SH-SY5Y cell lines expressing wild-type MARCKS and unphosphorylatable MARCKS, and cell morphology changes after cell stimulation were studied. PDBu induced lamellipodia formation on the neuroblastoma cell line SH-SY5Y and the morphology was sustained, whereas BK induced neurite outgrowth of the cells via lamellipodia-like actin accumulation that depended on transient MARCKS phosphorylation. Thus these findings show a novel BK signal cascade-that is, BK promotes neurite outgrowth through transient MARCKS phosphorylation involving the PKC-dependent RhoA/ROCK pathway and PP2A in a neuroblastoma cell line.     

Identification of small molecule proliferating cell nuclear antigen (PCNA) inhibitor that disrupts interactions with PIP-box proteins and inhibits DNA replication

We have discovered that 3,3′,5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC₅₀ of ∼1 μm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser¹³⁹)histone H2AX induction and cell growth inhibition was observed.