Early loss of proliferative potential of human peritoneal mesothelial cells in culture: the role of p16INK4a-mediated premature senescence.

Much has been learned about the mechanisms underlying cellular senescence. The pathways leading to senescence appear to vary, depending on the cell type and cell culture conditions. In this respect, little is known about senescence of human peritoneal mesothelial cells (HPMC). Previous studies have significantly differed in the reported proliferative lifespan of HPMC. Therefore, in the present study, we have examined how HPMC enter state of senescence under conditions typically used for HPMC culture. HPMC were isolated from omentum and grown into senescence. The cultures were assessed for the growth rate, the presence of senescence markers, activation of cell-cycle inhibitors, and the oxidative stress. HPMC were found to reach, on average, six population doublings before senescence. The terminal growth arrest was associated with decreased expression of Ki67 antigen, increased percentage of cells in the G1 phase, reduced early population doubling level cDNA-1 mRNA expression, and the presence of senescence-associated beta-galactosidase. Compared with early-passage cells, the late-passage HPMC exhibited increased expression of p16INK4a but not of p21Cip1. In addition, these cells generated more reactive oxygen species and displayed increased presence of oxidatively modified DNA (8-hydroxy-2'-deoxyguanosine). These results demonstrate that early onset of senescence in omentum-derived HPMC may be associated with oxidative stress-induced upregulation of p16INK4a.     

Cellular Distribution and Subcellular Localization of mCLCA1/2 in Murine Gastrointestinal Epithelia

mCLCA1/2 are members of the CLCA protein family that are widely expressed in secretory epithelia, but their putative physiological role still awaits elucidation. mCLCA1/2 have 95% amino acid identity, but currently no specific antibody is available. We have generated a rabbit polyclonal antibody (pAb849) against aa 424–443 of mCLCA1/2. In HEK293 cells transfected with mCLCA1; pAb849 detected two specific protein bands at ∼125 kDa and 90 kDa, representing full-length precursor and N-terminal cleavage product, respectively. pAb849 also immunoprecipitated mCLCA1 and labeled the protein by immunostaining. But pAb849 crossreacted with mCLCA3/4/6 despite ≤80% amino acid identity of the antigenic epitope. We therefore investigated the cellular localization of mCLCA1/2 in epithelial tissues, which do not express mCLCA3/4/6 (salivary glands, pancreas, kidney) or express mCLCA3/6 with known localization (mucus cells of stomach and small intestine; villi of small intestine). mCLCA1/2 mRNA and protein expression were found in both parotid and submandibular gland, and immunohistochemistry revealed labeling in parotid acinar cells, in the luminal membrane of parotid duct cells, and in the duct cells of submandibular gland. In exocrine pancreas, mCLCA1/2 expression was restricted to acinar zymogen granule membranes, as assessed by immunoblotting, immunohistochemistry, and preembedding immunoperoxidase and immunogold electron microscopy. Moreover, mCLCA1/2 immunolabeling was present in luminal membranes of gastric parietal cells and small intestinal crypt enterocytes, whereas in the kidney, mCLCA1/2 protein was localized to proximal and distal tubules. The apical membrane localization and overall distribution pattern of mCLCA1/2 favor a transmembrane protein implicated in transepithelial ion transport and protein secretion. (J Histochem Cytochem 58:653–668, 2010)     

Tissue and Cellular Expression Patterns of Porcine CFTR: Similarities to and Differences From Human CFTR

Emerging porcine models of cystic fibrosis (CF) are expected to mimic the human disease more closely than current mouse models do. However, little is known of the tissue and cellular expression patterns of the porcine CF transmembrane conductance regulator (pCFTR) and possible differences from human CFTR (hCFTR). Here, the expression pattern of pCFTR was systematically established on the mRNA and protein levels. Using specific anti-pCFTR antibodies, the majority of the protein was immunohistochemically detected on paraffin-embedded sections and on cryostate sections in the apical cytosol of intestinal crypt epithelial cells, nasal, tracheal, and bronchial epithelial cells, and other select, mostly glandular epithelial cells. Confocal laser scanning microscopy with co-localization of the Golgi marker 58K localized the protein in the cytosol between the Golgi apparatus and the apical cell membrane with occasional punctate or diffuse staining of the apical membrane. The tissue and cellular distribution patterns were confirmed by RT-PCR from whole tissue lysates or select cells after laser capture microdissection. Thus, expression of pCFTR was found to largely resemble that of hCFTR except for the kidney, brain, and cutaneous glands, which lack expression in pigs. Species-specific differences between pCFTR and hCFTR may become relevant for future interpretations of the CF phenotype in pig models. (J Histochem Cytochem 58:785–797, 2010)     

Crystal Structure Analysis of DNA Uridine Endonuclease Mth212 Bound to DNA

The reliable repair of pre-mutagenic U/G mismatches that originated from hydrolytic cytosine deamination is crucial for the maintenance of the correct genomic information. In most organisms, any uracil base in DNA is attacked by uracil DNA glycosylases (UDGs), but at least in Methanothermobacter thermautotrophicus ΔH, an alternative strategy has evolved. The exonuclease III homologue Mth212 from the thermophilic archaeon M. thermautotrophicus ΔH exhibits a DNA uridine endonuclease activity in addition to the apyrimidinic/apurinic site endonuclease and 3′ → 5′exonuclease functions. Mth212 alone compensates for the lack of a UDG in a single-step reaction thus substituting the two-step pathway that requires the consecutive action of UDG and apyrimidinic/apurinic site endonuclease.In order to gain deeper insight into the structural basis required for the specific uridine recognition by Mth212, we have characterized the enzyme by means of X-ray crystallography. Structures of Mth212 wild-type or mutant proteins either alone or in complex with DNA substrates and products have been determined to a resolution of up to 1.2 Å, suggesting key residues for the uridine endonuclease activity. The insertion of the side chain of Arg209 into the DNA helical base stack resembles interactions observed in human UDG and seems to be crucial for the uridine recognition. In addition, Ser171, Asn153, and Lys125 in the substrate binding pocket appear to have important functions in the discrimination of aberrant uridine against naturally occurring thymidine and cytosine residues in double-stranded DNA.     

Coordination chemistry of copper-molybdates with alkoxide ligands: The {Mo4O10(OMe)6} and [Mo2O4{RC(CH2O)3}2] clusters as building blocks

The reactions of the Keplerate super cluster [Mo₁₃₂O₃₇₂(CH₃CO₂)₃₀(H₂O)₇₂]⁴²⁻ with a Cu(II) source and an organonitrogen donor in methanol/DMF solutions yielded a series of bimetallic organic-inorganic oxide hybrid materials, including the molecular species [Cu(phen)₂MoO₄] (1) and [{Cu(terpy)}₂(MoO₄)₂] (2) and a series of materials constructed from the tetranuclear building block {Mo₄O₁₀(OMe)₆}²⁻: the molecular [{Cu₂(phen)₂(O₂CCH₃)₂ (MeOH)}Mo₄O₁₀(OMe)₆] (3), [{Cu(terpy)(O₂CCH₃)}₂Mo₄O₁₀(OMe)₆] (4) and [{Cu(terpy)Cl}₂Mo₄O₁₀(OMe)₆] (5), the one-dimensional phases [{Cu(bpy)(HOMe)₂}Mo₄O₁₀(OMe)₆] (6), [{Cu(bpy)(DMF)₂}Mo₄O₁₀(OMe)₆] (7), [{Cu(bpa)(DMF)₂}Mo₄O₁₀(OMe)₆] (8), [{Cu(phen)(DMF)₂}Mo₄O₁₀(OMe)₆] (9) and [{CuCl(dpa)}₂Mo₄O₁₀(OMe)₆] (10), and the two-dimensional material [{Cu₂(DMF)₂(pdpa)}{Mo₄O₁₀(OMe)₆}₂] (11). When methanol is replaced by the tridentate alkoxide tris-methoxypropane (trisp), the {Mo₂O₄(trisp)₂}²⁻ cluster building block is observed for [Cu(phen)Mo₂O₄(trisp)₂] (12), [Cu(bpa)(DMF)Mo₂O₄(trisp)₂] (13) and [{Cu(bpy)(NO₃)}₂Mo₂O₄(trisp)₂] (14).     

Tocopherol is the scavenger of singlet oxygen produced by the triplet states of chlorophyll in the PSII reaction centre

Recent developments on the role of tocopherol in the antioxidant network of the chloroplast and, in particular, in the protection of PSII in high light are summarized. The origin and conditions for singlet oxygen production in the reaction centre via P680 triplet formation are discussed, as well as the scavenging of this singlet oxygen by tocopherol. This is probably the obligatory function of tocopherol in the plant in high light acclimation. Furthermore, tocopherol is part of the modulation system of ROS in stress signalling.     

Molecular basis for the binding polyspecificity of an anti-cholera toxin peptide 3 monoclonal antibody

The onset of autoimmune diseases is proposed to involve binding promiscuity of antibodies (Abs) and T‐cells, an often reported yet poorly understood phenomenon. Here, we attempt to approach two questions: first, is binding promiscuity a general feature of monoclonal antibodies (mAbs) and second, what is the molecular basis for polyspecificity? To this end, the anti‐cholera toxin peptide 3 (CTP3) mAb TE33 was investigated for polyspecific binding properties. Screening of phage display libraries identified two epitope‐unrelated peptides that specifically bound TE33 with affinities similar to or 100‐fold higher than the wild‐type epitope. Substitutional analyses revealed distinct key residue patterns recognized by the antibody suggesting a unique binding mode for each peptide. A database query with one of the consensus motifs and a subsequent binding study uncovered 45 peptides (derived from heterologous proteins) that bound TE33. To better understand the structural basis of the observed polyspecificity we modeled the new cyclic epitope in complex with TE33. The interactions between this peptide and TE33 suggested by our model are substantially different from the interactions observed in the X‐ray structure of the wild‐type epitope complex. However, the overall binding conformation of the peptides is similar. Together, our results support the theory of a general polyspecific potential of mAbs. Copyright © 2005 John Wiley & Sons, Ltd.     

Newbouldiaquinone A: A naphthoquinone-anthraquinone ether coupled pigment, as a potential antimicrobial and antimalarial agent from Newbouldia laevis

The study of the chemical constituents of the roots of Newbouldia laevis (Bignoniaceae) has resulted in the isolation and characterization of a naphthoquinone-anthraquinone coupled pigment named newbouldiaquinone A (1) together with 14 known compounds: apigenin, chrysoeriol, newbouldiaquinone, lapachol, 2-methylanthraquinone, 2-acetylfuro-1,4-naphthoquinone, 2,3-dimethoxy-1,4-benzoquinone, oleanolic acid, canthic acid, 2-(4-hydroxyphenyl)ethyl triacontanoate, newbouldiamide, 5,7-dihydroxydehydroiso-alpha-lapachone, beta-sitosterol, and beta-sitosterol glucopyranoside. The structure elucidation of the isolated compounds was established based on spectroscopic studies, notably of the 2D NMR spectra. The antimalarial activity of compound (1) against Plasmodium falciparum in vitro shows moderate chemo suppression of parasitic growth. Its antimicrobial activity against a wide range of microorganisms was 13- and 24-fold more active against Candida gabrata and Enterobacter aerogens than the reference antibiotics nystatin and gentamycin.