SERPINA1 PiZ and PiS Heterozygotes and Lung Function Decline in the SAPALDIA Cohort

Background

Severe alpha1-antitrypsin (AAT) deficiency is a strong risk factor for COPD. But the impact of gene variants resulting in mild or intermediate AAT deficiency on the longitudinal course of respiratory health remains controversial. There is indication from experimental studies that pro-inflammatory agents like cigarette smoke can interact with these variants and thus increase the risk of adverse respiratory health effects. Therefore, we tested the effect of the presence of a protease inhibitor (Pi) S or Z allele (PiMS and PiMZ) on the change in lung function in different inflammation-exposed subgroups of a large, population-based cohort study.

Methodology and Principal Findings

The SAPALDIA population includes over 4600 subjects from whom SERPINA1 genotypes for S and Z alleles, spirometry and respiratory symptoms at baseline and after 11 years follow-up, as well as proxies for inflammatory conditions, such as detailed smoking history, obesity and high sensitivity C-reactive protein (hs-CRP), were available. All analyses were performed by applying multivariate regression models. There was no overall unfavourable effect of PiMS or PiMZ genotype on lung function change. We found indication that PiZ heterozygosity interacted with inflammatory stimuli leading to an accelerated decline in measures in use as indices for assessing mild airway obstruction. Obese individuals with genotype PiMM had an average annual decline in the forced mid expiratory flow (ΔFEF25-75%) of 58.4 ml whereas in obese individuals with PiMZ it amounted to 92.2 ml (p = 0.03). Corresponding numbers for persistent smokers differed even more strongly (66.8 ml (PiMM) vs. 108.2 ml (PiMZ), p = 0.005). Equivalent, but less strong associations were observed for the change in the FEV1/FVC ratio.

Conclusions

We suggest that, in addition to the well established impact of the rare PiZZ genotype, one Z allele may be sufficient to accelerate lung function decline in population subgroups characterized by elevated levels of low grade inflammation.     

Cellular concentrations of DDB2 regulate dynamic binding of DDB1 at UV-induced DNA damage

Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions.     

Ziram causes dopaminergic cell damage by inhibiting E1 ligase of the proteasome

The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated alpha-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target.     

The Pleistocene Ma U’Oi cave, northern Vietnam: palaeontology, sedimentology and palaeoenvironments

In November 2001, a Vietnamese-French team undertook the excavation of the Ma U’Oi cave in northern Vietnam. This limestone karst cave is located in the province of Hoà Binh, 70 km ESE from Hanoi and is typical of the northern Vietnam landscape. The site yielded an in situ mammalian fauna of a relatively modern composition. We also found a mixed fauna with a lower molar attributed to an archaic Homo(Demeter et al., in press). We estimate the age of Ma U’Oi fauna between 169 kyr, the age of Thum Wiman Nakin (Esposito et al., 1998) estimated by U/Th method and 80-60 kyr, the biochronological age of Lang Trang (Long et al., 1996), or even Holocene. The Ma U’Oi site is important because of the scarcity of Vietnamese sites of those particular levels. For that reason, it fills a gap in the biostratigraphy of Vietnam and permits new correlations with other sites of the mainland, especially those well documented from Thailand.     

Photo-crosslinking of proteins in intact cells reveals a dimeric structure of cyclooxygenase-2 and an inhibitor-sensitive oligomeric structure of microsomal prostaglandin E2 synthase-1

We have characterized the structures of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E₂ synthase-1 (mPGES-1) in intact cells using bifunctional and photo-activatable crosslinking agents. A dimeric complex was detected for COX-2 by both crosslinking approaches, consistent with the crystal structure of the enzyme. For mPGES-1, treatment of A549 cells with disuccinimidyl suberate yielded immunoreactive protein bands corresponding to a dimer (33 kDa) and a trimer (45 kDa), as observed for the isolated enzyme. Photo-crosslinking with photoactivatable methionine in intact cells generated complexes with molecular weights corresponding to the dimer (33 kDa) and two putative trimer forms (50 and 55 kDa). Treatment with the selective mPGES-1 inhibitor MF63 prevented the formation of the 50 and 55 kDa crosslinked complexes, while an inactive structural analogue had no effect. Our data indicate that COX-2 forms a dimer in intact cells and that mPGES-1 has an oligomeric structure that can be disrupted by a selective inhibitor.     

Middle Miocene elasmotheriine Rhinocerotidae from China and Mongolia: taxonomic revision and phylogenetic relationships

Eight elasmotheriine rhinocerotid species have been described from the Middle Miocene of China and Mongolia. In this paper a revised taxonomy is presented based on direct observation and comparison of the available material. A phylogenetic analysis based on 282 morphological characters has led to the reappraisal of Procoelodonta Matthew, 1931, Caementodon Heissig, 1972 and Huaqingtherium Huang & Yan, 1983. The genus Procoelodonta is split into three subgenera: P.  ( Procoelodonta ) Matthew, 1931, P.  ( Begertherium ) Beliaeva, 1971, and P.  ( Pasalarhinus subg. n). The genus Caementodon is split into two subgenera: C.  ( Caementodon ) Heissig, 1972 and C.  ( Beliajevina ) Heissig, 1974. Four species are assumed to have occurred in the Middle Miocene within the area studied: Procoelodonta ( Procoelodonta ) mongoliense (Osborn, 1924), ' P. ' ( Begertherium ) borissiaki (Beliaeva, 1971), ' Caementodon ' ( Beliajevina ) fangxianense (Yan, 1979) and Huaqingtherium lintungense (Zhai, 1978) (=' Caementodon tongxinensis ' Guan, 1988 =' Huaqingtherium qiui ' Guan, 1993 =' Hispanotherium tungurense ' Cerdeño, 1996). Shennongtherium hypsodontus Huang & Yan, 1983 is removed from the Elasmotheriina, owing to dental characters which suggest that it is a teleoceratine. The distribution of the main characters later seen in Elasmotherium is briefly discussed. The persistence and diversity of the Elasmotheriina throughout the Middle Miocene help explain how minute brachyodont animals gave rise to the mammoth-sized hypsodont Elasmotherium.     

Interferon Resistance of Hepatitis C Virus Genotype 1b: Relationship to Nonstructural 5A Gene Quasispecies Mutations

A 40-amino-acid sequence located in the nonstructural 5A (NS5A) protein of hepatitis C virus genotype 1b (HCV-1b) was recently suggested to be the interferon sensitivity-determining region (ISDR), because HCV-1b strains with an ISDR amino acid sequence identical to that of the prototype strain HCV-J were found to be resistant to alpha interferon (IFN-α) whereas strains with amino acid substitutions were found to be sensitive (N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, N. Izumi, F. Marumo, and C. Sato, J. Clin. Invest. 96:224–230, 1995; N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, Y. Ogura, N. Izumi, F. Marumo, and C. Sato, N. Engl. J. Med. 334:77–81, 1996). We used single-strand conformation polymorphism (SSCP) analysis, combined with cloning and sequencing strategies, to characterize NS5A quasispecies in HCV-1b-infected patients and determine the relationships between pre- and posttreatment NS5A quasispecies mutations and the IFN-α sensitivity of HCV-1b. The serine residues involved in phosphorylation of NS5A protein were highly conserved both in the various patients and in quasispecies in a given patient, suggesting that phosphorylation is important in NS5A protein function. A hot spot for amino acid substitutions was found at positions 2217 to 2218; it could be the result of either strong selection pressure or tolerance to these amino acid replacements. The proportion of synonymous mutations was significantly higher than the proportion of nonsynonymous mutations, suggesting that genetic variability in the region studied was the result of high mutation rates and viral replication kinetics rather than of positive selection. Sustained HCV RNA clearance was associated with low viral load and low nucleotide sequence entropy, suggesting (i) that the replication kinetics when treatment is started plays a critical role in HCV-1b sensitivity to IFN-α and (ii) that HCV-1b resistance to IFN-α could be conferred by numerous and/or related mutations that could be patient specific and located at different positions throughout the viral genome and could allow escape variants to be selected by IFN-α-stimulated immune responses. No NS5A sequence appeared to be intrinsically resistant or sensitive to IFN-α, but the HCV-J sequence was significantly more frequent in nonresponder quasispecies than in sustained virological responder quasispecies, suggesting that the balance between NS5A quasispecies sequences in infected patients could have a subtle regulatory influence on HCV replication.     

Mrp1 Multidrug Resistance-Associated Protein and P-Glycoprotein Expression in Rat Brain Microvessel Endothelial Cells

Abstract: Two membrane glycoproteins acting as energy-dependent efflux pumps, mdr -encoded P-glycoprotein (P-gp) and the more recently described multidrug resistance-associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P-gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood-brain barrier, but localization of MRP has not been well characterized yet. Using RT-PCR and immunoblot analysis, we have compared the expression of P-gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P-gp-encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [³H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P-gp, is not predominantly expressed in the blood-brain barrier endothelial cells and that Mrp1 and the mdr1b P-gp isoform may be present in other cerebral cells.     

Evaluation of soluble junctional adhesion molecule-A as a biomarker of human brain endothelial barrier breakdown

Background

An inducible release of soluble junctional adhesion molecule-A (sJAM-A) under pro-inflammatory conditions was described in cultured non-CNS endothelial cells (EC) and increased sJAM-A serum levels were found to indicate inflammation in non-CNS vascular beds. Here we studied the regulation of JAM-A expression in cultured brain EC and evaluated sJAM-A as a serum biomarker of blood-brain barrier (BBB) function.

Methodology/Principal Findings

As previously reported in non-CNS EC types, pro-inflammatory stimulation of primary or immortalized (hCMEC/D3) human brain microvascular EC (HBMEC) induced a redistribution of cell-bound JAM-A on the cell surface away from tight junctions, along with a dissociation from the cytoskeleton. This was paralleled by reduced immunocytochemical staining of occludin and zonula occludens-1 as well as by increased paracellular permeability for dextran 3000. Both a self-developed ELISA test and Western blot analysis detected a constitutive sJAM-A release by HBMEC into culture supernatants, which importantly was unaffected by pro-inflammatory or hypoxia/reoxygenation challenge. Accordingly, serum levels of sJAM-A were unaltered in 14 patients with clinically active multiple sclerosis compared to 45 stable patients and remained unchanged in 13 patients with acute ischemic non-small vessel stroke over time.

Conclusion

Soluble JAM-A was not suited as a biomarker of BBB breakdown in our hands. The unexpected non-inducibility of sJAM-A release at the human BBB might contribute to a particular resistance of brain EC to inflammatory stimuli, protecting the CNS compartment.