Disabled-2 protein facilitates assembly polypeptide-2-independent recruitment of cystic fibrosis transmembrane conductance regulator to endocytic vesicles in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical plasma membrane of fluid-transporting epithelia, where the plasma membrane abundance of CFTR is in part controlled by clathrin-mediated endocytosis. The protein networks that control CFTR endocytosis in epithelial cells have only been partially explored. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor critical for optimal clathrin coat formation. AP-2 is essential for recruitment of cargo proteins bearing the YXXΦ motif. Although AP-2 interacts directly with CFTR in vitro and facilitates CFTR endocytosis in some cell types, it remains unknown whether it is critical for CFTR uptake into clathrin-coated vesicles (CCVs). Disabled-2 (Dab2) is a clathrin-associated sorting protein (CLASP) that contributes to clathrin recruitment, vesicle formation, and cargo selection. In intestinal epithelial cells Dab2 was not found to play a direct role in CFTR endocytosis. By contrast, AP-2 and Dab2 were shown to facilitate CFTR endocytosis in human airway epithelial cells, although the specific mechanism remains unknown. Our data demonstrate that Dab2 mediates AP-2 independent recruitment of CFTR to CCVs in polarized human airway epithelial cells. As a result, it facilitates CFTR endocytosis and reduces CFTR abundance and stability in the plasma membrane. These effects are mediated by the DAB homology domain. Moreover, we show that in human airway epithelial cells AP-2 is not essential for CFTR recruitment to CCVs.     

Lipidic last breath of life in patients with alcoholic liver disease

Alcoholic liver disease (ALD) begins with the accumulation of lipid droplets in the liver. Lipids which accumulate in the liver can stimulate inflammation, and the fatty acid derivatives, hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs), may play an important role in this process. We evaluated the concentrations of linoleic and arachidonic acid derivatives in the plasma of patients with ALD, non-alcoholic fatty liver disease (NAFLD) and healthy individuals. The groups consisted of 173 subjects: 63 patients with ALD, 90 with NAFLD and 20 healthy volunteers. Plasma 12-, 15-, and 5-HETE as well as 9- and 13-HODE were assessed using HPLC and isoprostane 8-epi-PGF 2α III was evaluated with an ELISA. In addition the mRNA expression of lipoxygenases (5-LOX, 15-LOX-1, 15-LOX-2) in the liver samples of patients with ALD cirrhosis was measured. A significant difference between the plasma concentrations of the analyzed derivatives was found when divided according to gender. The most significant differences were found between healthy individuals and ALD patients, as well as ALD and NAFLD individuals regardless of gender. The increased plasma HODEs and HETEs concentrations were in line with the increase in 5- and 15-LOX-1 and 15-LOX-2 mRNA in liver samples from ALD cirrhosis patients. LOXs expression and peroxidation of polyunsaturated fatty acids by free radical-propagated chemical oxidation may be contributing factors in liver necroinflammatory injury in ALD.     

The structure of the O-polysaccharide from the lipopolysaccharide of Providencia alcalifaciens O36 containing 3-deoxy-d-manno-oct-2-ulosonic acid

An oligosaccharide that corresponds to the repeating unit of the O-polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O36. Structural studies of the oligosaccharide and O-deacylated lipopolysaccharide were performed using sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, and H-detected (1)H,(13)C HSQC and HMBC experiments. It was found that the O-polysaccharide is built up of linear trisaccharide repeating units containing 2-acetamido-2-deoxyglucose, 6-deoxy-l-talose (l-6dTal), and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and has the following structure. [structure: see text]     

The influence of modifications in imide fragment structure on 5-HT(1A) and 5-HT(7) receptor affinity and in vivo pharmacological properties of some new 1-(m-trifluoromethylphenyl)piperazines

New, flexible (7, 9, 11 and 13) and rigid (8, 10, 12 and 14) imides with a 1-(m-trifluorophenyl)piperazine fragment and a tetramethylene or a 1e,4e-cyclohexylene spacer, respectively, showed very high affinity (K(i)=0.3-34 nM) and agonistic in vivo activity for 5-HT(1A) receptors. Flexible new compounds and the previously described 5 also bound to 5-HT(7) receptors (K(i)=21-134 nM). Selected glutarimide derivatives, that is, the most potent postsynaptic 5-HT(1A) receptor agonist rigid compound 8 and its flexible analogue 7, as well as the previously described full agonist-rigid compound 6 and the partial agonist-its flexible counterpart 5 exhibited moderate affinity for alpha(1)-adrenoceptors (K(i)=85 - 268 nM), but were practically devoid of any affinity for dopamine D(2) sites. Those glutarimides demonstrated anxiolytic- (5 and 7) and antidepressant-like (5, 6 and 8) activity in the four-plate and the swim tests in mice, respectively; at the same time, however, they inhibited the locomotor activity of mice. The antidepressant-like effect of 8 was significantly stronger than that induced by imipramine used as a reference antidepressant.     

Effect of high hydrostatic pressure on hydration and activity of ribozymes

Formation and stabilization of RNA structure in the cell depends on its interaction with solvent and metal ions. High hydrostatic pressure (HHP) is a convenient tool in an analysis of the role of small molecules in the structure stabilization of biological macromolecules. Analysis of HHP effect and various concentrations of ions showed that water induce formation of the active ribozyme structure. So, it is clear that water is the driving force of conformational changes of nucleic acid.     

Bovine Coronavirus Nonstructural Protein 1 (p28) Is an RNA Binding Protein That Binds Terminal Genomic cis-Replication Elements

Nonstructural protein 1 (nsp1), a 28-kDa protein in the bovine coronavirus (BCoV) and closely related mouse hepatitis coronavirus, is the first protein cleaved from the open reading frame 1 (ORF 1) polyprotein product of genome translation. Recently, a 30-nucleotide (nt) cis-replication stem-loop VI (SLVI) has been mapped at nt 101 to 130 within a 288-nt 5′-terminal segment of the 738-nt nsp1 cistron in a BCoV defective interfering (DI) RNA. Since a similar nsp1 coding region appears in all characterized groups 1 and 2 coronavirus DI RNAs and must be translated in cis for BCoV DI RNA replication, we hypothesized that nsp1 might regulate ORF 1 expression by binding this intra-nsp1 cistronic element. Here, we (i) establish by mutation analysis that the 72-nt intracistronic SLV immediately upstream of SLVI is also a DI RNA cis-replication signal, (ii) show by gel shift and UV-cross-linking analyses that cellular proteins of ∼60 and 100 kDa, but not viral proteins, bind SLV and SLVI, (SLV-VI) and (iii) demonstrate by gel shift analysis that nsp1 purified from Escherichia coli does not bind SLV-VI but does bind three 5′ untranslated region (UTR)- and one 3′ UTR-located cis-replication SLs. Notably, nsp1 specifically binds SLIII and its flanking sequences in the 5′ UTR with ∼2.5 μM affinity. Additionally, under conditions enabling expression of nsp1 from DI RNA-encoded subgenomic mRNA, DI RNA levels were greatly reduced, but there was only a slight transient reduction in viral RNA levels. These results together indicate that nsp1 is an RNA-binding protein that may function to regulate viral genome translation or replication but not by binding SLV-VI within its own coding region.Coronaviruses (CoVs) (59) cause primarily respiratory and gastroenteric diseases in birds and mammals (35, 71). In humans, they most commonly cause mild upper respiratory disease, but the recently discovered human CoVs (HCoVs), HCoV-NL63 (65), HCoV-HKU1 (73), and severe acute respiratory syndrome (SARS)-CoV (40) cause serious diseases in the upper and lower respiratory tracts. The SARS-CoV causes pneumonia with an accompanying high (∼10%) mortality rate (69). The ∼30-kb positive-strand CoV genome, the largest known among RNA viruses, is 5′ capped and 3′ polyadenylated and replicates in the cytoplasm (41). As with other characterized cytoplasmically replicating positive-strand RNA viruses (3), translation of the CoV genome is an early step in replication, and terminally located cis-acting RNA signals regulate translation and direct genome replication (41). How these happen mechanistically in CoVs is only beginning to be understood.In the highly studied group 2 mouse hepatitis coronavirus model (MHV A59 strain) and its close relative the bovine CoV (BCoV Mebus strain), five higher-order cis-replication signals have been identified in the 5′ and 3′ untranslated regions (UTRs). These include two in the 5′ UTR required for BCoV defective interfering (DI) RNA replication (Fig. ​(Fig.1A)1A) described as stem-loop III (SLIII) (50) and SLIV (51). Recently, the SLI region in BCoV (15) has been reanalyzed along with the homologous region in MHV and is now described as comprising SL1 and SL2 (Fig. ​(Fig.1A),1A), of which SL2 has been shown to be a cis-replication structure in the context of the MHV genome (38). In the 3′ UTR, two higher-order cis-replication structures have been identified that function in both DI RNA and the MHV genome. These are a 5′-proximal bulged SL and adjacent pseudoknot that potentially act together as a unit (23, 27, 28, 72) and a 3′-proximal octamer-associated bulged SL (39, 76) (Fig. ​(Fig.1A).1A). In addition, the 5′-terminal 65-nucleotide (nt) leader and the 3′-terminal poly(A) tail have been shown to be cis-replication signals for BCoV DI RNA (15, 60).Open in a separate windowFIG. 1.RNA structures in the BCoV genome tested for nsp1 binding. (A) BCoV 5′-terminal and 3′-terminal cis-acting RNA SL structures and flanking sequences identified for BCoV DI RNA replication. Regions of the genome are identified and SL cis-replication elements are identified schematically. Open boxes at nt 100 and 211 identify AUG start codons for the short upstream ORF and ORF 1, respectively. A closed box at nt 124 identifies the UAG stop codon for the short upstream ORF. Shown below the SL structures are the RNA segments used as ³²P-labeled probes in the gel shift assays. BSL-PK, bulged SL-pseudoknot; 8mer-BSL, octamer-associated bulged SL. (B) Gel shift assays for probes when used with purified nsp1. Protein-RNA complexes identifying a shifted probe are labeled C.In CoVs, the 5′-proximal open reading frame (ORF) of ∼20 kb (called ORF 1) comprising the 5′ two-thirds of the genome is translated to overlapping polyproteins of ∼500 and ∼700 kDa, named pp1a and pp1ab (41). pp1ab is formed by a −1 ribosomal frameshift event at the ORF1a-ORF1b junction during translation (41). pp1a and pp1ab are proteolytically processed into potentially 16 nonstructural protein (nsp) end products or partial end products that are proposed to function together as the replicase (24). ORF 1a encodes nsps 1 to 11 which include papain-like proteases (nsp3), a 3C-like main protease (nsp5), membrane-anchoring proteins (nsps 4 and 6), a potential primase (nsp8), and RNA-binding proteins (nsp 7/nsp 8 complex and nsps 9 and 10) of imprecisely understood function (19, 20, 24, 25, 29, 43, 49, 77). ORF 1b encodes nsps 12 to 16 which function as an RNA-dependent RNA polymerase, a helicase, an exonuclease, an endonuclease, and a 2′-O-methyltransferase, respectively (6, 17, 24, 44). 3′ Proximal genomic ORFs encoding structural and accessory proteins are translated from a 3′-nested set of subgenomic mRNAs (sgmRNAs) (41).The N-terminal ORF 1a protein, nsp1, in the case of BCoV and MHV is also named p28 to identify the cleaved 28-kDa product (18). The precise role of nsp1 in virus replication has not been determined, but it is known that a sequence encoding an N-proximal nsp1 region in MHV (nt 255 to 369 in the 738-nt coding sequence) cannot be deleted from the genome without loss of productive infection (10). nsp1 also directly binds nsp7 and nsp10 (11) and by confocal microscopy is found associated with the membranous replication complex (10, 66) and virus assembly sites (11). The amino acid sequence of nsp1 is poorly conserved among CoVs, indicating that it may be a protein that interacts with cellular components (1, 58). In the absence of other viral proteins, MHV nsp1 induces general host mRNA degradation (79) and cell cycle arrest (16). The SARS-CoV nsp1 homolog, a 20-kDa protein, has been reported to cause mRNA degradation (30, 45), inhibition of host protein synthesis (30, 45, 70), inhibition of interferon signaling (70, 79), and cytokine dysregulation in lung cells (36).In this study, we examine the RNA-binding properties of BCoV nsp1 with the hypothesis that it is a potential regulator of translation or replication through its binding of SLVI mapping within its coding region. The rationale for this hypothesis stems from five observations. (i) In the BCoV DI RNA, the 5′-terminal one-third (approximately) of the nsp1 cistron and the entire nucleocapsid (N) protein cistron together comprise the single contiguous ORF in the DI RNA, and most of both coding regions appear required for DI RNA replication (15). (ii) The partial nsp1 cistron in the DI RNA must be translated in cis for DI RNA replication in helper virus-infected cells (12, 14). (iii) A similar part of the nsp1 cistron is found in the genome of all characterized naturally occurring group 1 and 2 CoV DI RNAs described to date (7, 8). (iv) A cis-acting SL named SLVI is found within the partial nsp1 cistron in the BCoV DI RNA (12). (v) Translation, which involves a 5′→3′ transit of ribosomes, and negative-strand synthesis, which involves a 3′→5′ transit of the RNA-dependent RNA polymerase, cannot simultaneously occur on the same molecule with a single ORF (4, 31). Thus, to enable genome replication an inhibition of translation at least early in infection for cytoplasmically replicating positive-strand RNA viruses is required (4, 5, 22, 32). Mechanisms of translation inhibition have been described for the Qβ viral genome, wherein the viral replicase autoregulates translation by binding an intracistronic cis-replication element (32), and for the polio virus genome, wherein genome circularization inhibits the early translation step (5, 22). Therefore, since nsp1 is synthesized early and also contains an intracistronic cis-replication element, we postulated that it is autoregulatory with RNA binding properties.Here, we do the following: (i) demonstrate by mutagenesis analysis that the 72-nt SLV, mapping immediately upstream of SLVI and within the partial nsp1 cistron, is also a cis-acting DI RNA replication element; (ii) show by gel shift and UV cross-linking analyses that there is likely no binding of an intracellular viral protein to SLV and SLVI (SLV-VI), but there is binding of unidentified cellular proteins of ∼60 and 100 kDa; and (iii) show by gel shift analysis that recombinant nsp1 purified from Escherichia coli does not bind SLV-VI but does bind SLs I to IV in the 5′ UTR and also the 3′-terminal bulged SL in the 3′ UTR, suggesting a possible regulatory role at these sites. Notably, specific binding with ∼2.5 μM affinity of nsp1 to SLIII and its flanking regions in the 5′ UTR was observed. Additionally, we show that, under conditions that would express nsp1 from a DI RNA-encoded sgmRNA, DI RNA levels are greatly reduced; viral RNA species levels, however, are reduced only slightly, and this reduction is transient. These results together indicate that nsp1 is an RNA-binding protein that may function as a regulator of viral translation or replication but not through its binding of cis-acting SLs V and VI within its own cistron.     

From macro to lab-scale: Changes in bacterial community led to deterioration of EBPR in lab reactor

A laboratory scale sequencing batch reactor (SBR), fed with synthetic wastewater containing a mixture of organic compounds, was operated for nearly six months. Despite maintaining the same operational conditions, a deterioration of enhanced biological phosphorus removal (EBPR) occurred after 40 days of SBR operation. The P_rel/C_upt ratio decreased from 0.28 to 0.06 P-mol C-mol^?1, and C requirements increased from 11 to 32 mg C h^?1 g^?1 of mixed liquor suspended solids. A FISH analysis showed that the percentage of Accumulibacter in an overall community of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) dropped from 93% to 13%. An increase in abundance of Gammaproteobacteria (from 2.6% to 22%) and Alphaproteobacteria (from 1.8% to 10%) was observed. The number of Competibacter increased from 0.5% to nearly 9%. Clusters 1 and 2 of Defluviicoccus-related GAOs, not detected before deterioration, constituted 35% and 27% of Alphaproteobacteria, respectively. We concluded that lab-scale experiments should not be extended implicitly to full-scale EBPR systems because some bacterial groups are detected mainlyin lab-scale reactors. Well-defined, lab-scale operational conditions reduce the number of ecological niches available to bacteria.