Topology of PhoE porin: the 'eyelet' region

A model for the topology of the PhoE porin has been proposed according to which the polypeptide traverses the outer membrane sixteen times mostly as amphipathic β-sheets, thereby exposing eight loops at the cell surface. Until now, no evidence has been obtained for the surface exposure of the third loop. Recently, the structure of porin of Rhodobacter capsulatus has been determined. The proposed model of PhoE is very similar to the structure of the R capsulatus porin, which has an ‘eyelet’ region, extending into the interior of the pore. The proposed third external loop of PhoE might form a similar ‘eyelet’ region. To determine the location of the predicted third external loop of PhoE, multiple copies of an oligonucleotide linker encoding an antigenic determinant of VP1 protein of foot-and-mouth disease virus (FMDV) were inserted. All hybrid proteins were properly inserted in the outer membrane. The monoclonal antibody MA11, directed against the linear FMDV epitope, was able to bind only to intact cells expressing a hybrid PhoE protein with at least three copies of the FMDV epitope present. Antibiotic sensitivity tests and single-channel conductance measurements revealed that the insertions influenced the channel size. These results are consistent with a location of the third loop of PhoE within the pore channel.     

Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs

Adult hematopoietic stem cells (HSCs) with serially transplantable activity comprise two subtypes. One shows a balanced output of mature lymphoid and myeloid cells; the other appears selectively lymphoid deficient. We now show that both of these HSC subtypes are present in the fetal liver (at a 1:10 ratio) with the rarer, lymphoid-deficient HSCs immediately gaining an increased representation in the fetal bone marrow, suggesting that the marrow niche plays a key role in regulating their ensuing preferential amplification. Clonal analysis of HSC expansion posttransplant showed that both subtypes display an extensive but variable self-renewal activity with occasional interconversion. Clonal analysis of their differentiation programs demonstrated functional and molecular as well as quantitative HSC subtype-specific differences in the lymphoid progenitors they generate but an indistinguishable production of multipotent and myeloid-restricted progenitors. These findings establish a level of heterogeneity in HSC differentiation and expansion control that may have relevance to stem cell populations in other hierarchically organized tissues.     

A putative alpha-helical porin from Corynebacterium glutamicum

The cell wall of Corynebacterium glutamicum contains a mycolic acid layer, which is a protective nonpolar barrier similar to the outer membrane of Gram-negative bacteria. The exchange of material across this barrier requires porins. Porin B (PorB) is one of them. Recombinant PorB has been produced in Escherichia coli, purified, crystallized and analyzed by X-ray diffraction, yielding 16 independent molecular structures in four different crystal forms at resolutions up to 1.8 Å. All 16 molecules have the same globular core, which consists of 70 residues forming four α-helices tied together by a disulfide bridge. The 16 structures vary greatly with respect to the 29 residues in the N- and C-terminal extensions. Since corynebacteria belong to the group of mycolata that includes some prominent human pathogens, the observed structure may be of medical relevance. Due to the clearly established solid structure of the core, the native porin has to be oligomeric, and the reported structure is one of the subunits. An α-helical porin in a bacterial outer envelope is surprising because all presently known structures of such porins consist of β-barrels. Since none of the four crystal packing arrangements was compatible with an oligomeric membrane channel, we constructed a model of such an oligomer that was consistent with all available data of native PorB. The proposed model is based on the required polar interior and nonpolar exterior of the porin, on a recurring crystal packing contact around a 2-fold axis, on the assumption of a simple C_n symmetry (a symmetric arrangement around an n-fold axis), on the experimentally established electric conductivity and anion selectivity and on the generally observed shape of porin channels.     

Inhibition of adenine nucleotide transport through the mitochondrial porin by a synthetic polyanion

The effect of a synthetic polyanion of Mr 10,000 (a copolymer of methacrylate, maleate and styrene in a 1:2:3 proportion) was studied on isolated rat liver mitochondria and on mitochondrial porin reconstituted into lipid bilayer membranes. Increasing concentrations of the polyanion inhibited the adenyl kinase located between both mitochondrial membranes in a dose-dependent fashion. Upon addition of the detergent digitonin in increasing concentrations the adenyl kinase activity was fully reversible. In reconstitution experiments with mitochondrial porin the polyanion increased the voltage dependence of the pore in such a way that the pore is switched into the closed state at much smaller voltages than in the absence of the polyanion. The asymmetric addition of the polyanion resulted in an asymmetric shift of the voltage-dependence of the pore. If the voltage is negative at the cis-side (the side of the addition of the polyanion) the pore closed rapidly whereas it was always open for potentials of opposite polarity. The results are discussed on the basis of a modification of the gate properties of the mitochondrial porin by the polyanion and by the assumption that the closed state of the pore is not permeable for nucleotides.     

Current recording from sensory cilia of olfactory receptor cells in situ. II. Role of mucosal Na+, K+, and Ca2+ ions

Action potential-driven current transients were recorded from sensory cilia and used to monitor the spike frequency generated by olfactory receptor neurons, which were maintained in their natural position in the sensory epithelium. Both basal and messenger-induced activities, as elicited with forskolin or cyclic nucleotides, were dependent on the presence of mucosal Na+. The spike rate decreased to approximately 20% when mucosal Na+ was lowered from 120 to 60 mM (replaced by N-methyl-D-glucamine+), without clear changes in amplitude and duration of the recorded action potential-driven transients. Mucosal Ca2+ and Mg2+ blocked spike discharge completely when increased from 1 to 10 mM in Ringer solution. Lowering mucosal Ca2+ below 1 mM increased the spike rate. These results can be explained by the presence of a cyclic nucleotide-dependent, Ca(2+)-sensitive cation conductance, which allows a depolarizing Na+ inward current to flow through the apical membrane of in situ receptor cells. A conductance with these properties, thought to provide the receptor current, was first described for isolated olfactory cells by Nakamura and Gold (1987. Nature (Lond.). 325:442-444). The forskolin-stimulated spike rate decreased when l-cis-diltiazem, a known blocker of the cyclic nucleotide-dependent receptor current, was added to the mucosal solution. Spike rate also decreased when the mucosal K+ concentration was lowered. Mucosal Ba2+ and 4-aminopyridine, presumably by means of cell depolarization, rapidly increased the spike rate. This suggests the presence of apical K+ channels that render the receptor cells sensitive to the K+ concentration of the olfactory mucus. With a slower time course, mucosal Ba2+ and 4-aminopyridine decreased the amplitude and caused rectification of the fast current transients (prolongation of action potentials). Abolishment of the apical Na+ current (by removal of mucosal Na+), as indicated by a strong decrease in spike rate, could be counteracted by adding 10 mM Ba2+ or 1 mM 4-aminopyridine to the mucosal solution, which re-established spiking. Similarly, blockage of the apical cation conductance with 10 mM Ca could be counteracted by adding 10 mM Ba2+ or by raising the mucosal K+ concentration. Thus mucosal concentrations of Na+, K+, and Ca2+ will jointly affect the sensitivity of odor detection.     

Biophysical Characterisation of Calumenin as a Charged F508del-CFTR Folding Modulator

The cystic fibrosis transmembrane regulator (CFTR) is a cyclic-AMP dependent chloride channel expressed at the apical surface of epithelial cells lining various organs such as the respiratory tract. Defective processing and functioning of this protein caused by mutations in the CFTR gene results in loss of ionic balance, defective mucus clearance, increased proliferation of biofilms and inflammation of human airways observed in cystic fibrosis (CF) patients. The process by which CFTR folds and matures under the influence of various chaperones in the secretory pathway remains incompletely understood. Recently, calumenin, a secretory protein, belonging to the CREC family of low affinity calcium binding proteins has been identified as a putative CFTR chaperone whose biophysical properties and functions remain uncharacterized. We compared hydropathy, instability, charge, unfoldability, disorder and aggregation propensity of calumenin and other CREC family members with CFTR associated chaperones and calcium binding proteins, wild-type and mutant CFTR proteins and intrinsically disordered proteins (IDPs). We observed that calumenin, along with other CREC proteins, was significantly more charged and less folded compared to CFTR associated chaperones. Moreover like IDPs, calumenin and other CREC proteins were found to be less hydrophobic and aggregation prone. Phylogenetic analysis revealed a close link between calumenin and other CREC proteins indicating how evolution might have shaped their similar biophysical properties. Experimentally, calumenin was observed to significantly reduce F508del-CFTR aggregation in a manner similar to AavLEA1, a well-characterized IDP. Fluorescence microscopy based imaging analysis also revealed altered trafficking of calumenin in bronchial cells expressing F508del-CFTR, indicating its direct role in the pathophysiology of CF. In conclusion, calumenin is characterized as a charged protein exhibiting close similarity with IDPs and is hypothesized to regulate F508del-CFTR folding by electrostatic effects. This work provides useful insights for designing optimized synthetic structural correctors of CFTR mutant proteins in the future.     

Brazilian Angiostrongylus cantonensis haplotypes,ac8 and ac9, have two different biological and morphological profiles

Angiostrongylus cantonensis is the etiologic agent of eosinophilic meningoencephalitis in humans. Cases have been recorded in many parts of the world, including Brazil. The aim of this study was to compare the differences in the biology and morphology of two different Brazilian haplotypes of A. : ac8 and ac9. A significantly larger number of L1 larvae eliminated in the faeces of rodents at the beginning of the patent period was observed for ac9 haplotype and compared to the total of L1 larvae eliminated, there was a significant difference between the two haplotypes. The ac9 haplotype showed a significant difference in the proportion of female and male specimens (0.6:1), but the same was not observed for ac8 (1.2:1). The morphometric analysis showed that male and female specimens isolated from ac8 haplotype were significantly larger with respect to body length, oesophagus length, spicule length (male) and distance from the anus to the rear end (female) compared to specimens from ac9. The morphological analysis by light microscopy showed little variation in the level of bifurcations at the lateral rays in the right lobe of the copulatory bursa between the two haplotypes. The biological, morphological and morphometric variations observed between the two haplotypes agree with the observed variation at the molecular level using the cytochrome oxidase subunit I marker and reinforce the possible influence of geographical isolation on the development of these haplotypes.     

Hemoglobin switching in sheep. Synthesis, cloning, and characterization of DNA sequences coding for the beta B, beta C, and gamma-globin mRNAs.

Synthetic double-stranded DNAs (sDNAs) were prepared from sheep globin mRNA templates isolated from reticulocytes producing either hemoglobin B (HbB) (alpha 2 beta B2), HbC (alpha 2 beta C2), or HbF (alpha 2 gamma 2). These DNAs were inserted into the Eco RI site of plasmid pMB9 by the homopolymer tailing method and used to transform Escherichia coli X1776 to tetracycline resistance. Recombinant clones were identified by colony hybridization and further characterized by molecular hybridization and restriction endonuclease analysis. All plasmids analyzed thus far contained either beta- or gamma-globin DNA sequences. Moreover, sDNAs used for cloning yielded restriction endonuclease fragments consistent with the presence of predominantly beta- or gamma-sDNA, indicating that formation of double-stranded alpha-sDNA proceeds much less efficiently under our conditions than the formation of non-alpha-sDNAs. Three recombinant plasmids, pS beta B2, pS beta C69, and pS gamma 56, were selected for detailed study. These were shown to contain, respectively, beta B-, beta C-, and gamma-DNA sequences by molecular hybridization and by protection of the appropriate cDNAs from S1 nuclease digestion. Each contained all of the restriction endonuclease sites defined for the synthetic sDNAs and protected at least 90% of the sequence length of homologous cDNA. Restriction endonuclease maps of the beta B- and beta C-globin genes were identical at all 12 sites that were mapped, whereas four differences were identified in the gamma gene compared to the two others; three of these corresponded to differences in amino acid sequence of the globins. A method was developed to isolate the anti-mRNA strand of the insert for use as a specific molecular hybridization probe analogous to complementary DNA.     

Voltage-dependent depolarization of bacterial membranes and artificial lipid bilayers by the peptide antibiotic nisin

The peptide antibiotic nisin is shown to disrupt valinomycin-induced potassium diffusion potentials imposed on intact cells of Staphylococcus cohnii 22. Membrane depolarization occurred rapidly at high diffusion potentials while at low potentials nisin-induced depolarization was slower suggesting that nisin requires a membrane potential for activity. This assumption was proven in experiments with planar lipid bilayers (black lipid membranes). Macroscopic conductivity measurements indicated a voltage-dependent action of nisin. The potential must have a trans-negative orientation with respect to the addition of nisin (added to the cis-side) and a sufficient magnitude (ca. -100 mV). With intact cells the threshold potential was lower (-50 to -80 mV at pH 7.5 and below -50 mV at pH 5.5). Single channel recordings resolved transient multistate pores, strongly resembling those introduced by melittin into artificial bilayers. The pores had diameters in the range of 0.2–1 nm, and lifetimes of few to several hundred milliseconds. The results indicate that nisin has to be regarded as a membrane-depolarizing agent which acts in a voltage-dependent fashion.Abbreviations BLM Black lipid membranes - CCCP carbonyl cyanide m-chlorophenylhydrazone - DOPC dioleoyl phosphatidylcholine - PS phosphatidylserine - TPP⁺ tetraphenylphosphonium cation     

Viola Zahnii Benz

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