Positions of the cytoplasmic end of BK α S0 helix relative to S1–S6 and of β1 TM1 and TM2 relative to S0–S6

The large-conductance, voltage- and Ca²⁺-gated K⁺ (BK) channel consists of four α subunits, which form a voltage- and Ca²⁺-gated channel, and up to four modulatory β subunits. The β1 subunit is expressed in smooth muscle, where it slows BK channel kinetics and shifts the conductance–voltage (G-V) curve to the left at [Ca²⁺] > 2 µM. In addition to the six transmembrane (TM) helices, S1–S6, conserved in all voltage-dependent K⁺ channels, BK α has a unique seventh TM helix, S0, which may contribute to the unusual rightward shift in the G-V curve of BK α in the absence of β1 and to a leftward shift in its presence. Such a role is supported by the close proximity of S0 to S3 and S4 in the voltage-sensing domain. Furthermore, on the extracellular side of the membrane, one of the two TM helices of β1, TM2, is adjacent to S0. We have now analyzed induced disulfide bond formation between substituted Cys residues on the cytoplasmic side of the membrane. There, in contrast, S0 is closest to the S2–S3 loop, from which position it is displaced on the addition of β1. The cytoplasmic ends of β1 TM1 and TM2 are adjacent and are located between the S2–S3 loop of one α subunit and S1 of a neighboring α subunit and are not adjacent to S0; i.e., S0 and TM2 have different trajectories through the membrane. In the absence of β1, 70% of disulfide bonding of W43C (S0) and L175C (S2–S3) has no effect on V₅₀ for activation, implying that the cytoplasmic end of S0 and the S2–S3 loop move in concert, if at all, during activation. Otherwise, linking them together in one state would obstruct the transition to the other state, which would certainly change V₅₀.     

Characteristics and application of S1–P1 nucleases in biotechnology and medicine

3′–nucleases/nucleotidases of the S1–P1 family (EC 3.1.30.1) are single–strand–specific or non-specific zinc–dependent phosphoesterases present in plants, fungi, protozoan parasites, and in some bacteria. They participate in a wide variety of biological processes and their current biotechnological applications rely on their single–strand preference, nucleotide non-specificity, a broad range of catalytic conditions and high stability. We summarize the present and potential utilization of these enzymes in biotechnology and medicine in the context of their biochemical and structure–function properties. Explanation of unanswered questions for bacterial and trypanosomatid representatives could facilitate development of emerging applications in medicine.     

Expression and secretion of β-glucuronidase and Pertussis toxin S1 by Streptomyces lividans

 Streptomyces lividans IAF18, obtained by homologous cloning, is capable of over-producing XlnA. To investigate the possibility of the expression of foreign genes, various coding regions of the xylanase A gene (xlnA) were analysed. Expression/secretion vectors were constructed containing the regulatory elements of xlnA with the coding region of the leader peptide with or without the truncated structural gene encoding the first 310 amino acids of the XlnA. The genes coding for the Escherichia coliβ-glucuronidase and subunit 1 of the Bordetella pertussis toxin (S1) were used and their expression analysed. S. lividans transformants where the β-glucuronidase gene was fused with the leader sequence produced up to 30 mg β-glucuronidase/culture filtrate whereas only fused XlnA/S1 was detected and its yield was estimated to be 1 mg/l. The disappearence of the B. pertussis toxin S1 and β-glucuronidase from the culture medium was due to the concomitant appearence of secreted proteases from S. lividans. Received: 19 July 1995/Received revision: 3 November 1995/Accepted: 20 November 1995     

Synthesis and SAR of 1,3-thiazolyl thiophene and pyridine derivatives as potent, orally active and S1P₃-sparing S1P₁ agonists

We have previously disclosed 1,2,4-oxadiazole derivative 3 as a potent S1P(3)-sparing S1P(1) agonist. Although compound 3 exhibits potent and manageable immunosuppressive efficacy in various in vivo models, recent studies have revealed that its 1,2,4-oxadiazole ring is subjected to enterobacterial decomposition. As provisions for unpredictable issues, a series of alternative compounds were synthesized on the basis of compound 3. Extensive SAR studies led to the finding of 1,3-thiazole 24c with the EC(50) value of 3.4 nM for human S1P(1), and over 5800-fold selectivity against S1P(3). In rat on host versus graft reaction (HvGR), the ID(50) value of 24c was determined at 0.07 mg/kg. The pharmacokinetics in rat and monkey is also reported. Compared to compound 3, 24c showed excellent stability against enterobacteria.     

The chaperone action of bovine milk αS1- and αS2-caseins and their associated form αS-casein

α_S-Casein, the major milk protein, comprises α_S1- and α_S2-casein and acts as a molecular chaperone, stabilizing an array of stressed target proteins against precipitation. Here, we report that α_S-casein acts in a similar manner to the unrelated small heat-shock proteins (sHsps) and clusterin in that it does not preserve the activity of stressed target enzymes. However, in contrast to sHsps and clusterin, α-casein does not bind target proteins in a state that facilitates refolding by Hsp70. α_S-Casein was also separated into α- and α-casein, and the chaperone abilities of each of these proteins were assessed with amorphously aggregating and fibril-forming target proteins. Under reduction stress, all α-casein species exhibited similar chaperone ability, whereas under heat stress, α-casein was a poorer chaperone. Conversely, α_S2-casein was less effective at preventing fibril formation by modified κ-casein, whereas α- and α_S1-casein were comparably potent inhibitors. In the presence of added salt and heat stress, α_S1-, α- and α_S-casein were all significantly less effective. We conclude that α_S1- and α-casein stabilise each other to facilitate optimal chaperone activity of α_S-casein. This work highlights the interdependency of casein proteins for their structural stability.     

Gαq-mediated plasma membrane translocation of sphingosine kinase-1 and cross-activation of S1P receptors

Sphingosine-1-phosphate (S1P), formed by sphingosine kinases (SphKs), regulates cellular proliferation and migration by acting as an agonist at specific receptors or intracellularly. Since S1P's effects are probably dependent on subcellular localization of its formation and degradation, we have studied the influence of G protein-coupled receptors on the localization of SphK1. Activation of G_q-coupled receptors induced a profound, rapid (half-life 3–5 s) and long-lasting (> 2 h) translocation of SphK1 to the plasma membrane. This was mimicked by expression of constitutively active G protein α-subunits specifically of the G_q family. Classical G_q signalling pathways, or phosphorylation at Ser²²⁵, phospholipase D and Ca²⁺/calmodulin were not involved in M₃ receptor-induced SphK1 translocation in HEK-293 cells. Translocation was associated with S1P receptor internalization, which was dependent on catalytic activity of SphK1 and S1P receptor binding and thus resulted from S1P receptor cross-activation. It is concluded that SphK1 is an important effector of G_q-coupled receptors, linking them via cross-activation of S1P receptors to G_i and G_12/13 signalling pathways.     

Synthesis and Anti-HBV Activity of Thiouracils Linked via S and N-1 to the 5-Position of Methyl β-D-Ribofuranoside

Abstract

Reverse nucleoside derivatives of 2-(methylsulfanyl)uracils 6a-d were prepared by treating of the sodium salt of 2-(methylsulfanyl)uracils (5a-d) with methyl 2,3-O-isopropylidene-5-O-p-toluenesulfonyl-β-D-ribofuranoside (2). The alkylation of 2-thiouracils 4a-d with methyl 5-deoxy-5-iodo-2,3-O-isopropylidene-D-ribofuranoside (3) afforded the corresponding S-ribofuranoside derivatives 8a-d. Deisopropylidenation of 6a-d and 8a-d afforded the corresponding deprotected derivatives 7a-d and 9a-d, respectively. The Anti-HBV activity of selected compounds was studied.     

Complete cloning of the chloroplast genome of safflower in λ EMBL3 and mapping of 23S and 16S rRNA genes

Summary A recombinant DNA library was constructed from partial BamHI or MboI digests of safflower (Carthamus tinctorius L.) chloroplast DNA, in the BamHI site of EMBL3. Seventeen recombinants, selected by chromosome walking, were found to contain overlapping fragments of the entire chloroplast genome. These clones were mapped using single and double digests of BamHI, EcoRI and HindIII. cDNAs synthesized from isolated 16S and 23S chloroplast rRNAs were used to map the ribosomal RNA genes relative to physical maps of the above restriction enzymes. The mapped positions of the rRNA genes for the safflower chloroplast DNA are in good agreement with previously published data for tobacco, spinach and several other higher plants.     

Characterization and Genetic Study of the Ovine α S2 -Casein (CSN1S2) Allele B

An interesting and quite complex protein pattern has been described at ovine milk proteins but the genetic control of the variation observed was assessed only in few cases. The aim of this work was to characterize the ovine α _s2 -casein (CSN1S2) B variant, first observed in the Italian Gentile di Puglia, a fine-wooled ovine breed, and to investigate its occurrence in two further breeds, the Sarda and Camosciata, which are the most widespread dairy breeds in Italy. The B variant differs from the most common form A with two amino acid exchanges: Asp₇₅ → Tyr₇₅ and Ile₁₀₅ → Val₁₀₅. The first substitution, resulting in a loss of a negative charge, is responsible for the higher isoelectric point of the B protein variant, which allows its detection by isoelectric focusing electrophoresis (IEF). The occurrence of CSN1S2*B in Sarda and Comisana was demonstrated. Since the Asp₇₅ → Tyr₇₅ substitution modifies the protein electric charge, milk properties may result affected to some extent.     

Cladistic analysis of 5S rRNA and 16S rRNA secondary and primary structure—The evolution of eukaryotes and their relation to archaebacteria

Summary The secondary structure of 5S rRNA has been elucidated by a cladistic analysis resulting in minimal models for eukaryotes, eubacteria, and halophilic-methanogenic archaebacteria, as well as for an ur-5S rRNA. This ancestor of all present-day 5S rRNA molecules is compared with an ur-tRNA and can be fitted into a tRNA-like structure allowing tertiary-structure interactions at the equivalent positions. A phylogenetic analysis of eukaryotic 5SrRNA and 16S rRNA sequences confirms particular monophyletic taxa: rhodophytes (red algae), chlorobionts (green algae and plants), metazoans (multicellular animals), euglenozoans (euglenids and trypanosomatids), a group of zygomycetes (excluding Kickxellales), a group of ascomycetes (excluding Protomycetales), two distinct groups of basidiomycetes, and a group consisting of phaeophyceans (brown algae) and oomycetes (water molds). The Euglenozoa show a distinct relation to the Eumycota (true fungi) and Metazoa. An analysis of archaebacterial sequences substantiates the paraphyletic nature of this third urkingdom defining the eubacteria as a sister group of the halophile-methanogens and defining the eukaryotes as a sister group of a particular lineage of the eocytes/sulfur-dependents. The latter fact implies that even the eocytes/sulfur-dependent archaebacteria are paraphyletic.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986Dedicated to the memory of Erik Huysmans who died on July 8, 1986, at the age of 29.     

Discovery of thiadiazole amides as potent, S1P₃-sparing agonists of sphingosine-1-phosphate 1 (S1P₁) receptor

High-throughput screening of GSK compound collection led to the discovery of a novel series of thiadiazole amides as potent and S1P(3)-sparing sphingosine-1-phosphate 1 (S1P(1)) receptor agonists. Synthesis, structure and activity relationship, selectivity, and some developability properties are described.     

Discovery, design and synthesis of novel potent and selective sphingosine-1-phosphate 4 receptor (S1P₄-R) agonists

High affinity and selective small molecule agonists of the S1P(4) receptor (S1P(4)-R) may have significant therapeutic utility in diverse disease areas including autoimmune diseases, viral infections and thrombocytopenia. A high-throughput screening (HTS) of the Molecular Libraries-Small Molecule Repository library identified 3-(2-(2,4-dichlorophenoxy)ethoxy)-6-methyl-2-nitropyridine as a moderately potent and selective S1P(4)-R hit agonist. Design, synthesis and systematic structure-activity relationships study of the HTS-derived hit led to the development of novel potent S1P(4)-R agonists exquisitely selective over the remaining S1P(1-3,5)-Rs family members. Remarkably, the molecules herein reported provide novel pharmacological tools to decipher the biological function and assess the therapeutic utility of the S1P(4)-R.     

Mössbauer, EPR, and MCD studies of the C9S and C42S variants of Clostridium pasteurianum rubredoxin and MCD studies of the wild-type protein

Rubredoxins contain a mononuclear iron tetrahedrally coordinated by four cysteinyl sulfurs. We have studied the wild-type protein from Clostridium pasteurianum and two mutated forms, C9S and C42S, in the oxidized and reduced states, with Mössbauer, integer-spin EPR, and magnetic circular dichroism (MCD) spectroscopies. The Mössbauer spectra of the ferric C42S and C9S mutant forms yielded zero-field splittings, D=1.2?cm^?1, that are about 40% smaller than the D-value of the wild-type protein. The ⁵⁷Fe hyperfine coupling constants were found to be ca. 8% larger than those of the wild-type proteins. The present study also revealed that the ferric wild-type protein has δ=0.24±0.01?mm/s at 4.2?K rather than δ=0.32?mm/s as reported in the literature. The Mössbauer spectra of both dithionite-reduced mutant proteins revealed the presence of two ferrous forms, A and B. These forms have isomer shifts δ=0.79?mm/s at 4.2?K, consistent with tetrahedral Fe²⁺(Cys)₃(O-R) coordination. The zero-field splittings of the two forms differ substantially; we found D=?7±1?cm^?1, E/D=0.09 for form A and D=+6.2±1.3?cm^?1, E/D=0.15 for form B. Form A exhibits a well-defined integer-spin EPR signal; from studies at X- and Q-band we obtained g _z =2.08±0.01, which is the first measured g-value for any ferrous rubredoxin. It is known from X-ray crystallographic studies that ferric C42S rubredoxin is coordinated by a serine oxygen. We achieved 75% reduction of C42S rubredoxin by irradiating an oxidized sample at 77?K with synchrotron X-rays; the radiolytic reduction produced exclusively form A, suggesting that this form represents a serine-bound Fe²⁺ site. Studies in different buffers in the pH?6–9 range showed that the A:B ratios, but not the spectral parameters of A and B, are buffer dependent, but no systematic variation of the ratio of the two forms with pH was observed. The presence of glycerol (30–50% v/v) was found to favor the B form. Previous absorption and circular dichroism studies of reduced wild-type rubredoxin have suggested d-d bands at 7400, 6000, and 3700?cm^?1. Our low-temperature MCD measurements place the two high-energy transitions at ca. 5900 and 6300?cm^?1; a third d-d transition, if present, must occur with energy lower than 3300?cm^?1. The mutant proteins have d-d transitions at slightly lower energy, namely 5730, 6100?cm^?1 in form A and 5350, 6380?cm^?1 in form B.     

The biodiversity and conservation of the birds of São Tomé and Príncipe

At present the majority of the endemic bird species occurring on São Tomé and Príncipe remain common, the rarer species being those largely confined to primary rainforest. In 1990 the dwarf olive ibis (Bostrychia bocagei), São Tomé fiscal shrike (Lanius newtoni), São Tomé short-tail (Amaurocichla bocagii) were rediscovered and in 1991 the São Tomé grosbeak (Neospiza concolor) was seen for the first time since 1888. Lowland primary forest is the only habitat on São Tomé in which all the endemic species are found. Primary forest on Príncipe remains largely unsurveyed since the beginning of the century. Due to the decline in the cocoa industry and poor infra-structure post-independence the extent of secondary forest is probably at its greatest since the late 1800s. This habitat is an important buffer zone against development for the remaining primary forest and also contains important populations of many of the endemic species, in particular the São Tomé scops owl (Otus hartlaubi), São Tomé oriole (Oriolus crassirostris) and giant weaver (Ploceus grandis). On both islands the remaining areas of primary forest need immediate protection and suitable boundaries have been designated under the Zona Ecológica plan. Fortunately, except for an important area around Lagôa Amélia, primary forest is not under immediate threat, although a variety of pressures are likely to increase. In conjunction with the protection of primary forest, a plant for managing the remaining timber resource on the islands will be a vital requirement, particularly if areas of shade forest, an important habitat for endemic bird species and a potentially valuable economic resource, are to be conserved and sustainably managed.     

Critical Role of S1PR1 and Integrin β4 in HGF/c-Met-mediated Increases in Vascular Integrity

Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met (1). We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.     

Aggregation and structural changes of α(S1)-, β- and κ-caseins induced by homocysteinylation

Elevated homocysteine levels are resulting in N-homocysteinylation of lysyl residues in proteins and they correlate with a number of human pathologies. However, the role of homocysteinylation of lysyl residues is still poorly known. In order to study the features of homocysteinylation of intrinsically unstructured proteins (IUP) bovine caseins were used as a model. α(S1)-, β- and κ-caseins, showing different aggregations and micelle formation, were modified with homocysteine-thiolactone and their physico-chemical properties were studied. Efficiency of homocysteine incorporation was estimated to be about 1.5, 2.1 and 1.3 homocysteyl residues per one β-, α(S1)-, and κ-casein molecule, respectively. Use of intrinsic and extrinsic fluorescent markers such as Trp, thioflavin T and ANS, reveal structural changes of casein structures after homocysteinylation reflected by an increase in beta-sheet content, which in some cases may be characteristic of amyloid-like transformations. CD spectra also show an increase in beta-sheet content of homocysteinylated caseins. Casein homocysteinylation leads in all cases to aggregation. The sizes of aggregates and aggregation rates were dependent on homocysteine thiolactone concentration and temperature. DLS and microscopic studies have revealed the formation of large aggregates of about 1-3μm. Homocysteinylation of α(S1)- and β-caseins results in formation of regular spheres. Homocysteinylated κ-casein forms thin unbranched fibrils about 400-800nm long. In case of κ-casein amyloidogenic effect of homocysteinylation was confirmed by Congo red spectra. Taken together, data indicate that N-homocysteinylation provokes significant changes in properties of native caseins. A comparison of amyloidogenic transformation of 3 different casein types, belonging to the IUP protein family, shows that the efficiency of amyloidogenic transformation upon homocysteinylation depends on micellization capacity, additional disulphide bonds and other structural features.     

Phenoxypropanolamine derivatives as selective inhibitors of the 20S proteasome β1 and β5 subunits

New series of thiophene-containing phenoxypropanolamines were synthesized and evaluated for their potency to inhibit the three proteolytic activities of the mammalian 20S proteasome. Noticeable inhibition of both ChT-L and PA activities was obtained with three compounds: one with unsubstituted phenoxypropanolamine group (7) and the two others with a p-Cl-substituted group (4 and 9). For three other compounds (3, 8 and 10), ChT-L activity alone was significantly inhibited. In silico docking performed on the β5 and β1 subunits bearing the respective ChT-L and PA catalytic sites showed features common to poses associated with active compounds. These features may constitute a selectivity criterion for structure-guided inhibitor design.     

Dual behavior of sodium dodecyl sulfate as enhancer or suppressor of insulin aggregation and chaperone-like activity of camel αS1-casein

Sodium dodecyl sulfate (SDS) at low concentrations considerably enhanced insulin aggregation and reduced the chaperone-like activity of purified camel αS₁-casein (αS₁-CN). These observed changes were the result of repulsive electrostatic interactions between both negative charged head groups of SDS and αS₁-CN, and the net negative charge of insulin molecules, resulting in the greater exposure of hydrophobic patches of insulin and its enhanced aggregation. In contrast, enhanced hydrophobic interactions were primarily responsible for the conformational changes observed in insulin and αS₁-CN at high SDS concentrations, resulting in increased binding of SDS and αS₁-CN to insulin and its reduced aggregation.