Observations on Bostrychia radicosa comb. nov. (Rhodomelaceae, Rhodophyta)

Only two genera in the Rhodomelaceae share the morphological character of transverse division of periaxial cells into two or more tier cells in which the pit connection is retained between the lower cell and the axial cell: Bostrychia and Rhodolachne. One species, Rhodolachne radicosa Itono, has been reported from mangroves, a common habitat for Bostrychia. Many collections of an entity similar to Rhodolachne radicosa have been made from localities around the Indo‐Pacific. Culture observations show a Polysiphonia‐type sexual life history in Malaysia and New Caledonia isolates that produce self‐compatible bisexual gametophytes. The New Caledonia isolate also has unisexual gametophytes. An isolate from New South Wales (Australia) reproduces asexually through successive generations of tetrasporophytes. The Thailand isolate has successive generations of mixed‐phase tetrasporophytes. The tetrasporangial stichidia also bear male spermatangial sectors, but female structures are lacking. Western Australia and Madagascar isolates do not reproduce in culture. Molecular evidence, based on sequencing of the rbcL and the large subunit ribosomal RNA genes, shows that these isolates belong to the genus Bostrychia. Low molecular weight carbohydrate analysis reveals high levels of digeneaside in all isolates. The sugar hexitol sorbitol, an osmolyte characteristic of Bostrychia, occurs in all isolates, whereas the Madagascar and New Caledonia isolates have very low levels of dulcitol. Molecular, low molecular weight carbohydrate and morphological evidence show that Rhodolachne radicosa belongs within the genus Bostrychia. We transfer Rhodolachne radicosa to Bostrychia radicosa (Itono) West, Zuccarello and Hommersand.     

microRNA‐379 couples glucocorticoid hormones to dysfunctional lipid homeostasis

In mammals, glucocorticoids (GCs) and their intracellular receptor, the glucocorticoid receptor (GR), represent critical checkpoints in the endocrine control of energy homeostasis. Indeed, aberrant GC action is linked to severe metabolic stress conditions as seen in Cushing's syndrome, GC therapy and certain components of the Metabolic Syndrome, including obesity and insulin resistance. Here, we identify the hepatic induction of the mammalian conserved microRNA (miR)‐379/410 genomic cluster as a key component of GC/GR‐driven metabolic dysfunction. Particularly, miR‐379 was up‐regulated in mouse models of hyperglucocorticoidemia and obesity as well as human liver in a GC/GR‐dependent manner. Hepatocyte‐specific silencing of miR‐379 substantially reduced circulating very‐low‐density lipoprotein (VLDL)‐associated triglyceride (TG) levels in healthy mice and normalized aberrant lipid profiles in metabolically challenged animals, mediated through miR‐379 effects on key receptors in hepatic TG re‐uptake. As hepatic miR‐379 levels were also correlated with GC and TG levels in human obese patients, the identification of a GC/GR‐controlled miRNA cluster not only defines a novel layer of hormone‐dependent metabolic control but also paves the way to alternative miRNA‐based therapeutic approaches in metabolic dysfunction.     

Online measurement of the respiratory activity in shake flasks enables the identification of cultivation phases and patterns indicating recombinant protein production in various Escherichia coli host strains

Escherichia coli is commonly used for recombinant protein production with many available host strains. Screening experiments are often performed in batch mode using shake flasks and evaluating only the final product concentration. This conventional approach carries the risk of missing the best strain due to limited monitoring capabilities. Thus, this study focuses on investigating the general suitability of online respiration measurement for selecting expression hosts for heterologous protein production. The oxygen transfer rate (OTR) for different T7‐RNA polymerase‐dependent Escherichia coli expression strains was compared under inducing and noninducing conditions. As model enzymes, a lipase A from Bacillus subtilis (BSLA) and a 3‐hydroxybutyryl‐CoA dehydrogenase from Thermus thermophilus (HBD) were chosen. Four strains were compared during expression of both enzymes in autoinduction medium. Additionally, four strains were compared during expression of the BSLA with IPTG induction. It was found that the metabolic burden during recombinant protein production induces a phase of constant OTR, while undisturbed cell growth with no or little product formation is indicated by an exponential increase. This pattern is independent of the host strain, expressed enzyme, and induction method. Furthermore, the OTR gives information about carbon source consumption, biomass formation, and the transition from production to noninduced second growth phase, thereby ensuring a fair comparison of different strains. In conclusion, online monitoring of the respiration activity is suited to qualitatively identify, if a recombinant protein is produced by a strain or not. Furthermore, laborious offline sampling is avoided. Thus, the technique is easier and faster compared to conventional approaches. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:315–327, 2018     

Crystal structure of human beta2-glycoprotein I: implications for phospholipid binding and the antiphospholipid syndrome

The high affinity of human plasma beta2-glycoprotein I (beta(2)GPI), also known as apolipoprotein-H (ApoH), for negatively charged phospholipids determines its implication in a variety of physiological pathways, including blood coagulation and the immune response. beta(2)GPI is considered to be a cofactor for the binding of serum autoantibodies from antiphospholipid syndrome (APS) and correlated with thrombosis, lupus erythematosus and recurrent fetal loss. We solved the beta(2)GPI structure from a crystal form with 84% solvent and present a model containing all 326 amino acid residues and four glycans. The structure reveals four complement control protein modules and a distinctly folding fifth C-terminal domain arranged like beads on a string to form an elongated J-shaped molecule. Domain V folds into a central beta-spiral of four antiparallel beta-sheets with two small helices and an extended C-terminal loop region. It carries a distinct positive charge and the sequence motif CKNKEKKC close to the hydrophobic loop composed of residues LAFW (313-316), resulting in an excellent counterpart for interactions with negatively charged amphiphilic substances. The beta(2)GPI structure reveals potential autoantibody-binding sites and supports mutagenesis studies where Trp316 and CKNKEKKC have been found to be essential for the phospholipid-binding capacity of beta(2)GPI.     

Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis

The members of the ABC transporter family transport a wide variety of molecules into or out of cells and cellular compartments. Apart from a translocation pore, each member possesses two similar nucleoside triphosphate-binding subunits or domains in order to couple the energy-providing reaction with transport. In the maltose transporter of several Gram-negative bacteria and the archaeon Thermo coccus litoralis, the nucleoside triphosphate-binding subunit contains a C-terminal regulatory domain. A dimer of the subunit is attached cytoplasmically to the translocation pore. Here we report the crystal structure of this dimer showing two bound pyrophosphate molecules at 1.9 A resolution. The dimer forms by association of the ATPase domains, with the two regulatory domains attached at opposite poles. Significant deviation from 2-fold symmetry is seen at the interface of the dimer and in the regions corresponding to those residues known to be in contact with the translocation pore. The structure and its relationship to function are discussed in the light of known mutations from the homologous Escherichia coli and Salmonella typhimurium proteins.     

Replication through an abasic DNA lesion: structural basis for adenine selectivity

Abasic sites represent the most frequent DNA lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. Although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by DNA polymerases, a phenomenon termed A‐rule. In this study, we present X‐ray structures of a DNA polymerase caught while incorporating a nucleotide opposite an abasic site. We found that a functionally important tyrosine side chain directs for nucleotide incorporation rather than DNA. It fills the vacant space of the absent template nucleobase and thereby mimics a pyrimidine nucleobase directing for preferential purine incorporation opposite abasic residues because of enhanced geometric fit to the active site. This amino acid templating mechanism was corroborated by switching to pyrimidine specificity because of mutation of the templating tyrosine into tryptophan. The tyrosine is located in motif B and highly conserved throughout evolution from bacteria to humans indicating a general amino acid templating mechanism for bypass of non‐instructive lesions by DNA polymerases at least from this sequence family.     

Novel nanomolar imidazo[4,5-b]pyridines as selective nitric oxide synthase (iNOS) inhibitors: SAR and structural insights

Inducible arginine oxidation and subsequent NO production by correspondent synthase (iNOS) are important cellular answers to proinflammatory signals. Prolonged NO production has been proved in higher organisms to cause stroke or septic shock. Several classes of potent NOS inhibitors have been reported, most of them targeting the arginine binding site of the oxygenase domain. Here we disclose the SAR and the rational design of potent and selective iNOS inhibitors which may be useful as anti-inflammatory drugs.     

Structure-function correlation in glycine oxidase from Bacillus subtilis

Structure-function relationships of the flavoprotein glycine oxidase (GO), which was recently proposed as the first enzyme in the biosynthesis of thiamine in Bacillus subtilis, has been investigated by a combination of structural and functional studies. The structure of the GO-glycolate complex was determined at 1.8 A, a resolution at which a sketch of the residues involved in FAD binding and in substrate interaction can be depicted. GO can be considered a member of the "amine oxidase" class of flavoproteins, such as d-amino acid oxidase and monomeric sarcosine oxidase. With the obtained model of GO the monomer-monomer interactions can be analyzed in detail, thus explaining the structural basis of the stable tetrameric oligomerization state of GO, which is unique for the GR(2) subfamily of flavooxidases. On the other hand, the three-dimensional structure of GO and the functional experiments do not provide the functional significance of such an oligomerization state; GO does not show an allosteric behavior. The results do not clarify the metabolic role of this enzyme in B. subtilis; the broad substrate specificity of GO cannot be correlated with the inferred function in thiamine biosynthesis, and the structure does not show how GO could interact with ThiS, the following enzyme in thiamine biosynthesis. However, they do let a general catabolic role of this enzyme on primary or secondary amines to be excluded because the expression of GO is not inducible by glycine, sarcosine, or d-alanine as carbon or nitrogen sources.     

Fluorescence emission properties of S-Layer enhanced green fluorescent fusion protein as a function of temperature, pH conditions, and guanidine hydrochloride concentration

The fluorescent properties of the S-layer enhanced green fluorescent fusion protein (rSbpA31-1068/EGFP) were investigated as a function of temperature, pH conditions, and guanidine hydrochloride concentration. These results were compared to the fluorescent properties of the recombinant enhanced green fluorescent protein (EGFP) and an equimolar mixture of the S-layer protein rSbpA and EGFP. The intensity of the fluorescence emission of the EGFP at 510 nm, after excitation at 490 nm, is not affected by the presence of rSbpA, either as a fusion partner or as a free protein in solution. In each of the three protein systems, the emission intensity at 510 nm reaches its maximum value between pH 7 and 9 at 20 degrees C and at 0 M guanidine hydrochloride. No fluorescence could be measured at pH 4 and 6 M guanidine hydrochloride. These results show that the S-layer fusion protein (rSbpA31-1068/EGFP) is a suitable candidate for future applications in nanobiotechonology at a wide range of pH, temperature, and guanidine hydrochloride concentrations.