Structural characterization and reactivity of gamma-octamolybdate functionalized by proline

The reaction of molybdate and dl-proline at pH 3.4 results in the formation of a Na₄[Mo₈O₂₆(proO)₂] · 22H₂O complex (pro = proline) in which two proline ligands are attached to molybdenum(VI) ions via monodentate coordination of the carboxylate groups. The structure of the complex was determined by single crystal X-ray diffraction and by combination of ¹H, ¹³C and ⁹⁵Mo NMR spectroscopy techniques in solution. The structure of the complex is strongly dependant on the pH. At native pH 3.4 the octamolybdate-type structure seems to be present in solution, but the increase of pH to 5.8 resulted in a rearrangement of the structure to a heptamolybdate-type structure. At physiological pH, the polyoxometalate framework was completely dissociated into the monomeric unit. The reactivity of the Na₄[Mo₈O₂₆(proO)₂] · 22H₂O towards the hydrolysis of ATP was tested at different pH values. While in solution at pH 3.4 the hydrolysis proceeded to yield AMP (adenosine monophosphate) and ADP (adenosine diphosphate) in nearly equal amounts, reaction mixture at pH 5.8 gave ADP as the only product of hydrolysis after 24 h of reaction. At neutral pH, the hydrolysis of ATP was slower, but it proceeded to yield 75% of ADP after 48 h of reaction.     

Indications towards a stereoselectivity of the salt-induced peptide formation reaction

Different reaction yields for l- and d-alanine in the salt-induced peptide formation (SIPF) reaction, differences in the circular dichroism spectra and the complex formation constants of the involved chlorocuprate complexes point at a stereoselective differentiation between the two stereoisomers in the SIPF reaction and give a possible explanation towards the origin of homochirality in the process of the origin of life. An explanation of the observed effects can for the time being only be based on assumptions but could possibly be related to the inherent chirality of the Cu^II ion as a central atom of the [CuCl(gly)(glyH₂)(H₂O)₂]⁺ complex due to parity violation in weak interactions and to amplification of chirality related to the structural properties of the complex.     

Easily polarizable proton transfer hydrogen bonds between the side chains of histidine and the carboxylic acid groups of glutamic and aspartic acid residues in proteins

The nature of the hydrogen bonds formed between glutamic acid and histidine residues between aspartic acid and histidine residues is studied by i.r. spectroscopy. These studies were performed with $\text{(}\text{l}\text{-Glu}\text{)}{}_{\mathrm{n}}\text{+(}\text{l}\text{-His}\text{)}{}_{\mathrm{n}}$ and with associates of monomeric Glu + His and Asp + His systems in solutions whereby these amino acids had protected α-amino and α-carboxylic groups. It is shown that the OH …N?^?O?H⁺N bonds are easily polarizable proton transfer hydrogen bonds. The residence time of the proton at the His is a little larger in the case of the Asp + His than in the case of the Glu + His systems. Polar environments shift this proton transfer equilibrium in favour of the proton limiting structure ^?O?H⁺N, and less polar ones in favour of the structure OH?N. These results demonstrate that the large proton polarizability of the hydrogen bonded system in the active centre of chymotrypsin is responsible for the charge shift caused by the substrate, and thus for the increase in reactivity of the serine residue and the catalytic activity of the enzyme.     

Functional analysis of TatA and TatB in Streptomyces lividans

Recently, genes encoding TatA, TatB, and TatC homologues were identified in Streptomyces lividans and the functionality of the twin-arginine translocation (Tat) pathway was demonstrated. Previously, we have shown that TatC is indispensable for Tat-dependent secretion in S. lividans. In the present work, we demonstrate that as TatB, S. lividans TatA is important but not essential for efficient secretion of xylanase C and tyrosinase. The results presented here indicate that in the presence of TatC, still partially functional translocation systems composed of TatAC or TatBC can be formed, suggesting that TatA and TatB have at least partially overlapping activities. However, the dissimilar effect caused by a tatA deletion or a tatB deletion on Tat-dependent secretion together with the fact that TatA cannot fully functionally substitute TatB and vice versa indicates that in S. lividans TatA and TatB are not functionally equivalent. Interestingly, soluble GST-tagged TatA and TatB were able to specifically bind Tat-dependent preproteins. The ability to bind Tat-dependent preproteins together with their cytoplasmic localization in S. lividans strongly suggests that both TatA and TatB, independently or associated, serve to recruit Tat-dependent preproteins to the translocase.     

P2Y12 receptor stimulation inhibits beta-adrenergic receptor-induced differentiation by reversing the cyclic AMP-dependent inhibition of protein kinase B

Cyclic AMP-dependent induction of differentiation by activation of the beta-adrenergic receptor is correlated with inhibition of protein kinase B activity concomitant with growth arrest and increase in glial fibrillary acidic protein (GFAP) synthesis in rat C6 glioma cells. Costimulation of the beta-adrenergic receptor with purinergic receptors activated by 2-methylthio-adenosine-5'-diphosphate (2MeSADP) increased protein kinase B (PKB) phosphorylation above the level measured in non-stimulated cells and abolished cAMP-dependent differentiation. Transfection of cells with constitutively active PKB confirmed that reactivation of PKB is involved in the 2MeSADP-dependent inhibition of GFAP synthesis. The P2Y(12) and P2Y(13) receptor antagonist AR-C69931MX [N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloro-methylene ATP] decreased PKB phosphorylation to the level in non-stimulated cells, whereas the P2Y(13) antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and P(1),P(3)-di(adenosine-5') tetraphosphate (Ap(4)A) did not alter the 2MeSADP-induced phosphorylation of PKB, showing that enhanced PKB activity and subsequent phosphorylation of glycogen synthase kinase-3 is due to stimulation of the P2Y(12) receptor. In addition, experiments in the presence of pertussis toxin and phosphatidylinositol 3-kinase (PI 3-K) activity assays demonstrated that the P2Y(12) receptor-mediated increase in PKB phosphorylation is G(i) protein- and PI 3-K-dependent. The presented data demonstrated that a cAMP-dependent inhibition of PKB induces differentiation of C6 glioma cells and that inhibition of adenylate cyclase and reactivation of the PI 3-K/PKB pathway by the P2Y(12) receptor reverses differentiation into enhanced proliferation.     

Proprotein convertases regulate insulin-like growth factor 1-induced membrane-type 1 matrix metalloproteinase in VSMCs via endoproteolytic activation of the insulin-like growth factor-1 receptor

The IGF-1 receptor (IGF-1R) and MT1-MMP are synthesized as larger precursor proproteins, which require endoproteolytic activation by the proprotein convertases (PCs) furin/PC5 to gain full biological activity. The aim of this study was to investigate the contribution of PCs to IGF-1R and/or MT1-MMP activation in vascular smooth muscle cells (VSMCs) as well as VSMC proliferation/migration, which are key elements in vascular remodeling. Furin and PC5 mRNAs and proteins were found in VSMCs. Inhibition of furin-like PCs with the specific pharmacological inhibitor dec-CMK inhibited IGF-1R endoproteolytic activation. Inhibition of IGF-1R maturation abrogated IGF-induced IGF-1R autophosphorylation, PI3-kinase and MAPK induction, as well as VSMC proliferation (p<0.05 vs. controls), whereas it had no effect of PDGF-stimulated signaling pathways or cell growth. Both, IGF-1 and PDGF-BB, induced MT1-MMP expression, but only IGF-1-mediated MT1-MMP induction was inhibited by dec-CMK. Induction of MMP-2 by IGF-1 was inhibited by the PI3-kinase inhibitor wortmannin, but not by the MEK-inhibitor PD98059. Dec-CMK inhibited VSMC chemotaxis comparable to the effects of the MMP-inhibitor GM6001 (both p<0.05 vs. controls), supporting that MMPs are involved. In conclusion, this study demonstrates that targeting furin-like PCs and thus inhibiting IGF-1R activation is a novel target to inhibit IGF-1-mediated signaling and cell functions, such as IGF-1-induced MT1-MMP/MMP-2 in VSMCs.     

Physiological basis of pre-harvest sprouting resistance in Sorghum bicolor (L.) Moench. ABA levels and sensitivity in developing embryos of sprouting-resistant and -susceptible varieties

Pre–harvest sprouting is a major problem in Sorghum cropswhich leads to losses in seed viability and produces importantdecreases in grain weight. In this paper we aimed to have aninsight into the physiological basis of pre-harvest sproutingresistance in this crop by assessing germinability, ABA embryoniccontent and embryonic sensitivity to ABA during seed developmentin three varieties presenting contrasting sprouting behaviour:Redland B2 (very susceptible), SC 650 (moderately resistant)and IS 9530 (very resistant). Redland B2 caryopses were ableto germinate with high germination indices from early stagesof development, while caryopses from IS 9530 did not presentgermination indices different from 0 until near physiologicalmaturity. SC 650 (moderately resistant) grains presented anintermediate pattern of behaviour. In all three varieties isolatedembryos were able to germinate with maximum germination indicesfrom as early as 15 d after pollination (DAP). Differences ingrain dormancy level were not paralleled by a consistently differentendogenous ABA content throughout maturation. However, whenABA embryonic content was measured in incubated 35 DAP caryopses,ABA level in B2 embryos after 24 h of incubation was found tobe less than half that observed in IS 9530 embryo after thesame period of incubation. In addition, B2 embryos were foundto be 10–fold less sensitive to the inhibitory effectof ABA than embryos from the other two varieties. These resultsexplain to a considerable extent differences in germinabilitybetween sproutingresistant and –susceptible varietiesand are consistent with differences in sprouting behaviour. Key words: Sorghum bicolor, abscisic acid, pre-harvest sprouting, seed development, germination     

Neuromuscular development in Patellogastropoda (Mollusca: Gastropoda) and its importance for reconstructing ancestral gastropod bodyplan features

Within Gastropoda, limpets (Patellogastropoda) are considered the most basal branching taxon and its representatives are thus crucial for research into evolutionary questions. Here, we describe the development of the neuromuscular system in Lottia cf. kogamogai. In trochophore larvae, first serotonin‐like immunoreactivity (lir) appears in the apical organ and in the prototroch nerve ring. The arrangement and number of serotonin‐lir cells in the apical organ (three flask‐shaped, two round cells) are strikingly similar to those in putatively derived gastropods. First, FMRFamide‐lir appears in veliger larvae in the Anlagen of the future adult nervous system including the cerebral and pedal ganglia. As in other gastropods, the larvae of this limpet show one main and one accessory retractor as well as a pedal retractor and a prototroch muscle ring. Of these, only the pedal retractor persists until after metamorphosis and is part of the adult shell musculature. We found a hitherto undescribed, paired muscle that inserts at the base of the foot and runs towards the base of the tentacles. An apical organ with flask‐shaped cells, one main and one accessory retractor muscle is commonly found among gastropod larvae and thus might have been part of the last common ancestor.     

Regulation of hexose transport in respiration deficient hamster lung fibroblasts

The transport of [3H]2-deoxy-D-glucose (2DG) and [3H]3-O-methyl-D-glucose (3-OMG) was elevated in a respiration deficient (NADH coenzyme Q [Co Q] reductase deficient) Chinese hamster lung fibroblast cell line (G14). This sugar transport increase was related to an increased Vmax for 2DG transport, 26.9 +/- 4.2 nmoles 2DG/mg protein/30 sec in the G14 cell line vs 9.5 +/- 0.6 nmoles 2DG/mg protein/30 sec in the parental V79 cell line. No differences were observed in their respective Km values for 2DG transport (3.9 +/- .6 vs. 3.0 +/- .13 mM). Factors which increase sugar transport (e.g., glucose deprivation, serum or insulin exposure) or decrease sugar transport (e.g., serum deprivation) in the parental V79 cell line had little effect on sugar transport in the G14 respiration deficient cell lines. Amino acid transport, specific 125I-insulin binding to cells, and insulin-stimulated DNA synthesis, however, were similar in both cell lines. Exposure of both cell lines to varying concentrations of cycloheximide (0.1-50 micrograms/ml) for 4 h resulted in differential effects on 2DG transport. In the parental cell line (V79) low cycloheximide concentrations resulted in decreased 2DG transport, while higher concentrations (greater than or equal to 1 microgram/ml) resulted in elevated 2DG transport. In the G14 cell line, 2DG transport decreased at all concentrations of cycloheximide (up to 50 micrograms/ml). The data indicate that the G14 mutant has been significantly and specifically affected in the expression of sugar transport activity and in the regulatory controls affecting sugar transport activity.