Multimode selective detection of mercury by chiroptical fluorescent sensors based on methionine/cysteine

Two multimode Hg(II) sensors, L‐MethBQA and L‐CysBQA, were obtained by fusing methionine or S‐methyl cysteine, into a bis‐quinolyl amine‐based chiral podand scaffold. Quinolyl groups serve as the fluorophore and possess nitrogen lone pairs capable of chelating metal ions. On exposure to Hg²⁺ or Zn²⁺, these sensors show signal enhancement in fluorescence. However, Cu²⁺ quenches their fluorescence in 30:70 acetontrile/water. L‐CysBQA complexes with Hg²⁺, producing an exciton‐coupled circular dichroism spectrum with the opposite sign to the one that is produced by Cu²⁺ or Zn²⁺ complexation. L‐CysBQA binds Hg²⁺ more strongly than Zn²⁺ and is shown to differentiate Hg²⁺ from other metal ions, such as Zn²⁺, Cu²⁺, Ni²⁺, and Pb²⁺, exceptionally well. The synergistic use of relatively soft sulfur, quinoline‐based chiral ligands and chiroptically enhanced fluorescence detection results in high sensitivity and selectivity for Hg²⁺. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Production of the potent antibacterial polyketide erythromycin C in Escherichia coli

An Escherichia coli strain capable of producing the potent antibiotic erythromycin C (Ery C) was developed by expressing 17 new heterologous genes in a 6-deoxyerythronolide B (6dEB) producer strain. The megalomicin gene cluster was used as the source for the construction of two artificial operons that contained the genes encoding the deoxysugar biosynthetic and tailoring enzymes necessary to convert 6dEB to Ery C. The reconstructed mycarose operon contained the seven genes coding for the enzymes that convert glucose-1-phosphate (G-1-P) to TDP-L-mycarose, a 6dEB mycarosyl transferase, and a 6dEB 6-hydroxylase. The activity of the pathway was confirmed by demonstrating conversion of exogenous 6dEB to 3-O-alpha-mycarosylerythronolide B (MEB). The reconstructed desosamine operon contained the six genes necessary to convert TDP-4-keto-6-deoxyglucose, an intermediate formed in the mycarose pathway, to TDP-D-desosamine, a desosamine transferase, a 6dEB 12-hydroxylase, and the rRNA methyltransferase ErmE; the last was required to confer resistance to the host cell upon production of mature macrolide antibiotics. The activity of this pathway was demonstrated by conversion of MEB to Ery C. When the mycarose and desosamine operons were expressed in an E. coli strain engineered to synthesize 6dEB, Ery C and Ery D were produced. The successful production of Ery C in E. coli shows the potentiality of this model microorganism to synthesize novel 6-deoxysugars and to produce bioactive glycosylated compounds and also establishes the basis for the future use of E. coli both in the production of new glycosylated polyketides and for the generation of novel bioactive compounds through combinatorial biosynthesis.     

Chronic kidney disease and automatic reporting of estimated glomerular filtration rate: a position statement

The systematic staging of chronic kidney disease (CKD) by glomerular filtration measurement and proteinuria has allowed the development of rational and appropriate management plans. One of the barriers to early detection of CKD is the lack of a precise, reliable and consistent measure of kidney function.The most common measure of kidney function is currently serum creatinine concentration. It varies with age, sex, muscle mass and diet, and interlaboratory variation between measurements is as high as 20%.The reference interval for serum creatinine concentration includes up to 25% of people (particularly thin, elderly women) who have an estimated glomerular filtration rate (eGFR) that is significantly reduced (< 60 mL/min/1.73 m). The recent publication of a validated formula (MDRD) to estimate GFR from age, sex, race and serum creatinine concentration, without any requirement for measures of body mass, allows pathology laboratories to "automatically" generate eGFR from data already acquired. Automatic laboratory reporting of eGFR calculated from serum creatinine measurements would help to identify asymptomatic kidney dysfunction at an earlier stage. eGFR correlates well with complications of CKD and an increased risk of adverse outcomes such as cardiovascular morbidity and mortality. We recommend that pathology laboratories automatically report eGFR each time a serum creatinine test is ordered in adults. As the accuracy of eGFR is suboptimal in patients with normal or near-normal renal function, we recommend that calculated eGFRs above 60 mL/min/1.73 m be reported by laboratories as "> 60 mL/min/1.73 m", rather than as a precise figure.     

Thrombin peptide (TP508) treatment of rat growth plate cartilage cells promotes proliferation and retention of the chondrocytic phenotype while blocking terminal endochondral differentiation

A synthetic peptide representing the receptor-binding domain of human thrombin (TP508, also known as Chrysalin) accelerates fracture repair in rats via endochondral ossification and promotes repair of rabbit cartilage defects. To understand how this peptide might stimulate cartilage and bone formation, we employed an established in vitro model of growth plate cartilage regulation. Rat costochondral cartilage resting zone and growth zone chondrocytes were treated with 0, 0.07, 0.7, or 7 microg/ml TP508 or a scrambled peptide, TP508-SP. Proliferation ([3H]-thymidine incorporation) was examined in pre-confluent cultures; effects on cell number, alkaline phosphatase activity, [35S]-sulfate incorporation, and responsiveness to vitamin D metabolites were tested using confluent cultures. TP508 did not affect proliferation of resting zone cells but it caused a dose-dependent increase in cell number and DNA synthesis of growth zone cells. Alkaline phosphatase specific activity of resting zone cells was reduced by TP508, whereas [35S]-sulfate incorporation was increased. Neither parameter was affected in growth zone cell cultures. TP508 treatment for 24 h did not induce resting zone cells to respond to 1alpha,25(OH)2D3, either with respect to alkaline phosphatase activity or proteoglycan production. In contrast, TP508 treatment reduced the stimulatory effect of 24R,25(OH)2D3 on alkaline phosphatase but it did not alter the stimulatory effect of 24R,25(OH)2D3 on [35S]-sulfate incorporation. In cultures treated for 48, 72, or 140 h with TP508, 1alpha,25(OH)2D3 restored alkaline phosphatase activity to control levels but did not stimulate activity over levels observed in untreated control cultures. The stimulatory effect of TP508 on [35S]-sulfate incorporation was evident up to 48 h post-confluence but at later time points, proteoglycan production was comparable to that seen in control cultures, control cultures challenged with 1alpha,25(OH)2D3, and cultures treated with TP508 followed by 1alpha,25(OH)2D3. TP508-SP had no effect on any of the parameters tested. These results indicate that TP508 exerts maturation specific effects on chondrocytes in the endochondral lineage, promoting cartilage extracellular matrix synthesis over endochondral differentiation in resting zone cells and proliferation over differentiation of growth zone cells.     

Differential arrangements of conserved building blocks among homologs of the Rad50/Mre11 DNA repair protein complex

Structural maintenance of chromosomes (SMC) proteins have diverse cellular functions including chromosome segregation, condensation and DNA repair. They are grouped based on a conserved set of distinct structural motifs. All SMC proteins are predicted to have a bipartite ATPase domain that is separated by a long region predicted to form a coiled coil. Recent structural data on a variety of SMC proteins shows them to be arranged as long intramolecular coiled coils with a globular ATPase at one end. SMC proteins function in pairs as heterodimers or as homodimers often in complexes with other proteins. We expect the arrangement of the SMC protein domains in complex assemblies to have important implications for their diverse functions. We used scanning force microscopy imaging to determine the architecture of human, Saccharomyces cerevisiae, and Pyrococcus furiosus Rad50/Mre11, Escherichia coli SbcCD, and S.cerevisiae SMC1/SMC3 cohesin SMC complexes. Two distinct architectural arrangements are described, based on the way their components were connected. The eukaryotic complexes were similar to each other and differed from their prokaryotic and archaeal homologs. These similarities and differences are discussed with respect to their diverse mechanistic roles in chromosome metabolism.     

Heterologous production of epothilone C and D in Escherichia coli

The epothilones are a family of polyketide natural products that show a high potential as anticancer drugs. They are synthesized by the action of a hybrid nonribosomal peptide synthetase/polyketide synthase in the myxobacterium Sorangium cellulosum. In this work, the genes encoding the entire cluster,epoA, epoB, epoC, epoD, epoE, and epoF, were redesigned and synthesized to allow for expression in Escherichia coli. The expression of the largest of the proteins, EpoD, also required the protein be separated into two polypeptides with compatible module linkers. Using a combination of lowered temperature, chaperone coexpression, and alternative promoters, we succeeded in producing a soluble protein from all genes in the epothilone cluster. The entire synthetic epothilone cluster was then expressed in a strain of E. coli modified to enable polyketide biosynthesis, resulting in the production of epothilones C and D. Furthermore, feeding a thioester of the normal substrate for EpoD to cells expressing the epoD, epoE, and epoF genes also led to the production of epothilones C and D. The design of the synthetic epothilone genes together with E. coli expression provides the ideal platform for both the biochemical investigation of the epothilone PKS and the generation of novel biosynthetic epothilone analogues.     

Hemicentin 2 and Fibulin 1 are required for epidermal-dermal junction formation and fin mesenchymal cell migration during zebrafish development

Hemicentin 1 (Hmcn1) and Hemicentin 2 (Hmcn2) belong to the fibulin family of extracellular matrix (ECM) proteins that play pivotal roles during development and homeostasis of a variety of vertebrate tissues. Recently, we have shown that mutations in zebrafish Hmcn1, also called Fibulin 6, lead to massive fin blistering, similar to the defects caused by the Fraser syndrome gene Fras1. In contrast, the role of Hmcn2 during vertebrate development has thus far been uncharacterized. In zebrafish, hmcn2, like fibulin 1 (fbln1), another member of the fibulin family, is predominantly expressed in fin mesenchymal cells and developing somites, contrasting the strict epithelial expression of hmcn1. While antisense morpholino oligonucleotide (MO)-based knockdown of hmcn2 did not yield any discernable defects, hmcn2/fbln1 double knockdown fish displayed blistering in the trunk, pointing to an essential contribution of these proteins from mesodermal sources for proper epidermal-dermal junction formation. In contrast, and unlike hmcn1 mutants, epidermal-dermal junctions in the fin folds of hmcn2/fbln1 double knockdown fish were only moderately affected. Instead, they displayed impaired migration of fin mesenchymal cells into the fin folds, pointing to a crucial role of Hmcn2 and Fbln1 to remodel the ECM of the fin fold interepidermal space, which is a prerequisite for fibroblast ingrowth. TEM analyses suggest that this ECM remodeling occurs at the level of actinotrichia, the collageneous migration substrate of mesenchymal cells, and at the level of cross fibers, which resemble mammalian microfibers. This work provides first insights into the role of Hmcn2 during vertebrate development, identifying it as an evolutionary conserved protein that acts in functional redundancy with Fbln1C and/or Fbln1D isoforms to regulate tissue adhesion and cell migration, while extending the current knowledge of the functions of vertebrate Fbln1.     

First is best

We experience the world serially rather than simultaneously. A century of research on human and nonhuman animals has suggested that the first experience in a series of two or more is cognitively privileged. We report three experiments designed to test the effect of first position on implicit preference and choice using targets that range from individual humans and social groups to consumer goods. Experiment 1 demonstrated an implicit preference to buy goods from the first salesperson encountered and to join teams encountered first, even when the difference in encounter is mere seconds. In Experiment 2 the first of two consumer items presented in quick succession was more likely to be chosen. In Experiment 3 an alternative hypothesis that first position merely accentuates the valence of options was ruled out by demonstrating that first position enhances preference for the first even when it is evaluatively negative in meaning (a criminal). Together, these experiments demonstrate a "first is best" effect and we offer possible interpretations based on evolutionary mechanisms of this "bound" on rational behavior and suggest that automaticity of judgment may be a helpful principle in clarifying previous inconsistencies in the empirical record on the effects of order on preference and choice.