Chain length dependence of apomyoglobin folding: structural evolution from misfolded sheets to native helices

Very little is known about how protein structure evolves during the polypeptide chain elongation that accompanies cotranslational protein folding. This in vitro model study is aimed at probing how conformational space evolves for purified N-terminal polypeptides of increasing length. These peptides are derived from the sequence of an all-alpha-helical single domain protein, Sperm whale apomyoglobin (apoMb). Even at short chain lengths, ordered structure is found. The nature of this structure is strongly chain length dependent. At relatively short lengths, a predominantly non-native beta-sheet conformation is present, and self-associated amyloid-like species are generated. As chain length increases, alpha-helix progressively takes over, and it replaces the beta-strand. The observed trends correlate with the specific fraction of solvent-accessible nonpolar surface area present at different chain lengths. The C-terminal portion of the chain plays an important role by promoting a large and cooperative overall increase in helical content and by consolidating the monomeric association state of the full-length protein. Thus, a native-like energy landscape develops late during apoMb chain elongation. This effect may provide an important driving force for chain expulsion from the ribosome and promote nearly-posttranslational folding of single domain proteins in the cell. Nature has been able to overcome the above intrinsic misfolding trends by modulating the composition of the intracellular environment. An imbalance or improper functioning by the above modulating factors during translation may play a role in misfolding-driven intracellular disorders.     

Functional Characterization of AbeD,an RND-Type Membrane Transporter in Antimicrobial Resistance in Acinetobacter baumannii

Background

Acinetobacter baumannii is becoming an increasing menace in health care settings especially in the intensive care units due to its ability to withstand adverse environmental conditions and exhibit innate resistance to different classes of antibiotics. Here we describe the biological contributions of abeD, a novel membrane transporter in bacterial stress response and antimicrobial resistance in A. baumannii.

Results

The abeD mutant displayed ~ 3.37 fold decreased survival and >5-fold reduced growth in hostile osmotic (0.25 M; NaCl) and oxidative (2.631 μM–6.574 μM; H₂O₂) stress conditions respectively. The abeD inactivated cells displayed increased susceptibility to ceftriaxone, gentamicin, rifampicin and tobramycin (~ 4.0 fold). The mutant displayed increased sensitivity to the hospital-based disinfectant benzalkonium chloride (~3.18-fold). In Caenorhabditis elegans model, the abeD mutant exhibited (P<0.01) lower virulence capability. Binding of SoxR on the regulatory fragments of abeD provide strong evidence for the involvement of SoxR system in regulating the expression of abeD in A. baumannii.

Conclusion

This study demonstrates the contributions of membrane transporter AbeD in bacterial physiology, stress response and antimicrobial resistance in A. baumannii for the first time.     

Sesquiterpene lactones and other constituents of Eupatorium lancifolium and E. semiserratum

The isolation of the cytotoxic and/or antileukaemic heliangolides eupacunolin, eupacunin, desacetyleupacunin, a new germacradienolide and a coumarin fr     

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients'' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.     

Genomic DNA amplification from a single bacterium

Genomic DNA was amplified about 5 billion-fold from single, flow-sorted bacterial cells by the multiple displacement amplification (MDA) reaction, using phi 29 DNA polymerase. A 662-bp segment of the 16S rRNA gene could be accurately sequenced from the amplified DNA. MDA methods enable new strategies for studying non-culturable microorganisms.     

Precursor development, characterisation and evaluation of sublimation enthalpies of novel volatile complexes of nickel

The nickel complexes namely bis(2-hydroxyacetophenoato)nickel(II), Ni(ohap)₂ (1), bis(2-hydroxyacetophenamine)nickel(II), Ni(ohapim)₂ (2) and bis(2-hydroxypropiophenamine)nickel(II), Ni(ohppim)₂ (3) were synthesised in a systematic approach for the development of volatile nickel precursors for MOCVD. Upon screening these complexes by dynamic TG in an inert N₂ purge gas environment, only complexes 2 and 3 were found to be completely volatile by 363 and 373 °C, respectively. These two were further characterized by FT-IR, C, H, and N and FAB-mass spectrometry. The molecular masses of the predominant Ni bearing vapour species were found to be 326 and 355 Da for 2 and 3, respectively. The complex 3 was analysed by single crystal XRD, ¹H and ¹³C NMR. The equilibrium vapour pressures (p_e) of the precursors 2 and 3 over the temperature ranges of 486-581 and 443-552 K were determined by a TG-based transpiration technique, yielding values of 130.2 (±7.2) and 113.3 (±7.5) kJ mol⁻¹, respectively, for their standard enthalpies of sublimation ().     

In Silico Metabolic Model and Protein Expression of Haemophilus influenzae Strain Rd KW20 in Rich Medium

The intermediary metabolism of Haemophilus influenzae strain Rd KW20 was studied by a combination of protein expression analysis using a recently developed direct proteomics approach, mutational analysis, and mathematical modeling. Special emphasis was placed on carbon utilization, sugar fermentation, TCA cycle, and electron transport of H. influenzae cells grown microaerobically and anaerobically in a rich medium. The data indicate that several H. influenzae metabolic proteins similar to Escherichia coli proteins, known to be regulated by low concentrations of oxygen, were well expressed in both growth conditions in H. influenzae. An in silico model of the H. influenzae metabolic network was used to study the effects of selective deletion of certain enzymatic steps. This allowed us to define proteins predicted to be essential or non-essential for cell growth and to address numerous unresolved questions about intermediary metabolism of H. influenzae. Comparison of data from in vivo protein expression with the protein list associated with a genome-scale metabolic model showed significant coverage of the known metabolic proteome. This study demonstrates the significance of an integrated approach to the characterization of H. influenzae metabolism.     

Synthesis,antimicrobial and antifungal activity of a new class of spiro pyrrolidines

A new class of spiro pyrrolidines, dispiro[oxindole-cyclohexanone]pyrrolidines, dispiro[oxindole-tetrahydronaphthalen-1-one]pyrrolidines, dispiro[oxindole-arylidene-cyclohexanone]pyrrolidines, dispiro[oxindole-hexahydro-indazole]pyrrolidines, and spiro[butenolide]pyrrolidines, have been screened for their antibacterial and antifungal activity against ten human pathogenic bacteria and four dermatophytic fungi. They were found to have antimicrobial and antifungal activity compounds against various pathogens except Bacillus subtilis. The spiro pyrrolidinines were synthesized by the regioselective 1,3-dipolar cycloaddition reaction of azomethine ylides generated either from isatin and sarcosine or from aziridine. The azomethine ylide so generated reacted with various dipolarophiles such as 2-arylidene-cyclohexanones, 2-arylidene-tetrahydronapthalen-1-ones, 2,6-bis(arylmethylidene)cyclohexanones and 3-arylidene-5-phenyl- butenolides.     

Generation of anti-c-met single domain antibody fragment based on human stable frameworks

The size reduction is an important issue in the biomedical application of antibody and single domain antibody fragment is recognized as very attractive tool. However, it is very time-consuming and laborious to generate the fragment antibody with targeted binding function. Here, we investigated the possibility to prepare single domain antibody (sdAb) by a simple grafting method based on stable human consensus framework sequences. The complementarity determining region sequences in V_H domain of anti-c-Met scFv from rabbit were grafted with the human V_H3 consensus framework sequences, which generated the anti-c-Met single domain antibody showing almost same binding activity to its scFv form. The generated single domain antibody could be produced as functional form in oxidizing cytoplasm of E. coli, but produced as inactive form in reducing cytoplasm. The structural analysis of the homology models gave us the insight on the stability of the single domain antibody. In this report, we have demonstrated that the very stable human consensus framework sequence can be used for the generation of active anti-c-Met sdAb via complementarity determining regions grafting. We expect that this kind of grafting method for the generation of sdAb may provide us with the opportunities to prepare sdAbs based on the known antibody sequences.     

G-Proteins mediate inhibition and activation of Ca2+-induced exocytosis from SLO-permeabilized peptidergic nerve endings

In SLO-permeabilized isolated nerve endings from the rat neurohypophysis, GTP, guanosine 5[y-thio]triphosphate (GTPyS) and guanosine 5(ßy-imido]triphosphate (GMPPNP) inhibit the Ca²⁺-evoked vasopressin release. Pretreatment with pertussis toxin enhances the inhibitory effects of both GTP-analogues. Omission of Mg²⁺ overcomes the effect of GMPPNP and reverses the inhibitory effect of GTP and GTPyS. In the absence of Mg²⁺, GTP and GTPyS now potentiate Ca²⁺-evoked secretion.