Structure and bactericidal activity of an antibiotic dodecapeptide purified from bovine neutrophils

Cytoplasmic granules of neutrophils store a variety of cationic polypeptides, which exert in vitro a potent antibacterial action and are potentially involved in host defense mechanisms. From an acid extract of bovine neutrophil granules we have purified over 2,000-fold a dodecapeptide exhibiting bactericidal activity against both Escherichia coli and Staphylococcus aureus at 10(-7)-10(-5) M concentration. The purification procedure involved only two steps of ion-exchange and reversed-phase chromatography. The peptide, named bactenecin, has the amino acid sequence, Arg-Leu-Cys-Arg-Ile-Val-Val-Ile-Arg-Val-Cys-Arg, maintained in a cyclic structure by a disulfide bond between the two cysteine residues. Computer modeling of the dodecapeptide resulted in a conformation in which the chain adopts an antiparallel extended structure forming a gamma turn at residue 7.     

Restriction of the T-cell receptor V delta gene repertoire is due to preferential rearrangement and is independent of antigen selection

To determine whether the limited V gene usage by the T-cell receptor delta (TCRD) chain is dictated by preferential rearrangement or by antigen selection, we characterized and compared the TCRDV gene repertoire of the productive with that of the unprotective allele in 80 human TCRG/TCRD clones. Six different V genes were found on the expressed allele; two of them, provisionally named DV7 and DV8, have not been described before on the surface of TCRG/TCRD T cells. Overall, six V genes and six non-V elements were isolated from the unproductive allele. Interestingly, the same set of genes was rearranged both in the productive and in the unproductive chromosome. These findings seem to suggest that antigen-independent mechanisms play a major role in the restriction of the TCRDV gene repertoire.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide database and have been assigned the accession numbers Z46643 (DV7-E2), Z46644 (DV8-E6), Z46645 (DV8-M1), Z46641 (AV12-E4), Z46642 (AV29-E5), Z46652 (DREC-E13), Z46637 (TCR-d), Z46638 (TCR-n), Z46639 (TCR-r), Z46653 (PSI-DVu), and Z46640 (TCR-w)     

Synergistic Stimulation of Interleukin 6 Release and Gene Expression by Phorbol Esters and Interleukin 1β in Rat Cortical Astrocytes: Role of Protein Kinase C Activation and Blockade

Abstract: The involvement of protein kinase C and its interaction with interleukin 1β in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C, and by the desensitization of protein kinase C. Interleukin 1β increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1β stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1β (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurosporine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C. Interleukin 1β stimulated interleukin 6 secretion via a mechanism that is also negatively modulated by a protein kinase C isoform or isoforms sensitive to staurosporine and desensitization. Finally, we showed that interleukin 1β and phorbol 12-myristate 13-acetate synergistically stimulated interleukin 6 release and its gene expression, operating in a manner insensitive to protein kinase C blockers and slightly reduced by protein kinase C desensitization.     

C5a fragment of bovine complement. Purification, bioassays, amino-acid sequence and other structural studies

C5a and des-Arg-C5a have been purified from bovine serum in milligram amounts. The progress of the purification was followed by measuring the chemotactic activity of the complement fragments. The two polypeptides induce activation of neutrophil-oriented locomotion and secretion with very similar dose/response effects. After preparing a rabbit antiserum to bovine C5a/des-Arg-C5a, a competitive enzyme-linked immunosorbent assay (ELISA) was set up for the detection of C5a from 5 ng/mol to 1 microgram/ml. The complete primary structure of bovine C5a, which consists of 74 amino acids, has been determined by sequence analysis of the tryptic peptides, aligned by peptides derived from a chymotryptic digest, and by partially sequencing the intact molecule. Bovine C5a has a sequence homology of 78% and 70% with porcine and human C5a, respectively, reacts with an antiserum to porcine C5a and is recognized by cell surface receptors on human neutrophils. Finally, the secondary structure of bovine C5a was investigated by circular dicroic spectroscopy and predicted from the amino acid sequence. A comparison of the content and distribution of alpha-helical and/or hydropathic regions, suggests that the three-dimensional structure of C5a might be modeled from the known crystal structure of the homologous C3a molecule.     

Regulated expression systems for the development of whole-cell biocatalysts expressing oxidative enzymes in a sequential manner

This work reports the preparation of two recombinant strains each containing two enzymatic activities mutually expressed through regulated systems for production of functionalized epoxides in one-pot reactions. One strain was Pseudomonas putida PaW340, containing the gene coding for styrene monooxygenase (SMO) from Pseudomonas fluorescens ST under the auto-inducing Ptou promoter and the TouR regulator of Pseudomonas sp. OX1 and the gene coding for naphthalene dihydrodiol dehydrogenase (NDDH) from P. fluorescens N3 under the Ptac promoter inducible by IPTG. The second strain was Escherichia coli JM109, in which the expression of SMO was under the control of the Pnah promoter and the NahR regulator of P. fluorescens N3 inducible by salicylate, while the gene expressing NDDH was under the control of the Plac promoter inducible by IPTG. SMO and NDDH activities were tested in bioconversion experiments using cinnamyl alcohol as reference substrate. The application that we selected is one example of the sequential use of the two enzymatic activities which require a temporal control of the expression of both genes.     

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry: αB-CRYSTALLIN DISSOCIATES β2-MICROGLOBULIN OLIGOMERS*

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β₂-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass spectrometry. Fibril formation of R3Aβ2m was potently prevented by αB-crystallin. αB-crystallin also prevented the unfolding and nonfibrillar aggregation of R3Aβ2m. From analysis of the NMR spectra collected at various R3Aβ2m to αB-crystallin molar subunit ratios, it is concluded that the structured β-sheet core and the apical loops of R3Aβ2m interact in a nonspecific manner with the αB-crystallin. Complementary information was derived from NMR diffusion coefficient measurements of wild-type β2m at a 100-fold concentration excess with respect to αB-crystallin. Mass spectrometry acquired in the native state showed that the onset of wild-type β2m oligomerization was effectively reduced by αB-crystallin. Furthermore, and most importantly, αB-crystallin reversibly dissociated β2m oligomers formed spontaneously in aged samples. These results, coupled with our previous studies, highlight the potent effectiveness of αB-crystallin in preventing β2m aggregation at the various stages of its aggregation pathway. Our findings are highly relevant to the emerging view that molecular chaperone action is intimately involved in the prevention of in vivo amyloid fibril formation.     

Point-of-care assays for tuberculosis: Role of nanotechnology/microfluidics

Tuberculosis (TB) remains one of the most devastating infectious diseases and its eradication is still unattainable given the limitations of current technologies for diagnosis, treatment and prevention. The World Health Organization's goal to eliminate TB globally by 2050 remains an ongoing challenge as delayed diagnosis and misdiagnosis of TB continue to fuel the worldwide epidemic. Despite considerable improvements in diagnostics for the last few decades, a simple and effective point-of-care TB diagnostic test is yet not available. Here, we review the current assays used for TB diagnosis, and highlight the recent advances in nanotechnology and microfluidics that potentially enable new approaches for TB diagnosis in resource-constrained settings.     

Collective Electronic Excitations in Thin Ag Films on Ni(111)

The collective electronic excitations in thin Ag films deposited onto the Ni(111) surface were studied by high-resolution electron energy loss spectroscopy. A broad loss peak at 7.7 eV was assigned to the Ag multipole plasmon, in excellent agreement with calculations based on s-d polarization model. Ag multipole plasmon was excited only at grazing incidence. Furthermore, a strong dependence on the impinging energy exists. Multipole plasmon could be measured only for a very strict range of primary electron beam energies and it was excited by electrons scattered at a reflection plane located just underneath the jellium edge. Such mode was found to be dramatically more sensible to the state of the surface with respect to ordinary surface plasmon. Moreover, we report experimental evidence of interference effects in surface plasmon excitation.