Peptide fragments of a beta-defensin derivative with potent bactericidal activity.

Beta-defensins are known to be both antimicrobial and able to chemoattract various immune cells. Although the sequences of paralogous genes are not highly conserved, the core defensin structure is retained. Defb14-1C(V) has bactericidal activity similar to that of its parent peptide (murine beta-defensin Defb14) despite all but one of the canonical six cysteines being replaced with alanines. The 23-amino-acid N-terminal half of Defb14-1C(V) is a potent antimicrobial while the C-terminal half is not. Here, we use a library of peptide derivatives to demonstrate that the antimicrobial activity can be localized to a particular region. Overlapping fragments of the N-terminal region were tested for their ability to kill Gram-positive and Gram-negative bacteria. We demonstrate that the most N-terminal fragments (amino acids 1 to 10 and 6 to 17) are potent antimicrobials against Gram-negative bacteria whereas fragments based on sequence more C terminal than amino acid 13 have very poor activity against both Gram-positive and -negative types. We further test a series of N-terminal deletion peptides in both their monomeric and dimeric forms. We find that bactericidal activity is lost against both Gram types as the deletion region increases, with the point at which this occurs varying between bacterial strains. The dimeric form of the peptides is more resistant to the peptide deletions, but this is not due just to increased charge. Our results indicate that the primary sequence, together with structure, is essential in the bactericidal action of this beta-defensin derivative peptide and importantly identifies a short fragment from the peptide that is a potent bactericide.

Peptide thioester synthesis through N-->S acyl-transfer: application to the synthesis of a beta-defensin.

Peptide thioesters readily prepared through N-->S acyl transfer of a specific C-terminal motif provide access to biologically active mini-proteins using native chemical ligation.

Isoleucine/leucine2 is essential for chemoattractant activity of beta-defensin Defb14 through chemokine receptor 6.

Beta-defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6. In order to identify other residues essential for functional interaction with CCR6 we used a library of peptide deletion derivatives based on Defb14. Loss of the initial two amino acids from the Defb14-1C(V) derivative destroys its ability to chemoattract cells expressing CCR6. As the second amino acid is an evolutionarily conserved leucine, we make full-length Defb14-1C(V) peptides with substitution of the leucine(2) for glycine (L2G), lysine (L2K) or isoleucine (L2I). Defb14-1C(V) L2G and L2K and are unable to chemoattract CCR6 expressing cells but the semi-conservative change L2I has activity. By circular dichroism spectroscopy we can see no evidence for a significant change in secondary structure as a consequence of these substitutions and so cannot attribute loss of chemotactic activity with disruption of the N-terminal helix. We conclude that isoleucine/leucine in the N-terminal alpha-helix region of this beta-defensin is essential for CCR6-mediated chemotaxis.

mc1r Pathway regulation of zebrafish melanosome dispersion.

Zebrafish rapidly alter their pigmentation in response to environmental changes. For black melanocytes, this change is due to aggregation or dispersion of melanin in the cell. Dispersion and aggregation are controlled by intracellular cyclic adenosine monophosphate (cAMP) levels, which increase upon stimulation by alpha melanocyte-stimulating hormone (alpha-MSH) or reduce with melanin-concentrating hormone (MCH). In mammals and birds, the melanocortin-1-receptor (MC1R) responds to MSH, and stimulates the synthesis of black eumelanin. While MSH-cAMP signaling stimulates melanogenesis in mammals, and melanosome dispersal in cold-blood vertebrates, the pathway components are highly conserved. However, it has only been assumed that mc1r mediates melanosome dispersal in fish. Here, using morpholino oligonucleotides designed to knockdown mc1r expression, we find that mc1r morphants are unable to disperse melanosomes when grown in dark conditions. We also use chemical modifiers of the cAMP pathway, and find an unexpected response to the specific phosphodiesterase 4 (PDE4) inhibitor, rolipram, in melanosome dispersal. When treated with the drug, melanosomes fail to fully disperse in dark conditions, despite presumed increased levels of cAMP, and in contrast to the effects of the nonselective PDE inhibitor, 3-isobutyl-1-methylxanthine. In conclusion, we demonstrate a direct role for mc1r in zebrafish melanosome dispersal in response to background, and use chemical modification of this pathway to uncover a possible new layer of regulation in melanosome dispersal in zebrafish.

Microcephalin coordinates mitosis in the syncytial Drosophila embryo.

Microcephalin (MCPH1) is mutated in primary microcephaly, an autosomal recessive human disorder of reduced brain size. It encodes a protein with three BRCT domains that has established roles in DNA damage signalling and the cell cycle, regulating chromosome condensation. Significant adaptive evolutionary changes in primate MCPH1 sequence suggest that changes in this gene could have contributed to the evolution of the human brain. To understand the developmental role of microcephalin we have studied its function in Drosophila. We report here that Drosophila MCPH1 is cyclically localised during the cell cycle, co-localising with DNA during interphase, but not with mitotic chromosomes. mcph1 mutant flies have a maternal effect lethal phenotype, due to mitotic arrest occurring in early syncytial cell cycles. Mitotic entry is slowed from the very first mitosis in such embryos, with prolonged prophase and metaphase stages; and frequent premature separation as well as detachment of centrosomes. As a consequence, centrosome and nuclear cycles become uncoordinated, resulting in arrested embryonic development. Phenotypic similarities with abnormal spindle (asp) and centrosomin (cnn) mutants (whose human orthologues are also mutated in primary microcephaly), suggest that further studies in the Drosophila embryo may establish a common developmental and cellular pathway underlying the human primary microcephaly phenotype.

Implementation and case-study results of potentially better practices to improve pain management of neonates.

OBJECTIVE: Collaborative quality improvement techniques were used to facilitate local quality improvement in the management of pain in infants. Several case studies are presented to highlight this process. METHODS: Twelve NICUs in the Neonatal Intensive Care Quality Improvement Collaborative 2002 focused on improving neonatal pain management and sedation practices. These centers developed and implemented evidence-based potentially better practices for pain management and sedation in neonates. The group introduced changes through plan-do-study-act cycles and tracked performance measures throughout the process. RESULTS: Strategies for implementing potentially better practices varied between centers on the basis of local characteristics. Individual centers identified barriers to implementation, developed tools for improvement, and shared their experience with the collaborative. Baseline data from the 12 sites revealed substantial opportunities for improved pain management, and local potentially better practice implementation resulted in measurable improvements in pain management at participating centers. CONCLUSIONS: The use of collaborative quality improvement techniques enhanced local quality improvement efforts and resulted in effective implementation of potentially better practices at participating centers.