David K. Smith

Effects of the anti-bacterial peptide cecropin B and its analogs, cecropins B-1 and B-2, on liposomes, bacteria, and cancer cells

Custom designed analogs of the natural anti-bacterial peptide cecropin B (CB) have been synthesized; cecropin B-1 (CB-1) was constructed by replacing the C-terminal segment (residues 26 to 35) with the N-terminal sequence of CB (positions 1 to 10 which include five lysine residues). The second analog, CB-2, is identical to CB-1 except for the insertion of a Gly-Pro residue pair between Pro-24 and Ala-25. These peptides were used to investigate their anti-liposome, anti-bacterial and anti-cancer activities. The strength of anti-liposome activity is reduced two- to three-fold when the analogs are used instead of natural CB based on DL50 analysis. Similarly, the potency of these analogs on certain bacteria is about two- to four-fold lower than those of CB based on LC measurements. In contrast, on leukemia cancer cells, the potency of CB-1 and CB-2 is about two- to three-fold greater than that of natural CB based on IC50 measurements. All CB, CB-1 and CB-2 peptides have comparable helix contents according to CD measurements. These results indicate that the designed cationic lytic peptides, having extra cationic residues, are less effective in breaking liposomes and killing bacteria but more effective in lysing cancer cells. The possible interpretations for these observations are discussed.

FoxM1c Counteracts Oxidative Stress-induced Senescence and Stimulates Bmi-1 Expression

The Forkhead box transcription factor FoxM1 is expressed in proliferating cells. When it was depleted in mice and cell lines, cell cycle defects and chromosomal instability resulted. Premature senescence was observed in embryonic fibroblasts derived from FoxM1 knock-out mice, but the underlying cause has remained unclear. To investigate whether FoxM1 can protect cells against stress-induced premature senescence, we established NIH3T3 lines with doxycycline-inducible overexpression of FoxM1c. Treatment of these lines with sublethal doses (20 and 100 μm) of H2O2 induced senescence with senescence-associated β-galactosidase expression and elevated levels of p53 and p21. Induction of FoxM1c expression markedly suppressed senescence and expression of p53 and p21. Consistent with down-regulation of the p19Arf-p53 pathway, p19Arf levels decreased while expression of the Polycomb group protein Bmi-1 was induced. That Bmi-1 is a downstream target of FoxM1c was further supported by the dose-dependent induction of Bmi-1 by FoxM1c at both the protein and mRNA levels, and FoxM1 and Bmi-1 reached maximal levels in cells at the G2/M phase. Depletion of FoxM1 by RNA interference decreased Bmi-1 expression. Using Bmi-1 promoter reporters with wild-type and mutated c-Myc binding sites and short hairpin RNAs targeting c-Myc, we further demonstrated that FoxM1c activated Bmi-1 expression via c-Myc, which was recently reported to be regulated by FoxM1c. Our results reveal a functional link between FoxM1c, c-Myc, and Bmi-1, which are major regulators of tumorigenesis. This link has important implications for the regulation of cell proliferation and senescence by FoxM1 and Bmi-1.

The Dependence of Membrane Permeability by the Antibacterial Peptide Cecropin B and Its Analogs, CB-1 and CB-3, on Liposomes of Different Composition

A natural antibacterial peptide, cecropin B (CB), and designed analogs, CB-1 and CB-3, were synthesized. The three peptides have different structural characteristics, with CB having one hydrophobic and one amphipathic α-helix, CB-1 having two amphipathic α-helices, and CB-3 having two hydrophobic α-helices. These differences were used as the rationale for a study of their efficacy in breaking liposomes with different combinations of phosphatidic acid and phosphatidylcholine. Biosensor binding measurements and encapsulating dye leakage studies showed that the higher binding affinity of CB and CB-1 to the polar heads of lipids is not necessary for the peptides to be more effective at lysing lipid bilayers, especially when liposomes have a higher phosphatidic acid content. Kinetic studies, by intrinsic and extrinsic fluorescence stopped-flow measurements, revealed two transitional steps in liposome breakage by CB and CB-1, although only one kinetic step was found for CB-3. Circular dichroism stopped-flow measurements, monitoring the formation of secondary structure in the peptides, found one kinetic step for the interaction of all of the peptides with the liposomes. Also, the α-helical motif of the peptides was maintained after interacting with the liposomes. Based on these results, the mechanisms of liposome lysis by CB, CB-1, and CB-3 are discussed.

Microscopic observations of the different morphological changes caused by anti-bacterial peptides on Klebsiella pneumoniae and HL-60 leukemia cells

Natural anti‐bacterial peptides cecropin B (CB) and its analogs cecropin B‐1 (CB‐1), cecropin B‐2 (CB‐2) and cecropin B‐3 (CB‐3) were prepared. The different characteristics of these peptides, with amphipathic/hydrophobic α‐helices for CB, amphipathic/amphipathic α‐helices for CB‐1/CB‐2, and hydrophobic/hydrophobic α‐helices for CB‐3, were used to study the morphological changes in the bacterial cell, Klebsiella pneumoniae and the leukemia cancer cell, HL‐60, by scanning and transmission electron microscopies. The natural and analog peptides have comparable secondary structures as shown by circular dichroism measurements. This indicates that the potency of the peptides on cell membranes is dependent of the helical characteristics rather than the helical strength. The microscopic results show that the morphological changes of the cells treated with CB are distinguishably different from those treated with CB‐1/CB‐2, which are designed to have enhanced anti‐cancer properties by having an extra amphipathic α‐helix. The morphological differences may be due to their different modes of action on the cell membranes resulting in the different potencies with lower lethal concentration and higher concentration of 50% inhibition (IC50) of CB on bacterium and cancer cell, respectively, as compared with CB‐1/CB‐2 (Chen et al. 1997. Biochim. Biophys. Acta 1336, 171–179). In contrast, CB‐3 has little effect on either the bacterium or the cancer cell. These results provide microscopic evidence that different killing pathways are involved with the peptides.

Solution structure of the C-terminal domain of the ciliary neurotrophic factor (CNTF) receptor and ligand free associations among components of the CNTF receptor complex.

The functional receptor complex of ciliary neurotrophic factor (CNTF), a member of the gp130 family of cytokines, is composed of CNTF, the CNTF receptor α (CNTFR), gp130, and the leukemia inhibitory factor receptor (LIFR). However, the nature of the receptor-mediated interactions in this complex has not yet been resolved. To address this issue we have determined the solution structure of the C-terminal or BC domain of CNTFR and studied the interactions of CNTFR with LIFR and gp130. We reported previously that the membrane distal cytokine-binding domain (CBD1) of LIFR could interact in vitro with soluble CNTFR (sCNTFR) in the absence of CNTF. Here we show that the CBD of human gp130 can also bind in vitro to sCNTFR in the absence of CNTF. In addition, the gp130 CBD could compete with the LIFR CBD1 for the binding of sCNTFR. Substitution of residues in the gp130 CBD, the LIFR CBD1, and the CNTFR BC domain that are expected to be involved in receptor-receptor interactions significantly reduced their interactions. An NMR chemical shift perturbation study of the interaction between the BC domains of CNTFR and gp130 further mapped the interaction surface. These data suggest that both the gp130 CBD and the LIFR CBD1 interact with CNTFR in a similar way and provide insights into the nature of the CNTF receptor complex.

A major component approach to presenting consensus sequences.

MOTIVATION Summarizing and displaying the information contained in a set of aligned sequences is an important aid to identifying patterns within the sequences. A variety of forms of consensus sequences have been used previously to provide this information. However, these methods can cause a loss of information or introduce ambiguities into the consensus sequence, and some graphical approaches may become difficult to interpret due to visual distortion. RESULTS We have developed a method to present a more precise and graphically clear view of a consensus sequence by using an approach based on defining the major components at each position in a sequence set. The major components are given in an ordered list and their frequencies are shown as histograms which can be colour coded to reflect conservative groupings. Minor components, a one-line character-based consensus sequence and information statistics can also be presented. As well as identifying the dominant sources of variation and conservation in the sequence set, the method also enables similarities and differences between subgroups of a sequence set to be readily assessed. AVAILIABILITY: On request from the authors. CONTACT bcsmith@usthk.ust.hk, hxue@usthk. ust.hk

Morphological differences observed by the different modes of action of the antibacterial peptides, cecropin B and its analogs CB-1, CB-2 and CB-3 on bacterium and cancer cell

Cecropins were originally discovered in the pupae of the cecropia moth (Hyalophora cecropia) [1] and are involved in humoral immune response against intruders such as non-pathogenic bacteria [2]. In addition to the cecropin’s anti-bacterial function, their inability to lyse healthy mammalian cells [3] while being capable of attacking transformed cells [4] may make them potentially useful as peptide anti-cancer drugs (a different strategy to the currently used chemical anti-cancer drugs). In this communication, we focus mainly on the area of morphological changes induced by cecropin B and its analogs, CB-1, CB-2 and CB-3 on the bacterium, Klebsiella Pneumoniae (KP), and the cancer cell, HL-60 leukemia (HL-60).