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A number of disorders and diseases are associated with conditions of high pH, and many conventional antibiotics lose their efficacy under these pH conditions, generating a need for novel antimicrobials. A potential solution to fulfill this need is Antimicrobial Peptides (AMPs) with high pH optima. This review shows that a variety of anionic and cationic AMPs with this pH dependency are produced by creatures across the eukaryotic kingdom, including rabbits, cattle, sheep, fish, crabs and frog. These AMPs exhibit activity against viruses, bacteria, and fungi that involve membrane interactions and appear to be facilitated by a variety of mechanisms that generally promote passage across membranes to attack intracellular targets, such as DNA or protein synthesisand/or membrane lysis. Some of these mechanisms are unknown, but those elucidated include the use of bacterial pores and transporters, the self-promoted uptake pathway, and established models of membrane interaction, such as the carpet mechanism, toroidal pore formation, the adoption of tilted peptide, and the SHM model. A variety of potential roles have been proposed for these AMPs, including use as antivirals, antibacterials, antifungals, adjuvants to antimicrobial therapy, biomarkers of disease, and probes for pathogenic microbes. In this review, these properties are described and discussed, emphasizing the antimicrobial mechanisms used by these AMPs and the pH dependency of these mechanisms.

Anionic antimicrobial peptides (AAMPs) with net charges ranging from -1 to -8 have been identified in frogs, toads, newts and salamanders across Africa, South America and China. Most of these peptides show antibacterial activity and a number of them are multifunctional, variously showing antifungal activity, anticancer action, neuropeptide function and the ability to potentiate conventional antibiotics. Antimicrobial mechanisms proposed for these AAMPs, include toroidal pore formation and the Shai-Huang-Matsazuki model of membrane interaction along with pH dependent amyloidogenesis and membranolysis via tilted peptide formation. The potential for therapeutic and biotechnical application of these AAMPs has been demonstrated, including the development of amyloid-based nanomaterials and antiviral agents. It is concluded that amphibian AAMPs represent an untapped potential source of biologically active agents and merit far greater research interest.

Purpose Up to 25% of chronic hepatitis B (CHB) patients eventually develop hepatocellular carcinoma (HCC), a disease with poor prognosis unless detected early. This study identifies a blood-based RNA biomarker panel for early HCC detection in CHB. Materials and methods A genome-wide RNA expression study was performed using RNA extracted from blood samples from Malaysian patients (matched HCC, CHB, controls). Genes differentiating HCC from controls were selected for further testing using quantitative real-time polymerase chain reaction. Finally, a 6-gene biomarker panel was identified and characterized using a training set (cohort I = 126), and tested against 2 test sets (cohort II = 222; cohort III = 174). The total number of samples used for each group is: HCC + CHB = 143, CHB = 211, control = 168. Results Our gene panel displays a consistent trend distinguishing HCC from controls in our test sets, with an area under receiver-operating characteristic curve of 0.9 in cohort III. Our independent test set (cohort III) showed that the gene panel had a sensitivity of 70% with a specificity of 92%. The biomarker profile for HCC was consistently detected in a small subgroup of CHB patients, thus potentially predicting early, preclinical cases of cancer that should be screened more intensively. Conclusion The biomarkers identified in this study can be used as the basis of a blood-based test for the detection of early HCC in CHB.

• Approximately 2% of all cancers are glioma, themselves the most common primary tumours of the central nervous system.

Host defense peptides show great potential for development as new antimicrobial agents with novel mechanisms of action. However, a small number of resistance mechanisms to their action are known, and here, we report a novel bacterial resistance mechanism mediated by a lipid receptor. Maximin H5 from Bombina maxima bound anionic and zwitterionic membranes with low affinity (Kd225 μM) while showing a strong ability to lyse (55%) and penetrate (π6.0 mN m(-1)) these membranes. However, the peptide bound Escherichia coli and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) membranes with higher affinity (Kd65 μM) and showed a very low ability for bilayer lysis (8%) and partitioning (π1.0 mN m(-1)). Increasing levels of membrane DMPE correlated with enhanced binding by the peptide (R(2) = 0.96) but inversely correlated with its lytic ability (R(2) = 0.98). Taken with molecular dynamic simulations, these results suggest that maximin H5 possesses membranolytic activity, primarily involving bilayer insertion of its strongly hydrophobic N-terminal region. However, this region was predicted to form multiple hydrogen bonds with phosphate and ammonium groups within PE head-groups, which in concert with charge-charge interactions anchor the peptide to the surface of E. coli membranes, inhibiting its membranolytic action.

Fungal infections with multiple resistance to conventional antifungals are increasingly becoming a medical problem, and there is an urgent need for new antifungal compounds with novel mechanisms of action. Here, we show that aurein 2.5, a naturally occurring peptide antibiotic, displays activity against the fungal strains: Rhodotorula rubra and Schizosaccharomyces pombe (MICs 130 μM). The peptide adopted high levels of membrane-interactive α-helical structure ( 65%) in the presence of lipid membranes derived from these organisms and showed strong propensities to penetrate (π ≥ 13 mN m(-1) ) and lyse them ( 70%). Based on these data, we suggest that aurein 2.5 kills yeasts via membranolytic mechanisms and may act as a template for the development of therapeutically useful antifungal agents.

Antimicrobial peptides (AMPs) are potent antibiotics of the innate immune system that have been extensively investigated as a potential solution to the global problem of infectious diseases caused by pathogenic microbes. A group of AMPs that are increasingly being reported are those that utilise pH dependent antimicrobial mechanisms and here, we review research into this area. This review shows that these antimicrobial molecules are produced by a diverse spectrum of creatures, including vertebrates and invertebrates, and are primarily cationic although a number of anionic examples are known. Some of these molecules exhibit high pH optima for their antimicrobial activity but in most cases, these AMPs show activity against microbes that present low pH optima, which reflects the acidic pH generally found at their sites of action, particularly the skin. The modes of action used by these molecules are based on a number of major structure / function relationships, which include metal ion binding, changes to net charge and conformational plasticity, and primarily involve the protonation of histidine, aspartic acid and glutamic acid residues at low pH. The pH dependent activity of pore forming antimicrobial proteins involves mechanisms that generally differ fundamentally to those used by pH dependent AMPs, which can be described by the carpet, toroidal pore and barrel-stave pore models of membrane interaction. A number of pH dependent AMPs and antimicrobial proteins have been developed for medical purposes and have successfully completed clinical trials, including kappacins, LL-37, histatins and lactoferrin, along with a number of their derivatives. Major examples of the therapeutic application of these antimicrobial molecules include wound healing as well as the treatment of multiple cancers and infections due to viruses, bacteria and fungi. In general, these applications involve topical administration, such as the use of mouth washes, cream formulations and hydrogel delivery systems. Nonetheless, many pH dependent AMPs and antimicrobial proteins have yet to be fully characterized and these molecules, as a whole, represent an untapped source of novel biologically active agents that could aid fulfillment of the urgent need for alternatives to conventional antibiotics, helping to avert a return to the pre-antibiotic era.

Maximin H5 (MH5) is an amphibian antimicrobial peptide specifically targeting Staphylococcus aureus. At pH 6, the peptide showed an improved ability to penetrate (ΔΠ = 6.2 mN m(-1)) and lyse (lysis = 48%) Staphylococcus aureus membrane mimics, which incorporated physiological levels of lysylated phosphatidylglycerol (Lys-PG, 60%), compared to that at pH 7 (ΔΠ = 5.6 mN m(-1) and lysis = 40% at pH 7) where levels of Lys-PG are lower (40%). The peptide therefore appears to have optimal function at pH levels known to be optimal for the organism's growth. MH5 killed S. aureus (minimum inhibitory concentration of 90 μM) via membranolytic mechanisms that involved the stabilization of α-helical structure (approximately 45-50%) and showed similarities to the "Carpet" mechanism based on its ability to increase the rigidity (Cs(-1) = 109.94 mN m(-1)) and thermodynamic stability (ΔGmix = -3.0) of physiologically relevant S. aureus membrane mimics at pH 6. On the basis of theoretical analysis, this mechanism might involve the use of a tilted peptide structure, and efficacy was noted to vary inversely with the Lys-PG content of S. aureus membrane mimics for each pH studied (R(2) ∼ 0.97), which led to the suggestion that under biologically relevant conditions, low pH helps mediate Lys-PG-induced resistance in S. aureus to MH5 antibacterial action. The peptide showed a lack of hemolytic activity (2% hemolysis) and merits further investigation as a potential template for development as an antistaphylococcal agent in medically and biotechnically relevant areas.

Exogenous 5-aminolevulinic acid (ALA) is taken up directly by bacteria, yeasts, fungi, and some parasites, which then induces the accumulation of protoporphyrin IX (PPIX). Subsequent light irradiation of PPIX leads to the inactivation of these organisms via photodamage to their cellular structures. ALA uptake and light irradiation of PPIX produced by host cells leads to the inactivation of other parasites, along with some viruses, via the induction of an immune response. ALA-mediated PPIX production by host cells and light irradiation result in the inactivation of other viruses via either the induction of a host cell response or direct photodynamic attack on viral particles. This ALA-mediated production of light-activated PPIX has been extensively used as a form of photodynamic therapy (PDT) and has shown varying levels of efficacy in treating conditions that are associated with microbial infection, ranging from acne and verrucae to leishmaniasis and onychomycosis. However, for the treatment of some of these conditions by ALA-based PDT, the role of an antimicrobial effect has been disputed and in general, the mechanisms by which the technique inactivates microbes are not well understood. In this study, we review current understanding of the antimicrobial mechanisms used by ALA-based PDT and its role in the treatment of microbial infections along with its potential medical and nonmedical applications.

In vitro chemosensitivity testing of short term primary glioma cultures derived from brain biopsies is still in the research phase and has not yet found a place in clinical use. The main reasons for this slow progression are the small amounts of tissue available and the lack of a suitably sensitive assay capable of use in the clinical setting. This study examines whether the MTS and ATP cell survival assays, which determine cytotoxicity via colorimetric and luminescence analysis respectively, could potentially fulfill this role. Primary glioma cultures were tested for chemosensitivity using the MTS and ATP assays and were found to be generally sensitive to cisplatin and paclitaxel but relatively resistant to carmustine and etoposide. For both assays, LD50 values lay in the range 2 - 130 μg/ml but in the vast majority of cases, those obtained by the ATP assay were markedly lower those obtained by the MTS assay. Moreover, at cell numbers less than 2000 in the cases of paclitaxel and carmustine and less than 4500 in the case of cisplatin, these drugs were generally indicated as ineffective against the glioma cultures tested by the MTS assay but effective against these cultures by the ATP assay. These data clearly demonstrate that the ATP assay is more sensitive when estimating small cell numbers generated by primary glioma cultures from brain biopsies and more reliably detects higher kill rates by anticancer drugs. This study also supports the feasibility of using the ATP assay for chemosensitivity testing in a clinical setting.

Cationic defence peptides show high therapeutic potential as antimicrobial and anticancer agents. Some of these peptides carry a C-terminal amide moiety which has been shown to be required for antimicrobial activity. However, whether this is a general requirement or whether C-terminal amidation is required for the anticancer activity of defence peptides is unclear. In response, this study analyses the toxicity of a series of C-terminally amidated defence peptides and their non-amidated isoforms to normal fibroblast cells, a variety of tumour cells and bacterial cells. The toxicities of these peptides to microbial and cancer cells were generally200 microM. Peptides were either unaffected by C-terminal amidation or showed up to 10-fold decreases or increases in efficacy. However, these peptides all showed toxicity to normal fibroblast cells with levels (generally150 microM) that were comparable to those of their antimicrobial and anticancer activities. In contrast to previous claims which have been based on analysis of single amidation events, the results of this study clearly show that the C-terminal amidation of defence peptides has a variable effect on their antimicrobial and anticancer efficacy and no clear effect on their selectivity for these cell types.

Aurein 2.5 (GLFDIVKKVVGAFGSL-NH(2)) is an uncharacterised antimicrobial peptide. At an air/water interface, it exhibited strong surface activity (maximal surface pressure 25mNm(-1)) and molecular areas consistent with the adoption of alpha-helical structure orientated either perpendicular (1.72nm(2)molecule(-1)) or parallel (3.6nm(2)molecule(-1)) to the interface. Aurein 2.5 was strongly antibacterial, exhibiting a minimum inhibitory concentration (MIC) of 30microM against Bacillus subtilis and Escherichia coli. The peptide induced maximal surface pressure changes of 9mNm(-1) and 5mNm(-1), respectively, in monolayers mimicking membranes of these organisms whilst compression isotherm analysis of these monolayers showed DeltaG(Mix)0, indicating destabilisation by Aurein 2.5. These combined data suggested that toxicity of the peptide to these organisms may involve membrane invasion via the use of oblique orientated alpha-helical structure. The peptide induced strong, comparable maximal surface changes in monolayers of DOPG (7.5mNm(-1)) and DOPE monolayers (6mNm(-1)) suggesting that the membrane interactions of Aurein 2.5 were driven by amphiphilicity rather than electrostatic interaction. Based on these data, it was suggested that the differing ability of Aurein 2.5 to insert into membranes of B. subtilis and E. coli was probably related to membrane-based factors such as differences in lipid packing characteristics. The peptide was active against both sessile E. coli and Staphylococcus aureus with an MIC of 125microM. The broad-spectrum antibacterial activity and non-specific modes of membrane action used by Aurein 2.5 suggested use as an anti-biofilm agent such as in the decontamination of medical devices.

Currently, antimicrobial drug resistance is a global problem that threatens to precipitate a ‘Post-antibiotic era’ in which the ability of common infections and minor injuries to kill is a very real possibility. A potential solution to this problem is the development of host defence peptides, which are endogenous antibiotics that kill microbes via membranolytic action, based in part on the belief that microbes were unlikely to develop resistance to this action. However, the incidence of microbes exhibiting resistance to the action of host defence peptides is growing and an increasingly diverse spectrum of mechanisms is being reported to underpin this resistance. These mechanisms can be broadly categorized as those that either: destroy these peptides, such as through the production of bacterial proteases; intercept/shield these peptides, such as by the release of host cell proteoglycans by bacterial enzymes; or export these peptides, such as via the use of bacterial efflux pumps. Here we give an overview of these mechanisms, with a focus on recent developments in this area, and then discuss the potential of inhibitors of these resistance mechanisms to treat infections due to bacterial pathogens.

The barley yellow dwarf virus movement protein (BYDV-MP) requires its N-terminal sequence to promote the transport of viral RNA into the nuclear compartment of host plant cells. Here, graphical analysis predicts that this sequence would form a membrane interactive amphiphilic α-helix. Confirming this prediction, NT1, a peptide homologue of the BYDV-MP N-terminal sequence, was found to be α-helical (65%) in the presence of vesicles mimics of the nuclear membrane. The peptide increased the fluidity of these nuclear membrane mimics (rise in wavenumber of circa 0.5–1.0 cm −1 ) and induced surface pressure changes of 2 mN m −1 in lipid monolayers with corresponding compositions. Taken with isotherm analysis these results suggest that BYDV-MP forms an N-terminal amphiphilic α-helix, which partitions into the nuclear membrane primarily through thermodynamically stable associations with the membrane lipid headgroup region. We speculate that these associations may play a role in targeting of the nuclear membrane by BYDM-MP.

Cancer is a major cause of premature death and there is an urgent need for new anticancer agents with novel mechanisms of action. Here we review recent studies on a group of peptides that show much promise in this regard, exemplified by arthropod cecropins and amphibian magainins and aureins. These molecules are alpha-helical defence peptides, which show potent anticancer activity (alpha-ACPs) in addition to their established roles as antimicrobial factors and modulators of innate immune systems. Generally, alpha-ACPs exhibit selectivity for cancer and microbial cells primarily due to their elevated levels of negative membrane surface charge as compared to non-cancerous eukaryotic cells. The anticancer activity of alpha-ACPs normally occurs at micromolar levels but is not accompanied by significant levels of haemolysis or toxicity to other mammalian cells. Structure/function studies have established that architectural features of alpha-ACPs such as amphiphilicty levels and hydrophobic arc size are of major importance to the ability of these peptides to invade cancer cell membranes. In the vast majority of cases the mechanisms underlying such killing involves disruption of mitochondrial membrane integrity and/or that of the plasma membrane of the target tumour cells. Moreover, these mechanisms do not appear to proceed via receptor-mediated routes but are thought to be effected in most cases by the carpet/toroidal pore model and variants. Usually, these membrane interactions lead to loss of membrane integrity and cell death utilising apoptic and necrotic pathways. It is concluded that that alpha-ACPs are major contenders in the search for new anticancer drugs, underlined by the fact that a number of these peptides have been patented in this capacity.

Atherosclerotic coronary heart disease and other forms of cardiovascular disease (CVD) are the major cause of mortality in type II diabetes (T2DM) as well as a major contributor to morbidity and lifetime costs. The purpose of this article is the identification of the biochemical parameters in plasma, which may serve as predisposition factors to CVD in T2DM patients of different ages. The variability of hyperglycemia, dyslipidemia, and inflammation with age progression was also studied for comparison. Four different diabetic groups allocated on the basis of the subjects' age (Group A: 15-25 years old; Group B: 26-40 years old; Group C: 40-60 years old; Group D: 60-80 years old) and consisting of 10 patients each, in parallel with 10 healthy controls matched for age, sex, and ethnic origin were screened for glucose, insulin, lipid profile (total cholesterol, triglycerides, LDL, and HDL), and inflammatory mediators (CRP, IL-6, and TNF-alpha). Significant differences were observed among the expressions of biochemical markers among different age groups. Hyperglycemia showed no variability with age whereas dyslipidemia correlated positively with age progression, as well as obesity, low physical activity, and family history of heart disease or diabetes. Marked inflammation was prominent only in Groups C and D. This article indicates that different biochemical parameters may be used for the assessment of CVD risk in T2DM patients of variable age.

Phenothiazinium based photosensitisers (PhBPs) possess planar heteroaromatic ring structures that give the parent molecules photosensitising properties. PhBPs show potential application in photodynamic therapy (PDT) as antitumour agents, and in photodynamic chemotherapy (PACT) as antimicrobial compounds. PhBPs show selectivity for tumour and microbial cells, which appears to be based on electrostatic interactions between the positive charge generally carried by these molecules and the negative charge found on the outer surface of these target cells. In some cases, a site of action for photoactivated PhBPs is the outer membrane / envelope of the target cell. Such action can involve the modification of membrane lipid and / or lipopolysaccharide, and the inactivation of essential proteins and enzymes, with these effects usually leading to cell lysis and death. However, more often, PhBPs are internalised by target cells, promoted by a variety of factors, including low pH and enzymatic reduction, and upon photoactivation, internalised, PhBPs are able to inflict damage on a number of intracellular targets. In tumour cells, PhBPs can photodamage DNA and the membranes of organelles, thereby inducing necrosis and / or apoptosis. In bacterial cells, whilst DNA is generally a primary target of PhBPs, these compounds can exhibit multiple sites of action within a given cell and show different sites of action between different bacterial species. This variable targeting makes PhBPs attractive propositions as alternatives to conventional antibiotics in that the emergence of bacterial strains with acquired resistance to these compounds appears to be highly unlikely.

"Comet assay" showed light activated (3.15 Jcm-2 over 30 min) phenothiazinium based photosensitisers (PhBPs) to induce photo-damage of Staphylococcus aureus DNA, as indicated by DNA "tails" between 80 and 120 microm. In general, PhBPs exhibited significant singlet oxygen yields (Phi(DeltaPhBP)0.7), suggesting the use of type II mechanisms of photo-oxidation. However, the photodynamic action of PhBPs on DNA showed generally insignificant production of 7,8-dihydro-8-oxo-2'-deoxyguanosine, normally a major product of type II DNA photo-oxidation. These combined results show DNA to be a major site of action of PhBPs and suggest that this action may involve type II attack on a nucleoside(s) other than guanosine.

This study investigated the effects of streptozotoci n (STZ) -induced diabetes mellitus (DM) on protein and cation levels in ocular tissues (lenses, cornea, lacrimal glands and retina with sclera) of rats. Diabetic rats and their lacrimal glands weighed significantly less (p < 0.05) than age -matched controls. Diabetic animals also had significantly (p < 0.05) elevated blood glucose and significantly reduced (p < 0.05) plasma insulin compared to controls. Total protein concentrations in the cornea, lens, lacrimal gland and retina with sclera were markedly reduced compared to controls (circa 50%-90%). Diabetic cornea, lenses, lacrimal glands, and retina with sclera contained more Ca 2+ (p < 0.05) than age-matched controls (2-3 fold). Levels of Zn 2+ were also elevated (p < 0.05) in the cornea and retina with sclera of diabetic rats, as compared to control rats (2-3 fold), but were unaffected in lenses and lacrimal glands. In contrast, levels of Cu 2+ , Mg 2+ , Na + and K + were significantly reduced (p < 0.05) in all ocular tissues of diabetic rats when compared to control animals (circa 30%-70%). These results show that STZ-induced DM is generally associated with significant physiochemical changes in ocular tissues of rats with changes observed in body weight, blood glucose, and insulin levels and protein and cation concentrations compared to healthy agematched controls. Based on these data, it has been speculated that diabetes may induce changes in ocular tissues that include: higher protein turnover through increased protease activity and changes in Na + / K + channel function. It is suggested that these changes may be associated with diabetic retinopathy, diabetic cornea and sight impairment. (Int J Diabetes Metab 13:154-158, 2005)

Calpain inhibitors show the potential to serve as non-surgical alternatives in treating diabetic cataract and other types of these disorders. Here, we have tested the recently developed calpain inhibitor, SJA6017, for its ability to inhibit cataractogenesis in porcine lenses. These lenses were incubated in increasing levels of extralenticular calcium (Ca2+; 5–30 mM). Atomic absorption spectroscopy was used to determine total internal lens Ca2+ and a correlation between porcine lens Ca2+ uptake and levels of lens opacification were found with a total internal lens Ca2+ level of 5.8 μM Ca2+ g−1 wet lens weight corresponding to the onset of catarctogenesis. A total internal lens Ca2+ level of 8.0 μM Ca2+ g−1 wet lens weight corresponded to cataract occupying approximately 70% of the lens cell volume. This degree of cataract was reduced by approximately 40%, when SJA6017 (final concentration 0.8 μM) was included in the extralenticular medium, suggesting that the Ca2+-mediated activation of calpains may be involved in the observed opacification. Supporting this suggestion atomic absorption spectroscopy showed that the effect of SJA6017 (final concentration 0.8 μM) on lens opacification was not due to the compound restricting porcine lens Ca2+ uptake. The results indicate that calpain-induced cataractogenesis is dependent on extracellular Ca2+ and the calpain inhibitor SJA6017 (0.8 μM) had no significant effect on Ca2+ uptake by lens. Its inhibitory effect on lens opacification may be due to a direct action on the activity of calpain. (Mol Cell Biochem 261: 169–173, 2004)

There is emerging evidence to suggest that the unregulated Ca(2+)-mediated proteolysis of essential lens proteins by calpains might be a major contributor to some forms of cataract in both animals and humans. Moreover, recently solved calpain structures have revealed molecular-level details of the activation mechanism used by these proteases, enabling the structure-based design of potent calpain inhibitors with the potential to act as anti-cataract agents. These agents offer the first real hope of an urgently needed alternative to the surgical treatment of at least some forms of cataract and relief from a life-depreciating condition on a global scale.

Phenothiazinium dyes, and derivatives, were tested for toxicity to Escherichia coli and Staphylococcus aureus. The dyes were generally lipophilic (log P1) and showed inherent dark toxicity (minimum lethal concentrations: 3.1-1000 microM). Dye illumination (total light dose of 3.15 J cm(-1) over 30 min) led to up to eight-fold reductions in minimum lethal concentrations. Most of the illuminated dyes showed significant relative singlet oxygen yields (phi'delta: 0.18-1.35) suggesting a type II mechanism of generating a phototoxic response. Although generally up to six-fold more effective against S. aureus, the dyes tested efficiently killed E. coli and may be of particular use in combating Gram-negative pathogens.

Escherichia coli low molecular mass penicillin-binding proteins (PBPs) include PBP4, PBP5, PBP6 and PBP6b. Evidence suggests that these proteins interact with the inner membrane via C-terminal amphiphilic α-helices. Nonetheless, the membrane interactive mechanisms utilized by the C-terminal anchors of PBP4 and PBP6b show differences to those utilized by PBP5 and PBP6. Here, hydrophobic moment-based analyses have predicted that, in contrast to the PBP4 and PBP6b C-termini, those of PBP5 and PBP6 are candidates to form oblique orientated α-helices. Consistent with these predictions, Fourier transform infrared spectroscopy (FTIR) has shown that peptide homologs of the PBP4 and PBP5 C-terminal regions, P4 and P5, respectively, both possessed the ability to adopt α-helical structure in the presence of lipid. However, whereas P4 appeared to show a preference for interaction with the surface regions of dimyristoylglycerophosphoethanolamine and dimyristoylglycerophosphoglycerol membranes, P5 appeared to show deep penetration of both these latter membranes and dimyristoylglycerophosphocholine membranes. Based on these results, we have suggested that in contrast to the membrane anchoring of the PBP4 and PBP6b C-terminal α-helices, the PBP5 and PBP6 C-terminal α-helices may possess hydrophobicity gradients and penetrate membranes in an oblique orientation.

The protease, m‐calpain, has been implicated in a number of pathological conditions. The enzyme is a calcium‐dependent heterodimer whose activity appears to be modulated by membrane interaction involving a segment, TAMRIL, located in domain V of the protein's small subunit. Based on a sequence analysis of m‐calpain, using DWIH and hydrophobic moment plot based methodologies, we have shown that this segment may contribute to a lipid interactive, oblique orientated, α‐helical region. Our results could form a basis for future studies on the postulated lipid modulation of m‐calpain activity.

The Escherichia coli low molecular mass penicillin-binding proteins (PBP4, PBP5 and PBP6) are a group of penicillin-sensitive enzymes involved in the final stages of cell wall assembly. It has been suggested that these proteins may interact with the periplasmic face of the inner membrane via C-terminal amphiphilic alpha-helices. Theoretical analysis has predicted that these C-terminal helical regions may be membrane interactive. We have tested this hypothesis by assaying PBP C-terminal homologues (P4, P5 and P6) for haemolytic activity. Our results show that the PBP5 and PBP6 C-terminal homologues readily lyse sheep erythrocytes in a pH-dependent manner with LD50's of 3.5 x 10(-6) M and 6.8 x 10(-7) M respectively at pH 7. These results appear to support the present model for the membrane anchoring of PBP5 and PBP6. The PBP4 C-terminal homologue shows no evidence of haemolytic activity which could imply a different means of membrane association for PBP4.

A series of related 2-arylindoles have been evaluated for their anticancer activity against a range of glioblastoma cell lines using a number of different cell-based assays to determine cell viability after treatment with the compounds. The best indoles, which showed comparable activity to cisplatin against a U87MG cell line in the MTS assay, were taken forward and initial studies suggest that their mechanism of action is consistent with the generation of reactive oxygen species followed by autophagic cell death. Furthermore, activity was also observed in glioblastoma short-term cell cultures for the best lead compound and in some cases gave low micromolar IC50s.

Host defense peptides (HDPs) are components of the innate immune system with activity against a broad range of microbes. In some cases, it appears that this activity is mediated by the ability of these peptides to permeabilize microbial membranes via the formation of amyloid associated structures. Recent evidence suggests that the naturally occurring function of the Aβ40 and Aβ42 peptides, which are causative agents of Alzheimer's disease, may be to serve as amyloidogenic HDPs. Here, it is hypothesized that the neurotoxicity of these peptides is related to aberrant use of their amyloid-mediated antimicrobial mechanisms, which provides the as yet unexplored paradigm of a relationship among HDPs, neurodegenerative disorders, and other conditions that could contribute to their understanding and remediation.—Harris, F., Dennison, S. R., Phoenix, D. A. Aberrant action of amyloidogenic host defense peptides: a new paradigm to investigate neurodegenerative disorders?

Theoretical analysis indicates that peptide VP1 forms a membrane interactive amphiphilic alpha-helix with antibacterial properties. Fourier transform infra-red based analyses showed VP1 to be alpha-helical (45%) in the presence of vesicle mimics of membranes from Staphylococcus aureus and to induce increases in the fluidity of these vesicles, as indicated by a rise in wavenumber of circa 0.5 to 1.0 cm(-1). The peptide induced surface pressure increases of 5 mN m(-1) in monolayer mimics of S. aureus membranes confirm the formation of a membrane interactive alpha-helix. These interactions appeared to involve significant hydrophobic and electrostatic contributions as VP1 induced comparable surface pressure changes in anionic (5.5 mN m(-1)) and zwitterionic (4 mN m(-1)) lipid monolayers. It is suggested that whilst efficacy requires further sequence specific information, the peptides generic structure provides the basis for its broad antimicrobial activity.

Type 2 diabetes mellitus (T2DM) can lead to death without treatment and it has been predicted that the condition will affect 215 million people worldwide by 2010. T2DM is a multifactorial disorder whose precise genetic causes and biochemical defects have not been fully elucidated, but at both levels, calpains appear to play a role. Positional cloning studies mapped T2DM susceptibility to CAPN10, the gene encoding the intracellular cysteine protease, calpain 10. Further studies have shown a number of noncoding polymorphisms in CAPN10 to be functionally associated with T2DM while the identification of coding polymorphisms, suggested that mutant calpain 10 proteins may also contribute to the disease. Here we review recent studies, which in addition to the latter enzyme, have linked calpain 5, calpain 3, and its splice variants, calpain 2 and calpain 1 to T2DM-related metabolic pathways along with T2DM-associated phenotypes, such as obesity and impaired insulin secretion, and T2DM-related complications, such as epithelial dysfunction and diabetic cataract.

The antimicrobial activity of the anionic peptide, AP1 (GEQGALAQFGEWL), was investigated. AP1 was found to kill Staphylococcus aureus with an MLC of 3mM and to induce maximal surface pressure changes of 3.8 mN m(-1) over 1200s in monolayers formed from lipid extract of S. aureus membranes. FTIR spectroscopy showed the peptide to be alpha-helical (100%) in the presence of vesicles formed from this lipid extract and to induce increases in their fluidity (Deltanu circa 0.5 cm(-1)). These combined data show that AP1 is able to function as an alpha-helical antimicrobial peptide against Gram-positive bacteria and suggest that the killing mechanism used by the peptide involves interactions with the membrane lipid headgroup region. Moreover, this killing mechanism differs strongly from that previously reported for AP1 against Gram-negative bacteria, indicating the importance of considering the effects of membrane lipid composition when investigating the structure/function relationships of antimicrobial peptides.

The ability of phenothiazinium-based photosensitizers to induce photodamage to Escherichia coli membranes is investigated. Phenothiazinium-based photosensitizers were found to be somewhat lipophilic (log P>0.7) and to induce surface-pressure changes (3-12 mN m(-1)) in lipid monolayers mimetic of bacterial membranes, implying that these molecules are able to penetrate biological membranes. Under dark and light conditions (3.15 J cm(-1) for 30 min), phenothiazinium-based photosensitizers were incubated with E. coli cells. These cells showed levels of dark bacteriolysis that ranged between 6% and 13%, with light conditions leading to no significant increase in these levels. Gas chromatography-based analyses showed such incubations to produce no significant changes in the levels of C(16) and C(18) fatty acid chain saturation found in E. coli whole lipid-extracts. It is concluded that the phenothiazinium-based photosensitizers studied may not use E. coli membranes as their primary photodynamic target, but may inflict photodamage on cytoplasmic targets, possibly DNA.

Phenothiazinium-based molecules exhibit phototoxicity against a broad range of bacteria. In general, these photosensitizers use several cellular uptake pathways, coupled to type II mechanisms of photo-oxidation, to inflict bacterial damage. These molecules show potential to act as novel alternatives to conventional antibiotics.

Escherichia coli penicillin-binding protein 5 (PBP5) is anchored to the periplasmic face of the inner membrane via a C-terminal amphiphilic alpha-helix. The results of washing experiments have suggested an electrostatic contribution to the anchoring mechanism which may involve the cationic region of the C-terminal alpha-helix. Similarities between this anchor domain and some surface active agents, such as melittin, suggest that the cationic region of the PBP5 anchor may require the presence of anionic phospholipids for membrane interaction. Washing experiments performed on membranes of HDL11, an E. coli mutant in which the expression of the major anionic phospholipids is under lac control, found no such requirement. The results are discussed in relation to the hypothesis that the cationic region may interact with other sources of negative charge, possibly arising from a PBP complex.