Isolation, pharmacology and gene organization of KPSFVRFamide: A neuropeptide from Caenorhabditis elegans

To date, 53 peptides with C-terminal RFamides have been identified by the genome sequencing project in the nematode, Caenorhabditis elegans. In this study the FMRFamide-related peptide (FaRP) KPSFVRFamide (879.90 Da [MH](+)) was structurally characterized from extracts of the nematode, Caenorhabditis elegans. Two copies of KPSFVRFamide are encoded by a gene designated flp-9. RT-PCR identified a single cDNA product which was confirmed as flp-9 by sequence determination. Flp-9 cDNA was isolated from larval stages of C. elegans but was not detected-in adult worms, indicating that its expression is may be developmentally regulated. KPSFVRFamide displays sequence homology to the nematode peptide, KPNFIRFamide (PF4). The physiological effects of KPSFVRFamide, PF4 and the chimeras, KPNFVRFamide and KPSFIRFamide, were measured on body wall muscle and the vagina vera of the parasitic nematode, Ascaris suum. KPNFVRFamide and KPNFIRFamide had Cl--dependent inhibitory activity on innervated and denervated muscle-preparations, whereas KPSFVRFamide and KPSFIRFamide did not elicit a detectable physiological effect. Although all 4 peptides had inhibitory effects on the vagina vera, KPSFVRFamide and KPSFIRFamide (threshold, greater than or equal to 0.1 mu M) were less potent than KPNFVRFamide and KPNFIRFamide (threshold, greater than or equal to 10 nM). (C) 1999 Academic Press.

Comparison of the binding specificity of two bacterial metalloproteases, LasB of Pseudomonas aeruginosa and ZapA of Proteus mirabilis, using N-alpha mercaptoamide template-based inhibitor analogues

The metalloproteases ZapA of Proteus mirabilis and LasB of Pseudomonas aeruginosa are known to be virulence factors their respective opportunistic bacterial pathogens, and are members of the structurally related serralysin and thermolysin families of bacterial metalloproteases respectively. Secreted at the site of infection, these proteases play a key role in the infection process, contributing to tissue destruction and processing of components of the host immune system. Inhibition of these virulence factors may therefore represent an antimicrobial strategy, attenuating the virulence of the infecting pathogen. Previously we have screened a library of N-alpha mercaptoamide dipeptide inhibitors against both ZapA and LasB, with the aim of mapping the S1' binding site of the enzymes, revealing both striking similarities and important differences in their binding preferences. Here we report the design, synthesis, and screening of several inhibitor analogues, based on two parent inhibitors from the original library. The results have allowed for further characterization of the ZapA and LasB active site binding pockets, and have highlighted the possibility for development of broad-spectrum bacterial protease inhibitors, effective against enzymes of the thermolysin and serralysin metalloprotease families.

Pharmacological Activity of Ibuprofen Release from Measoporous Silica

Novel drug delivery systems (DDS) to improve the pharmacokinetic profile of hydrophobic drugs following oral administration are an area of keen interest in drug research. An ideal DDS should not adversely affect drug activity, be capable of delivering a therapeutic dose of drug, and allow homogenous drug loading and drug release. Mesoporous silica has been proposed for this application, with ibuprofen employed as the model drug. It was hypothesised that mesoporous silica MCM-41 is capable of delivering a pharmacologically therapeutic dose of ibuprofen. Ibuprofen-loaded MCM-41 can be prepared reproducibly at a drug to carrier ratio of 30% (wt/wt). The release profile was seen to be 90% within 2 h. Initial assessment of COX-1 inhibitory activity suggests the absence of adverse effects attributable to drug-carrier interaction. The results of this study provide further evidence in support of the proposed use of mesoporous silica in drug delivery.

Terahertz detection of illegal objects

This paper describes the main parameters - contrast, spatial resolution, and thermal sensitivity - which define the performance of any stand-off imaging system. The origin of the signature for both metal and dielectric objects hidden under clothing in the frequency range from 100 GHz to 500 GHz is discussed. At 100 GHz the signature is dominated by reflection whilst at 500 GHz it is dominated by emission. A 94-GHz-passive millimetre-wave imaging system has been designed and fabricated to image objects under clothing. This imager is based on a Schmidt camera folded using polarisation techniques.

Photodynamic Antimicrobial Chemotherapy (PACT) in combination with antibiotics for treatment of Burkholderia cepacia complex infection

This study aimed to determine if Photodynamic Antimicrobial Chemotherapy (PACT) was effective in the treatment of Burkholderia cepacia complex infection and whether a synergistic effect was evident if PACT was used in combination with antibiotics. The susceptibility of both planktonic and biofilm cultures of B. cepacia complex strains to methylene blue (MB) and meso-tetra(n-methyl-4-pyridyl)porphine tetra-tosylate (TMP)-mediated PACT was determined alone and in combination with antibiotics used in the treatment of Cystic Fibrosis pulmonary infection caused by these bacteria. When B. cepacia complex strains were grown planktonically, high levels of kill of were achieved with both TMP and MB-mediated PACT with strain and photosensitizer specific differences apparent. When strains were grown in biofilm, antibiotic treatment alone was bactericidal in 17/36 (47%) strain/antibiotic combinations tested. When antibiotic treatment was combined with PACT, bactericidal activity was apparent for 33/36 (92%) strain/antibiotic combinations. No antagonism was detected between PACT and antibiotic treatment with the combination synergistic for 6/36 (17%) and indifferent for 30/36 (83%) strain/antibiotic combinations. PACT could be a viable treatment option, either alone or in combination with antibiotics for treatment of B. cepacia complex pulmonary infection.

Surface localisation of photosensitisers on intraocular lens biomaterials for prevention of infectious endophthalmitis and retinal protection

Cataract surgery is one of the most commonly-practiced surgical procedures in Western medicine, and, while complications are rare, the most serious is infectious postoperative endophthalmitis. Bacteria may adhere to the implanted intraocular lens (IOL) and subsequent biofilm formation can lead to a chronic, difficult to treat infection. To date, no method to reduce the incidence of infectious endophthalmitis through bacterial elimination, while retaining optical transparency, has been reported. In this study we report a method to optimise the localisation of a cationic porphyrin at the surface of suitable acrylate copolymers, which is the first point of contact with potential pathogens. The porphyrin catalytically generates short-lived singlet oxygen, in the presence of visible light, which kills adherent bacteria indiscriminately. By restricting the photosensitiser to the surface of the biomaterial, reduction in optical transparency is minimised without affecting efficacy of singlet oxygen production. Hydrogel IOL biomaterials incorporating either methacrylic acid (MAA) or methyl methacrylate (MMA) co-monomers allow tuning of the hydrophobic and anionic properties to optimise the localisation of porphyrin. Physiochemical and antimicrobial properties of the materials have been characterised, giving candidate materials with self-generating, persistent anti-infective character against Gram-positive and Gram-negative organisms. Importantly, incorporation of porphyrin can also serve to protect the retina by filtering damaging shortwave visible light, due to the Soret absorption (?max) 430 nm). © 2012 Elsevier Ltd. All rights reserved.

The chains of the heterodimeric amphibian skin antimicrobial peptide, distinctin, are encoded by separate messenger RNAs

Using a primer to a conserved nucleotide sequence of previously-cloned skin peptides of Phyllomedusa species, two distinct cDNAs were “shotgun” cloned from a skin secretion-derived cDNA library of the frog, Phyllomedusa burmeisteri. The two ORFs separately encode chains A and B of an analog of the previously-reported heterodimeric peptide, distinctin. LC-MS/MS analysis of native versus dithiotreitol reduced crude venom, confirmed the predicted primary sequences as well as the cystine link between the two monomers. Distinctin predominantly exists in the venom as a heterodimer (A-B), neither of the constituent peptides were detected as monomer, whereas of the two possible homodimers (A-A or B-B), only B-B was detected in comparatively low quantity. In vitro dimerization of synthetic replicates of the monomers demonstrated that besides heterodimer, both homodimers are also formed in considerable amounts. Distinctin is the first example of an amphibian skin dimeric peptide that is formed by covalent linkage of two chains that are the products of different mRNAs. How this phenomenon occurs in vivo, to exclude significant homodimer formation, is unclear at present but a “favored steric state” type of interaction between chains is most likely.

Development and validation of an ultrasensitive fluorescence planar waveguide biosensor for the detection of paralytic shellfish toxins in marine algae

Marine dinoflagellates of the genera Alexandrium are well known producers of the potent neurotoxic paralytic shellfish toxins that can enter the food web and ultimately present a serious risk to public health in addition to causing huge economic losses. Direct coastal monitoring of Alexandrium spp. can provide early warning of potential shellfish contamination and risks to consumers and so a rapid, sensitive, portable and easy-to-use assay has been developed for this purpose using an innovative planar waveguide device. The disposable planar waveguide is comprised of a transparent substrate onto which an array of toxin-protein conjugates is deposited, assembled in a cartridge allowing the introduction of sample, and detection reagents. The competitive assay format uses a high affinity antibody to paralytic shellfish toxins with a detection signal generated via a fluorescently labelled secondary antibody. The waveguide cartridge is analysed by a simple reader device and results are displayed on a laptop computer. Assay speed has been optimised to enable measurement within 15 min. A rapid, portable sample preparation technique was developed for Alexandrium spp. in seawater to ensure analysis was completed within a short period of time. The assay was validated and the LOD and CCß were determined as 12 pg/mL and 20 pg/mL respectively with an intra-assay CV of 11.3% at the CCß and an average recovery of 106%. The highly innovative assay was proven to accurately detect toxin presence in algae sampled from the US and European waters at an unprecedented cell density of 10 cells/L. © 2012 Elsevier B.V. All rights reserved.

Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis

Introduction: Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic autoimmune diseases with variable clinical outcomes. We investigated whether the synovial fluid (SF) proteome could distinguish a subset of patients in whom disease extends to affect a large number of joints.

Methods: SF samples from 57 patients were obtained around time of initial diagnosis of JIA, labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression verified by immunochemical methods. Protein glycosylation status was confirmed by hydrophilic interaction liquid chromatography.

Results: A truncated isoform of vitamin D binding protein (VDBP) is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p < 0.05). Furthermore, sialylated forms of immunopurified synovial VDBP were significantly reduced in extended oligoarticular patients (p < 0.005).

Conclusion: Reduced conversion of VDBP to a macrophage activation factor may be used to stratify patients to determine risk of disease extension in JIA patients.

KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1α survival pathways

KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1a (HIF-1a). HIF-1a is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1a. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1a in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1a levels in KNK437-treated cells. This suggested that the absence of HIF-1a in hypoxic cells was not due to the enhanced protein degradation. HIF-1a is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1a mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1a levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.