Structural and functional studies of bacterial protein tyrosine kinases

While protein tyrosine kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. Studies of close to 20 homologs of bacterial protein tyrosine (BY) kinases have inaugurated a blooming new field of research, all since just the end of the last decade. These kinases are key regulators in the polymerization and exportation of the virulence-determining polysaccharides which shield the bacterial from the non-specific defenses of the host. This research is aimed at furthering our understanding of the BY kinases through the use of X-ray crystallography and various in vitro and in vivo experiments. We reported the first crystal structure of a bacterial PTK, the C-terminal kinase domain of E. coli tyrosine kinase (Etk) at 2.5Å resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting evidences, we proposed a unique activation mechanism for BY kinases in Gram-negative bacteria. The phosphorylation of tyrosine residue Y574 at the active site and the specific interaction of P-Y574 with a previously unidentified key arginine residue, R614, unblock the Etk active site and activate the kinase. Both in vitro kinase activity and in vivo antibiotics resistance studies utilizing structure-guided mutants further support the novel activation mechanism. In addition, the level of phosphorylation of their C-terminal Tyr cluster is known to regulate the translocation of extracellular polysaccharides. Our studies have significantly clarified our understanding of how the phosphorylation status on the C-terminal tyrosine cluster of BY kinases affects the oligomerization state of the protein, which is likely the machinery of polysaccharide export regulation. In summary, this research makes a substantial contribution to the rapidly progressing research of bacterial tyrosine kinases.

A polysaccharide extracted from sphagnum moss as antifungal agent in archaeological conservation

On the basis of the well-known preservative properties of Sphagnum moss, a potential opportunity to use moss polysaccharides (Sphagnan) in art conservation was tested. Polysaccharides were extracted from the moss (S. palustre spp.) in the amount of 4.1% of the Sphagnum plant dry weight. All lignocelluloses were removed from this extract as a result of the treatment of the moss cellulose with sodium chlorite. The extracted polysaccharide possessed a strong acidic reaction (pH 2.8) and was soluble in water and organic solvents. The extract was tested on laboratory bacterial cultures by the disk-diffusion method. The antibacterial effect was demonstrated for E. coli and P. aeruginosa (both gram-negative) while Staphylococcus aurelus (gram-positive) was shown to be insensitive to Sphagnum polysaccharides. The antifungal effect of Sphagnum extract was tested by the disk-diffusion method on the spores of seventeen fungal species. These fungi were isolated from ethnographic museum objects and from archaeological objects excavated in the Arctic. Twelve of these isolates appeared susceptible to the extract. The inhibiting effect of the extract was also tested by the modified broth-dilution method on the most typical isolate (Aspergillus spp.). In this experiment, in one ml of the nutritious broth, 40µl of 3% solution of polysaccharides in water killed 10,000 fungal spores in 6 hours. The inhibiting effect was not connected to the acidity or osmotic effect of Sphagnum polysaccharides. As an example of the application of Sphagnum polysaccharides in art conservation, they were added as preservative agents to conservation waxes. After three weeks of exposure of microcrystalline wax to test fungi (Aspergillus spp.), 44% of wax was consumed. When, however, ~ 0.1% (w/w) of Sphagnum extract was mixed with wax, the weight loss of wax was only 4% in the same time interval. On the basis of this study it was concluded that Sphagnum moss and Sphagnum products can be recommended for use in art conservation as antifungal agents.

Amphibian skin peptides and their corresponding cDNAs from single lyophilized secretion samples: identification of novel brevinins from three species of Chinese frogs

Brevinins are peptides of 24 amino acid residues, originally isolated from the skin of the Oriental frog, Rana brevipoda porsa, by nature of their microbicidal activity against a wide range of Gram-positive and Gram-negative bacteria and against strains of pathogenic fungi. cDNA libraries were constructed from lyophilized skin secretion of three, unstudied species of Chinese frog, Odorrana schmackeri, Odorrana versabilis and Pelophylax plancyi fukienensis, using our recently developed technique. In this report, we describe the “shotgun” cloning of novel brevinins by means of 3'-RACE, using a “universal” degenerate primer directed towards a highly conserved nucleic acid sequence domain within the 5'-untranslated region of previously characterized frog skin peptide cDNAs. Novel brevinins, deduced from cloned cDNA open-reading frames, were subsequently identified as mature peptides in the same samples of respective species skin secretions. Bioinformatic analysis of both prepro-brevinin nucleic acid sequences and translated open-reading frame amino acid sequences revealed a highly conserved signal peptide domain and a hypervariable anti-microbial peptide-encoding domain. The experimental approach described here can thus rapidly provide robust structural data on skin anti-microbial peptides without harming the donor amphibians.

Scientifi c discourses on chronic pain without organic cause Incorporating gender perspective for pain resignifyng-repoliticizing

Chronic pain, without any organic or physical cause (DC), which in psycho-medical terminology is known as fi bromyalgia, (FM), is diagnosed each year to a considerable number of women in capitalistic societies. Our main interest in the following paper is to go in depth in the elaboration of this symptom, its treatment and the psychosocial effects, both in the social order as well as in the lives of the people who suffer from it. Our main goal in the following paper is to look deeper in the elaboration (conceptualization) of this symptom, its treatment and psychological affects, both in the social order as well as in the lives of the people who suffer from it, we are using linked speeches in Spanish magazines publications. The result has been the emergence of three hegemonic discourse positions: One position “scientist”, one “therapeutic of the conformity” position and one “economic and legalistic” position. Each of these has a specifi c feature, but on the whole, is enhanced, producing effects such as the absence of social context to explain the disease; disregard of gender differences in the management and treatment; the instrumentalization of pain to legitimize their practices and the subjection of women to the “psycho-biomedical” paradigm. In that way, a new signifi cance and politicization of the concept of pain is proposed.

Reduced sperm yield from testicular biopsies of vasectomized men is due to increased apoptosis

Objective: To compare sperm yields, apoptotic indices, and sperm DNA fragmentation from vasectomized men and fertile men undergoing vasectomy.

Design: Testicular biopsies from vasectomized (n 26) and fertile men (n 46), were milked to calculate sperm/gram and also formalin-?xed to determine the numbers of developing sperm and incidence and intensities of testicular FasL, Fas, Bax, and Bcl-2. Testicular sperm DNA fragmentation was assessed using the alkaline Comet assay.

Setting: An ART unit.

Patient(s): Twenty-six men attending for intracytoplasmic sperm injection (ICSI) and 46 men attending for vasectomies.

Main Outcome Measure(s): Spermatocyte, spermatid and sperm yields, Fas, FasL, and Bax staining.

Result(s): Sperm yields from men vasectomized 5 years previously were markedly reduced compared to fertile men. Increased intensities of FasL and Bax staining were observed in the seminiferous tubules of vasectomy men. FasL positivity (percentage) also increased in Sertoli cells, and both FasL and Fas positivity (percentage) increased in primary spermatocytes and round spermatids of vasectomized men. Sperm DNA fragmentation, an end point marker of apoptosis, increased signi?cantly in vasectomized men compared to fertile men.

Conclusion(s): Reduced sperm yields after vasectomy are associated with increased apoptosis through the Fas–FasL and Bax pathways. Sperm after vasectomy displayed increased DNA fragmentation. (Fertil Steril 2007;87:834–41. ©2007 by American Society for Reproductive Medicine.)

Inactivation of Human beta-Defensins 2 and 3 by Elastolytic Cathepsins

beta-Defensins are antimicrobial peptides that contribute to the innate immune responses of eukaryotes. At least three defensins, human beta-defensins 1, 2, and 3 (HBD-1, -2, and -3), are produced by epithelial cells lining the respiratory tract and are active toward Gram-positive (HBD-3) and Gram-negative (HBD-1, -2, and -3) bacteria. It has been postulated that the antimicrobial activity of defensins is compromised by changes in airway surface liquid composition in lungs of patients with cystic fibrosis (CF), therefore contributing to the bacterial colonization of the lung by Pseudomonas and other bacteria in CF. In this report we demonstrate that HBD-2 and HBD-3 are susceptible to degradation and inactivation by the cysteine proteases cathepsins B, L, and S. In addition, we show that all three cathepsins are present and active in CF bronchoalveolar lavage. Incubation of HBD-2 and -3 with CF bronchoalveolar lavage leads to their degradation, which can be completely (HBD-2) or partially (HBD-3) inhibited by a cathepsin inhibitor. These results suggest that beta-defensins are susceptible to degradation and inactivation by host proteases, which may be important in the regulation of beta-defensin activity. In chronic lung diseases associated with infection, overexpression of cathepsins may lead to increased degradation of HBD-2 and -3, thereby favoring bacterial infection and colonization.

Functional domains of the human epididymal protease inhibitor, eppin

Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram-negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin’s domains are involved in the protein’s antibacterial activity, only the Kunitz domain is required for selective protease inhibition.

Textile wastewater treatment using granular activated carbon adsorption in fixed beds

This work involved the treatment of industrial wastewater from a nylon carpet printing plant which currently receives no treatment and is discharged to sea. As nylon is particularly difficult to dye, acid dyes are required for successful coloration and cause major problems with the plant's effluent disposal in terms of color removal. Granular activated carbon Filtrasorb 400 was used to treat a ternary solution of acid dyes and the process plant effluent containing the dyes in a fixed-bed column system. Experimental data were correlated using the bed depth service time (BDST) model to previously published work by the authors for single dye adsorption. The results were expressed in terms of the BDST adsorption capacity, in milligrams of adsorbate per gram of adsorbent, and indicated that there was a 12-25% decrease iri adsorption capacity in the ternary system compared to the single component system; This reduction has been attributed to competitive adsorption occurring in the ternary component system. Dye adsorption from the process plant effluent showed an approximate 65% decrease in adsorption capacity compared to the ternary solution system. This has been attributed to interference caused by the other colorless textile effluent pollutants found in the process wastewater. A chemical oxygen demand analysis on these components indicated that the dyes accounted for only 14% of the total oxygen demand.

Multi-Study Assessment of Vaginal Cytokines and Microflora in the Safety Evaluation of Intravaginal Rings in Pig-Tailed Macaques

BACKGROUND: HIV microbicide trials have emphasized the need to evaluate the safety of topical microbicides and delivery platforms in an animal model prior to conducting clinical efficacy trials. An ideal delivery device should provide sustainable and sufficient concentrations of effective products to prevent HIV transmission while not increasing transmission risk by either local mucosal inflammation and/or disruption of the normal vaginal microflora.

METHODS: Safety analyses of macaque-sized elastomeric silicone and polyurethane intravaginal rings (IVRs) loaded with candidate antiretroviral (ARV) drugs were tested in four studies ranging in duration from 49 to 73 days with retention of the IVR being 28 days in each study. Macaques were assigned to 3 groups; blank IVR, ARV-loaded IVR, and naïve. In sequential studies, the same macaques were used but rotated into different groups. Mucosal and systemic levels of cytokines were measured from vaginal fluids and plasma, respectively, using multiplex technology. Changes in vaginal microflora were also monitored. Statistical analysis (Mann-Whitney test) was used to compare data between two groups of unpaired samples (with and without IVR, and IVR with and without ARV) for the groups collectively, and also for individual macaques.

RESULTS: There were few statistically significant differences in mucosal and systemic cytokine levels measured longitudinally when the ring was present or absent, with or without ARVs. Of the 8 proinflammatory cytokines assayed a significant increase (p = 0.015) was only observed for IL8 in plasma with the blank and ARV loaded IVR (median of 9.2 vs. 5.7 pg/ml in the absence of IVR). There were no significant differences in the prevalence of H2O2-producing lactobacilli or viridans streptococci, or other microorganisms indicative of healthy vaginal microflora. However, there was an increase in the number of anaerobic gram negative rods in the presence of the IVR (p= < 0.0001).

CONCLUSIONS: IVRs with or without ARVs neither significantly induce the majority of potentially harmful proinflammatory cytokines locally or systemically, nor alter the lactobacillus or G. vaginalis levels. The increase in anaerobic gram negative rods alone suggests minimal disruption of normal vaginal microflora. The use of IVRs as a long-term sustained delivery device for ARVs is promising and preclinical studies to demonstrate the prevention of transmission in the HIV/SHIV nonhuman primate model should continue.

Comparison of techniques to examine the diversity of fungi in adult patients with cystic fibrosis

This study compares conventional and molecular techniques for the detection of fungi in 77 adult cystic fibrosis (CF) patients. Three different methods were investigated, i.e., (1) conventional microbiological culture (including yeasts and filamentous fungi), (2) mycological culture with CF-derived fungal specific culture media, and (3) Non-culture and direct DNA extraction from patient sputa. Fungi isolated from environmental air samples of the CF unit were compared to fungi in sputa from CF patients. Fungi (n = 107) were detected in 14/77(18%) of patients by method 1, in 60/77 (78%) of patients by method 2 and with method 3, in 77/77(100%) of the patients. The majority of yeasts isolated were Candida albicans and C. dubliniensis. Exophiala (Wangiella) dermatitidis, Scedosporiumapiospermum, Penicillium spp., Aspergillus fumigatus, and Aspergillus versicolor were also identified by sequence analysis of the rDNA short internal transcribed spacer (ITS2) region. Conventional laboratory analysis failed to detect fungi in 63 patients mainly due to overgrowth by Gram-negative organisms. Mycological culture with antibiotics dramatically increased the number of fungi that could be detected. Molecular techniques detected fungi such as Saccharomyces cerevisiae, Malassezia spp., Fuscoporia ferrea, Fusarium culmorum, Acremonium strictum, Thanatephorus cucumeris and Cladosporium spp. which were not found with other methods. This study demonstrates that several potentially important fungi may not be detected if mycological culture methods alone are used. A polyphasic approach employing both enhanced mycological culture with molecular detection will help determine the presence of fungi in the sputa of patients with CF and their healthcare environment.

Development of selective media for the isolation of yeasts and filamentous fungi from the sputum of adult patients with cystic fibrosis (CF)

Yeasts and filamentous fungi are beginning to emerge as significant microbial pathogens in patients with cystic fibrosis (CF), particularly in relation to allergic-type responses, as seen in patients with allergic bronchopulmonary aspergillosis (ABPA), Aspergillus bronchitis and in invasive fungal disease in lung transplant patients. Four fungal media were compared in this study, including Sabouraud Dextrose Agar (SDA) and Medium B, with and without the addition of selective antibiotics, where antibiotic-supplemented media were designated with (+). These media were compared for their ability to suppress contaminating, mainly Gram-ve pathogens, in CF sputa (Pseudomonas aeruginosa, Burkholderia cepacia complex [BCC] organisms) and to enhance the growth of fungi present in CF sputum. Medium B consisted of glucose (16.7 g/l), agar (20 g/l), yeast extract (30 g/l) and peptone (6.8 g/l) at pH 6.3 and both SDA(+) and Medium B+ were supplemented with cotrimethoxazole, 128 mg/l; chloramphenicol, 50 mg/l; ceftazidime, 32 mg/l; colistin, 24 mg/l). Employment of SDA(+) or Medium B+ allowed an increase in specificity in the detection of yeasts and moulds, by 42.8% and 39.3%, respectively, over SDA when used solely. SDA(+) had a greater ability than Medium B+ to suppress bacterial growth from predominantly Gram-ve co-colonisers. This is a significant benefit when attempting to detect and isolate fungi from the sputum of CF patients, as it largely suppressed any bacterial growth, with the exception of the BCC organisms, thus allowing for an increased opportunity to detect target fungal organisms in sputum and represented a significant improvement over the commercial medium (SDA), which is currently used. Overall, both novel selective media were superior in their ability to suppress bacteria in comparison with the commercially available SDA medium, which is routinely employed in most clinical microbiology diagnostic laboratories presently. Alternatively, Medium B+ had a great ability to grow fungi than SDA(+) and when employed together, the specificity of combined use was 82%, with a sensitivity for yeasts, filamentous fungi, and combined overall fungi of 96.0%, 92.3% and 96.0%, respectively. Overall, when employing one fungal selective medium for the routine detection of yeasts and filamentous fungi in the sputum of CF patients, we would recommend employment of Medium B+. However, we would recommend the combined employment of SDA(+) and Medium B+, in order to synergistically isolate and detect the greatest number of fungi present in CF sputa. (C) 2008 European Cystic Fibrosis Society. Published by Elsevier B.V All rights reserved.

Drug delivery strategies for photodynamic antimicrobial chemotherapy: From benchtop to clinical practice

Light and photosensitizer-mediated killing of many pathogens, termed photodynamic antimicrobial chemotherapy (PACT), has been extensively investigated in vitro. A wide range of organisms from the Gram-positive Staphylococcus aureus to the Gram-negative Pseudomonas aeruginosa have been proven to be susceptible to PACT. Multidrug-resistant strains are just as susceptible to this treatment as their naive counterparts. Both enveloped and non-enveloped viruses have demonstrated susceptibility in vitro, in addition to fungi and protozoa. Significantly, however, no clinical treatments based on PACT are currently licensed. This paper provides a comprehensive review of work carried out to date on delivery of photosensitizers for use in PACT, including topical, intranasal and oral/buccal delivery, as well as targeted delivery. We have also reviewed photo-antimicrobial surfaces. It is hoped that, through a rational approach to formulation design and subsequent success in small-scale clinical trials, more widespread use will be made of PACT in the clinic, to the benefit of patients worldwide. (C) 2009 Elsevier B.V. All rights reserved.

Antifungal photodynamic therapy

In photodynamic antimicrobial chemotherapy (PACT), a combination of a sensitising drug and visible light causes selective destruction of microbial cells. The ability of light-drug combinations to kilt microorganisms has been known for over 100 years. However, it is only recently with the beginning of the search for alternative treatments for antibiotic-resistant pathogens that the phenomenon has been investigated in detail. Numerous studies have shown PACT to be highly effective in the in vitro destruction of viruses and protozoa, as well as Gram-positive and Gram-negative bacteria and fungi. Results of experimental investigations have demonstrated conclusively that both dermatomycetes and yeasts can be effectively killed by photodynamic action employing phenothiazinium, porphyrin and phthatocyanine photosensitisers. Importantly, considerable setectivity for fungi over human cells has been demonstrated, no reports of fungal resistance exist and the treatment is not associated with genotoxic or mutagenic effects to fungi or human cells. In spite of the success of cell culture investigations, only a very small number of in vivo animal. and human trials have been published. The present paper reviews the studies published to date on antifungal applications of PACT and aims to raise awareness of this area of research, which has the potential to make a significant impact in future treatment of fungal infections. (c) 2007 Elsevier GmbH. All rights reserved.

“Shotgun” cloning of skin peptide precursors from skin secretion-derived cDNA libraries of the Fujian large-headed frog, Limnonectes fujianensis

Amphibian skin secretions are rich sources of biologically-active peptides and several studies involving molecular cloning of their biosynthetic precursors have revealed that many exhibit highly-conserved domain architectures with an associated high degree of primary structural conservation of the signal peptides. This conservation of primary structure is reflected at the level of nucleotide sequence — a finding that has permitted our group to design primers to these sites facilitating “shotgun” cloning using cDNA libraries from uninvestigated species. Here we describe the results of such an approach using a skin secretion-derived cDNA library from the Fujian large-headed frog, Limnonectes fujianensis, a completely unstudied species. In over 50 clones studied by this approach, 12 were found to encode peptides of different primary structure. Representatives of 5 different families of antimicrobial peptides derived from the skins of ranid frogs were found and these were brevinin-1 (n = 3), the ranatuerin-2 (n = 3), esculentin-2 (n = 1), temporin (n = 1) and chensinin (n = 1). Three clones encoded peptides that were novel with no homologues present in contemporary on-line databases. These included two related 16-mer peptides, named peptides SC-16a and b, and an unrelated 24-mer, named peptide AG-24. Preliminary biological characterisation of SC-16a has demonstrated an antimicrobial activity against Gram-negative bacteria with a minimal inhibitory concentration of 35 µM with no observable haemolysis up to 200 µM. This finding may suggest that this peptide represents a novel class of antimicrobial with little effect on eukaryotic membranes.

A potent novel antimicrobial peptide related to caerin 1.12 from the skin secretion of the Australian frog, Litoria aurea

Skin secretions from Australian frogs of the genus Litoria have been extensively studied for many years and are known to contain a large array of antimicrobial peptides that often bear their specific names — caerins (L. caerulea), aureins (L. aurea), citropins (L. citropa) and maculatins (L. genimaculata) — and each group displays distinct primary structural attributes. During a systematic transcriptome cloning study using a cDNA library derived from skin secretion of L. aurea, a series of identical clones were identified that encoded a novel 25-mer antimicrobial peptide that displayed 92% structural identity with caerin 1.12 from L. caerulea, differing in amino acid sequence at only two positions — Arg for Gly at position 7 and Leu amide for Ser amide at the C-terminus. The novel peptide had conserved Pro residues at positions 15 and 19 that flank a flexible hinge region which previous studies have suggested are important for effective orientation of the two alpha-helices within the bacterial membrane resulting in lysis of cells. As the two substitutions in the novel peptide serve to increase both positive charge and hydrophobicity, we synthesised a replicate and determined its minimal inhibitory concentration (MIC) against Gram positive Staphylococcus aureus and Gram negative Escherichia coli. The MICs for these organisms were 3 µM and 4 µM, respectively, indicating a high potency and haemolysis was