The crude skin secretion of the pepper frog leptodactylus labyrinthicus is rich in metallo and serine peptidases

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology.

The effect of ß-alanine on buffering capacity and exercise performance during and following high-intensity exercise in healthy males

Introduction Intense exercise induced acidosis occurs from the accumulation of hydrogen ions as by-products of anaerobic metabolism. Oral ingestion of ß-alanine, a limiting precursor of the intracellular physiochemical buffer carnosine in skeletal muscle, may counteract any detrimental effect of acidosis and benefit performance. The aim of this study was to investigate the effect of ß-alanine as an ergogenic aid during high intensity exercise performance in healthy males. Methods Five males ingested either ß-alanine (BAl) (4.8 g.d-1 for 4wk, then 6.4 g.d-1 for 2wk) or placebo (Pl) (CaCO3) in a crossover design with 6 wk washout between. Following supplementation, participants performed two different intense exercise protocols over consecutive days. On the first day a repeated sprint ability (RSA) test of 5 x 6s, with 24s rest periods, was performed. On the second day a cycling capacity test measuring the time to exhaustion (TTE) was performed at 110% of their max workload achieved in a pre supplementation max test (CCT110%). Non-invasive quantification of carnosine, prior to, and following each supplementation, with magnetic resonance spectrometry was performed in the soleus and gastrocnemius. Time to fatigue (CCT110%), peak and mean power (RSA), blood pH, and plasma lactate were measured. Results Muscle carnosine concentration was not different prior to ß-alanine supplementation and increased 18% in the soleus and 26% in the gastrocnemius, respectively with 6 wk supplementation. There was no difference in the measured performance variables during the RSA test (peak and average power output). TTE during the CCT110% was significantly enhanced following the ingestion of BAl (155s ± 19.03) compared to Pl (134s ± 26.16). No changes were observed in blood pH during either exercise protocol and during the recovery from exercise. Plasma lactate in the BAl condition was significantly higher than Pl only from the 15th minute following exercise during the CCT110%. FIG. 1: Changes in carnosine concentration in the gastrocnemius prior and post 6 week chronic supplementation of placebo and β-alanine. Values expressed as mean.* p<0.05 from Pl at 6 weeks, # p<0.05 from pre supplementation. Conclusion/Discussion Greater muscle carnosine content following 6wk supplementation of ß-alanine enhanced the potential for intracellular buffering capacity. However, this only translated into enhanced performance during the CCT110% high intensity cycling exercise protocol, with no change observed during the RSA test. No differences in post exercise and recovery plasma lactates and blood pH, indicates that 6wks ß-alanine supplementation has no effect on anaerobic metabolism during multiple bout high intensity exercise. Changes in plasma lactate during recovery supports that ß-alanine supplementation may affect anaerobic metabolism however during single bout high intensity.

The effect of ß-alanine on buffering capacity and exercise performance during and following high-intensity exercise in healthy males

Intense exercise induced acidosis occurs after accumulation of hydrogen ions as by-products of anaerobic metabolism. Oral ingestion of ß-alanine, a limiting precursor of the intracellular physiochemical buffer carnosine in skeletal muscle, may counteract detrimental effects of acidosis and benefit performance. This study aimed to investigate the effect of ß-alanine as an ergogenic aid during high intensity exercise performance. Five healthy males ingested either ß-alanine or placebo (Pl) (CaCO3) in a crossover design with 6 wk washout between. Participants performed two different intense exercise protocols over consecutive days. On the first day a repeated sprint ability (RSA) test was performed. On the second day a cycling capacity test measuring the time to exhaustion (TTE) was performed at 110% of maximum workload achieved in a pre supplementation max test (CCT110%). Non-invasive quantification of carnosine, prior to, and following each supplementation, with in vivo magnetic resonance spectrometry was performed in the soleus and gastrocnemius muscle. Time to fatigue (CCT110%), peak and mean power (RSA), blood pH, and plasma lactate were measured. Muscle carnosine concentration was not different prior to ß-alanine supplementation and increased 18% in the soleus and 26% in the gastrocnemius, respectively after supplementation. There was no difference in the measured performance variables during the RSA test (peak and average power output). TTE during the CCT110% was significantly enhanced following the ingestion of BAl (155s ± 19.03) compared to Pl (134s ± 26.16). No changes were observed in blood pH during either exercise protocol and during the recovery from exercise. Plasma lactate after BAI was significantly higher than Pl only from the 15th minute following exercise during the CCT110%. Greater muscle carnosine content following 6wk supplementation of ß-alanine enhanced the potential for intracellular buffering capacity. This translated into enhanced performance during the CCT110% high intensity cycling exercise protocol but not during the RSA test. The lack of change in plasma lactate or blood pH indicates that 6wks ß-alanine supplementation has no effect on anaerobic metabolism during multiple-bout high-intensity exercise. Changes measured in plasma lactate during recovery support the hypothesis that ß-alanine supplementation may affect anaerobic metabolism particularly during single bout high intensity.

A high cell count cascade full bridge converter for wide bandwidth ultrasonic transducer excitation

A nine level modular multilevel cascade converter (MMCC) based on four full bridge cells is shown driving a piezoelectric ultrasonic transducer at 71 and 39 kHz, in simulation and experimentally. The modular cells are small stackable PCBs, each with two fully integrated surface mount 22 V, 40 A MOSFET half-bridge converters, and include all control signal and power isolation. In this work, the bridges operate at 12 V and 384 kHz, to deliver a 96 Vpp 9 level waveform with an effective switching frequency of 3 MHz. A 9 pH air cored inductor forms a low pass filter in conjunction with the 3000 pF capacitance of the transducer load. Eight equally phase-displaced naturally sampled pulse width modulation (PWM) drive signals, along with the modulating sinusoid, are generated using phase accumulation techniques in a dedicated FPGA. Experimental time domain and FFT plots of the multilevel and transducer output waveforms are presented and discussed.

Dispersion of zero valent iron nanoparticles onto bentonites and the decolourization of orange II

Nano Zero valent iron (Fe0) were reported as an effective material for azo dye removal, however, similar to other nano-materials, ultra-fine powder has a strong tendency to agglomerate into larger particles, resulting in an adverse effect on both effective surface area and catalyst performance. Here we report nano sized Fe0 particles dispersed onto the surface of natural bentonites. X-ray diffraction was used to study the sample phases. Scanning electron microscopy and transmission electron microscopy were applied to study the morphology and morphological changes. Spherical individual Fe0 particles were observed after dispersion onto bentonites, and these samples were used for orange II (OII) decolourization with wide working pH range. Higher reactivity is attributed to good dispersion of Fe0 particles on clay minerals’ surface. This study is significant for providing novel modified clay based catalyst materials for the decolourization of azo dye contaminants from wastewater.

Encapsulation of Hydrocortisone and Mesalazine in Zein Microparticles

Zein was investigated for use as an oral-drug delivery system by loading prednisolone into zein microparticles using coacervation. To investigate the adaptability of this method to other drugs, zein microparticles were loaded with hydrocortisone, which is structurally related to prednisolone; or mesalazine, which is structurally different having a smaller LogP and ionizable functional groups. Investigations into the in vitro digestibility, and the electrophoretic profile of zein, and zein microparticles were conducted to shed further insight on using this protein as a drug delivery system. Hydrocortisone loading into zein microparticles was comparable with that reported for prednisolone, but mesalazine loading was highly variable. Depending on the starting quantities of hydrocortisone and zein, the average amount of microparticles equivalent to 4 mg hydrocortisone, (a clinically used dose), ranged from 60-115 mg, which is realistic and practical for oral dosing. Comparatively, an average of 2.5 g of microparticles was required to deliver 250 mg of mesalazine (a clinically used dose), so alternate encapsulation methods that can produce higher and more precise mesalazine loading are required. In vitro protein digestibility revealed that zein microparticles were more resistant to digestion compared to the zein raw material, and that individual zein peptides are not preferentially coacervated into the microparticles. In combination, these results suggest that there is potential to formulate a delivery system based on zein microparticles made using specific subunits of zein that is more resistant to digestion as starting material, to deliver drugs to the lower gastrointestinal tract.

Straminipilan protists : a study of their taxonomic position, morphology and abiotic stress-mediated gene expression

Thraustochytrids have become of considerable industrial and scientific interest in the past decade due to their health benefits. They have been proven to be the principle source in marine and estuarine fish diets with high percentage of long chain (LC) or polyunsaturated fatty acids (PUFA). Therefore, the oil extracted from fish for human document.forms[0].elements[13].select();consumption is rich in PUFA with high omega-3 fatty acid content. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of all of the omega-3 fatty acids, are considered beneficial essential oils for humans with a wide range of health benefits. These include brain and neural development in infants, general wellbeing of adults and drug delivery through precursor molecules. They have become one of the most extensively studied organisms for industrial oil preparations as PUFA extraction from fish becomes less profitable. Many forms of these Thraustochytrid oils are being trialled for human consumption all over the world. In Australia, there has been little research performed on these organisms in the past ten years. A few Australian studies have been conducted in the form of comparative studies related to PUFA production within the related genera, but not focussed on their identification or cellular and genomic characterisation. Therefore, the main aim of this study was to investigate the morphological and genetic characteristics of Australian Thraustochytrids in order to aid in their identification and characterisation, as well as to better understand the effect of environmental conditions in the regulation of PUFA production. It was also noted that there was a knowledge gap in the preservation and total genomic DNA extraction of these organisms for the purposes of scientific research. The cryopreservation of these organisms for studies around the world follows existing generic methods. However, it is well understood that many of these generic methods attract not only high costs for chemicals, but also uses considerable storage space and other resources, all of which can be improved with new or modified approaches. In this context, a simple and inexpensive bead preservation method is described, without compromising the storage shelf life. We also describe, for the first time, the effects of culture age on the successful cryopreservation of Thraustochytrids. It was evident in the literature that DNA and RNA extractions for molecular and genetic studies of Thraustochytrids follow the classical phenol-chloroform extraction methods. It was also observed that modern protocols failed to avoid the use of phenol-chloroform rather than improving preparation and cell disruption. In order to provide a high quantity and quality DNA extraction, a modified protocol has been introduced that employs the use of modern commercial extraction kits and standard laboratory equipment. Thraustochytrids have been shown to be highly conserved in their 18S rDNA gene sequences, which is used as the current standard for identification. It was demonstrated that the 18S rDNA gene sequence limits the recognition of closely related genera or within the genera from each member. Therefore, it was proposed that another profile, such as a randomly amplified polymorphic DNA (RAPD) based profiling system, be tested for use in the characterisation of Thraustochytrids. The RAPD profiles were shown to provide a unique DNA fingerprint for each isolate and small variations in their genome were able to be detected. This method involved the use of a minimum number of standard arbitrary primers and with an increase in the number of different primers used, a very high discrimination between organisms could be achieved. However, the method was not suitable for taxonomic purposes because the results did not correlate with other taxonomic features such as morphology. Another knowledge gap was found with respect to Australian Thraustochytrid growth characteristics, in that these had not been recorded and published. In order to rectify this, a record of colony and microscopic features of 12 selected isolates was performed. The results of preliminary studies indicated that further microbiological and biochemical studies are needed for full characterisation of these organisms. This information is of great importance to bio-prospecting of new Thraustochytrids from Australian ecosystems and would allow for their accurate identification, and so permit the prediction of their PUFA capability by comparison with related genera/species. It was well recognized that environmental stress plays a role in the PUFA production and is mainly due to the reactive oxygen species as abiotic stress (Chiou et al., 2001; Okuyama et al., 2008; Shabala et al., 2009; Shabala et al., 2001). In this aspect, this study makes the first attempt towards better understanding of this phenomenon by way of the use of real-time PCR for the detection of environmental effects on the regulation of PUFA production. Three main environmental conditions including temperature, pH and oxygen availability were monitored as stress inducers. In summary, this study provides novel approaches for the preservation and handling of Thraustochytrids, their molecular biological features, taxonomy, characterisation and responses to environmental factors with respect to their oil production enzymes. The information produced from this study will prove to be vital for both industrial and scientific investigations in the future.

Enhancement of bacterial mutagenicity of bifunctional alkylating agents by expression of mammalian glutathione s-transferase

Recently, we inserted the plasmid vector pKK233-2 containing rat GSH S-transferase (GST) 5-5 cDNA into Salmonella typhimurium TA1535 and found that these bacteria [GST 5-5(+)] expressed the protein and produced mutations when ethylene or methylene dihalides were added [Thier, R., Taylor, J. B., Pemble, S. E., Ketterer, B., Persmark, M., Humphreys, W. G., and Guengerich, F. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8576-8580]. After exposure to the known GST 5-5 substrate 1,2-epoxy-3-(4′-nitrophenoxy)propane, the GST 5-5(+) strain showed fewer mutants than the bacteria transfected with the cDNA clone in a reverse orientation [GST 5-5(-)], suggesting a protective role of GST 5-5. However, mutations were considerably enhanced in the GST 5-5(+) strain [as compared to GST 5-5(-)] when 1,2,3,4-diepoxybutane (butadiene diepoxide) or 1,2-epoxy-4-bromobutane was added. The GST 5-5(+) and GST 5-5(-) bacterial stains showed similar responses to 1,2-epoxypropane, 3,4-epoxy-1-butene, and 1,4-dibromobutane. The results suggest that some bifunctional activated butanes are transformed to mutagenic products through GSH conjugation. We also found that the GST 5-5(+) strain showed enhanced mutagenicity with 1,4-dibromo-2,3-epoxybutane, 1,2-epoxy-3-bromopropane (epibromohydrin), and (±)-1,4-dibromo-2,3-dihydroxybutane. The possibility was considered that a 5-membered thialonium ion may be involved in the mutagenicity. Model thialonium compounds were rather stable to hydrolysis in aqueous solution at pH 7.4 and slowly alkylated 4-(4-nitrobenzyl)pyridine. The presence of a hydroxyl group β to the sulfur did not enhance reactivity. Mechanisms involving episulfonium ions are considered more likely. Potential oxidation products of the toxic pesticide 1,2-dibromo-3-chloropropane (DBCP) were also considered in this system. DBCP itself gave rather similar results in the two strains. Others have reported that oxidation of DBCP is required for mutagenicity, along with GST-catalyzed GSH conjugation [Simula, T. P., Glancey, M. J., Söderlund, E. J., Dybing, E., and Wolf, C. R. (1993) Carcinogenesis 14, 2303-2307]. The putative oxidation product 1,2-dibromopropional did not show a difference between the two strains. However, 1,3-dichloroacetone, a model for the putative oxidation product 1-bromo-3-chloroacetone, was considerably more mutagenic in the GST 5-5(+) strain.

Development of a polymer theranostic for prostate cancer

Theranostics offers an improved treatment strategy for prostate cancer by facilitating simultaneous targeting of tumour cells with subsequent drug delivery and imaging. In this report we describe the synthesis of hyperbranched polymers that are biocompatible, can specifically target and be internalised by prostate cancer cells (through targeting of prostate-specific membrane antigen – PSMA) and ultimately facilitate controlled delivery of a model drug. The theranostic also incorporates a far-red fluorescent dye that allows tracking of the polymer via optical imaging. Controlled synthesis of the polymer is achieved via reversible addition fragmentation chain transfer polymerisation of polyethylene glycol monomethyl methacrylate, with ethylene glycol dimethacrylate as the branching agent. Incorporation of 20 mol% of an hydrazide-methacrylate monomer allows post-ligation of a model drug, fluorene-2-carboxaldehyde, through a hydrolytically-degradable hydrazone linkage. The rate of degradation of this particular linker was enhanced at endosomal pH (pH = 5.5) where [similar]95% of the model drug was released in 4 hours compared to less than 5% released over the same period at physiological pH. The theranostic showed high uptake into prostate cancer cells expressing prostate-specific membrane antigen, while minimal uptake was observed in PC3 cells negative for PSMA, highlighting the enhanced efficacy of the targeting ligand.

A study, by fire-assay and ICP-MS, of the distribution of rhodium, platinum, and gold in iron-age pottery

Forty-six archaeological specimens were treated by fire-assay and subsequently analysed by ICP-MS for selected precious metals: Ph, Pt and Au. The investigation was prompted by the possibility that archaeological samples could serve as "indicators" of the precious metal composition of the clays from the excavated sites. Therefore, the experimentally obtained concentrations were carefully studied to determine if there were anomalous levels of these precious metals in the deposits from which the specimens originated. Furthermore, the analytical data were used to establish if it was feasible to distinguish ancient potsherds based on precious metal concentrations, for employment as a basis in provenance studies.

Available evidence does not support serosorting as an HIV risk reduction strategy

Recent data highlighted the association between penetration of antiretrovirals in the central nervous system (CNS) and neurocognitive impairment in HIVpositive patients. Existing antiretrovirals have been ranked according to a score of neuropenetration, which was shown to be a predictor of anti-HIVactivity in the CNS and improvement of neurocognitive disorders [1]. Main factors affecting drug penetration are known to be protein binding, lipophilicity and molecular weight [2]. Moreover, active translation by membrane transporters (such as p-glycoprotein) could be a key mechanism of passage [3]. The use of raltegravir (RGV), a novel antiretroviral drug targeted to inhibit the HIV preintegrase complex, is increasing worldwide due to its efficacy and tolerability. However, penetration of RGV in the CNS has not been yet elucidated. In fact, prediction of RGV neuropenetration according to molecular characteristics is controversial. Intermediate protein binding (83%) and large volume of distribution (273 l) could suggest a high distribution beyond extracellular spaces [4]. On the contrary, low lipophilicity (oil/water partition coefficient at pH 7.4 of 2.80) and intermediate molecular weight (482.51 Da) suggest a limited diffusion. Furthermore, in-vitro studies suggest that RGV is substrate of p-glycoprotein, although this efflux pump has not been identified to significantly affect plasma pharmacokinetics [5]. In any case, no data concerning RGV passage into cerebrospinal fluid of animals or humans have yet been published.

Tunable electric field enhancement and redox chemistry on TiO2 island films via covalent attachment to Ag or Au nanostructures

Abstract Ag-TiO2 and Au-TiO2 hybrid electrodes were designed by covalent attachment of TiO2 nanoparticles to Ag or Au electrodes via an organic linker. The optical and electronic properties of these systems were investigated using the cytochrome b5 (Cyt b5) domain of sulfite oxidase, exclusively attached to the TiO2 surface, as a Raman marker and model redox enzyme. Very strong SERR signals of Cyt b 5 were obtained for Ag-supported systems due to plasmonic field enhancement of Ag. Time-resolved surface-enhanced resonance Raman spectroscopic measurements yielded a remarkably fast electron transfer kinetic (k = 60 s -1) of Cyt b5 to Ag. A much lower Raman intensity was observed for Au-supported systems with undefined and slow redox behavior. We explain this phenomenon on the basis of the different potential of zero charge of the two metals that largely influence the electronic properties of the TiO2 island film. © 2013 American Chemical Society.

Highly sensitive electrochemical sensor for nitric oxide using the self-assembled monolayer of 1,8,15,22-tetraaminophthalo-cyanatocobalt(II) on glassy carbon electrode

Glassy carbon (GC) electrode modified with a self-assembled monolayer (SAM) of 1,8,15,22-tetraaminophthalocyanatocobalt(II) (4α-CoIITAPc) was used for the selective and highly sensitive determination of nitric oxide (NO). The SAM of 4α-CoIITAPc was formed on GC electrode by spontaneous adsorption from DMF containing 1 mM 4α-CoIITAPc. The SAM showed two pairs of well-defined redox peaks corresponding to CoIII/CoII and CoIIIPc−1/CoIIIPc−2 in 0.2 M phosphate buffer (PB) solution (pH 2.5). The SAM modified electrode showed excellent electrocatalytic activity towards the oxidation of nitric oxide (NO) by enhancing its oxidation current with 310 mV less positive potential shift when compared to bare GC electrode. In amperometric measurements, the current response for NO oxidation was linearly increased in the concentration range of 3×10−9 to 30×10−9 M with a detection limit of 1.4×10−10 M (S/N=3). The proposed method showed a better recovery for NO in human blood serum samples.

Determination of l-dopa using electropolymerized 3,3′,3″,3‴-tetraaminophthalocyanatonickel(II) film on glassy carbon electrode

Electropolymerized film of 3,3′,3″,3‴-tetraaminophthalocyanatonickel(II) (p-NiIITAPc) on glassy carbon (GC) electrode was used for the selective and stable determination of 3,4-dihydroxy-l-phenylalanine (l-dopa) in acetate buffer (pH 4.0) solution. Bare GC electrode fails to determine the concentration of l-dopa accurately in acetate buffer solution due to the cyclization reaction of dopaquinone to cyclodopa in solution. On the other hand, p-NiIITAPc electrode successfully determines the concentration of l-dopa accurately because the cyclization reaction was prevented at this electrode. It was found that the electrochemical reaction of l-dopa at the modified electrode is faster than that at the bare GC electrode. This was confirmed from the higher heterogeneous electron transfer rate constant (k0) of l-dopa at p-NiIITAPc electrode (3.35 × 10−2 cm s−1) when compared to that at the bare GC electrode (5.18 × 10−3 cm s−1). Further, it was found that p-NiIITAPc electrode separates the signals of ascorbic acid (AA) and l-dopa in a mixture with a peak separation of 220 mV. Lowest detection limit of 100 nM was achieved at the modified electrode using amperometric method. Common physiological interferents like uric acid, glucose and urea does not show any interference within the potential window of l-dopa oxidation. The present electrode system was also successfully applied to estimate the concentration of l-dopa in the commercially available tablets.