Moving pieces in a proteomic puzzle: mass fingerprinting of toxic fractions from the venom of Tityus serrulatus (Scorpiones, Buthidae).

Scorpion venoms are very complex mixtures of molecules, most of which are peptides that display different kinds of biological activity. These venoms have been studied in the light of their pharmacological targets and their constituents are able to bind specifically to a variety of ionic channels located in prey tissues, resulting in neurotoxic effects. Toxins that modulate Na(+), K(+), Ca(++) and Cl(-) currents have been described in scorpion venoms. Mass spectrometry was employed to analyze toxic fractions from the venom of the Brazilian scorpion Tityus serrulatus in order to shed light on the molecular composition of this venom and to facilitate the search for novel pharmacologically active compounds. T. serrulatus venom was first subjected to gel filtration to separate its constituents according to their molecular size. The resultant fractions II and III, which account for 90 and 10% respectively of the whole venom toxic effect, were further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), on-line liquid chromatography/electrospray mass spectrometry (LC/ESMS) and off-line LC/MALDI-TOFMS in order to establish their mass fingerprints. The molecular masses in fraction II were predominantly between 6500 and 7500 Da. This corresponds to long-chain toxins that mainly act on voltage-gated Na(+) channels. Fraction III is more complex and predominantly contained molecules with masses between 2500 and 5000 Da. This corresponds to the short-chain toxin family, most of which act on K(+) channels, and other unknown peptides. Finally, we were able to measure the molecular masses of 380 different compounds present in the two fractions investigated. To our knowledge, this is the largest number of components ever detected in the venom of a single animal species. Some of the toxins described previously from T. serrulatus venom could be detected by virtue of their molecular masses. The interpretation of this large set of data has provided us with useful proteomic information on the venom, and the implications of these findings are discussed.     

Cardenolides content in wild Sardinian Digitalis purpurea L. populations

In order to quantify the amounts of digitoxigenin and gitoxigenin in wild Sardinian Digitalis purpurea L. an easy extraction method and an high performance liquid chromatography (HPLC) analytical technique have been set up. The analyzed samples, stemming from six different locations, showed a great variability in glycoside content. The HPLC analyses carried out on 2-year-old plants of D. purpurea showed that the amounts of digitoxigenin and gitoxigenin ranged between 11.34 and 240.59 mg kg(-1) and 4.05 and 178.07 mg kg(-1), respectively, calculated on fresh material. Chemometric analyses, carried out considering different morphological characters, showed that correlations between morphological variations and glycoside content are poor.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and high-performance liquid chromatography study of quantitative and qualitative variation in tarantula spider venoms

Animal venoms are important sources of novel pharmacological tools, useful in biochemical characterization of their receptors. Venom quality control, batch-to-batch homogeneity and high reproducibility of venom fractionation and toxin purification are crucial issues for biochemical and pharmacological studies. To address these issues, a study of the variability of tarantula spider venom samples was undertaken. Venom profiles of samples collected from individuals of different age and sex, and from sibling spiders of the same species, were generated by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and analyzed to assess venom variability and method accuracy. Sex-linked venom variation was studied on eight species. Clear qualitative differences were observed for six out of eight species, as well as quantitative differences. Age-related variation studied in Poecilotheria rufilata showed essentially age-related quantitative differences between adults of both sexes and immature juveniles. The venoms of nine siblings and three wild-collected Pterinochilus murinus were studied for individual variation, showing only very minor quantitative differences. On the same samples, the quality of MALDI-TOFMS venom fingerprinting was demonstrated to be highly reproducible. Our results show that tarantula venom peptide fingerprinting is a highly reliable identification method, that pooled batches of venom from several animals can be used for venom purification, that venom composition does not appear to be qualitatively related to ontogenesis in the spiders studied, and that qualitative sex-linked variation occurs across most species and may be important in activity studies.     

Mass spectrometric analysis of the individual variability of Bothrops jararaca venom peptide fraction. Evidence for sex-based variation among the bradykinin-potentiating peptides

Variation in the snake venom proteome is well documented and it is a ubiquitous phenomenon at all taxonomical levels. However, variation in the snake venom peptidome is so far not described. In this work we used mass spectrometry [liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOFMS)] to explore sex-based differences among the venom peptides of eighteen sibling specimens of Bothrops jararaca of a single litter born and raised in the laboratory. MALDI-TOFMS analyses showed individual variability among the bradykinin-potentiating peptides (BPPs), and, interestingly, four new peptides were detected only in female venoms and identified by de novo sequencing as cleaved BPPs lacking the C-terminal Q-I-P-P sequence. Similar results were obtained with venom from wild-caught adult non-sibling specimens of B. jararaca and in this case we were able to identify the gender of the specimen by analyzing the MALDI-TOF profile of the peptide fraction and finding the cleaved peptides only in female venoms. Synthetic replicates of the cleaved BPPs were less potent than the full-length BPP-10c in potentiating the bradykinin hypotensive effect, suggesting that the C-terminus is critical for the interaction of the BPPs with their mammalian molecular targets. This work represents a comprehensive mass spectrometric analysis of the peptide fraction of B. jararaca venom and shows for the first time sex-based differences in the snake venom peptidome of sibling and non-sibling snakes and suggests that the BPPs may follow distinct processing pathways in female and male individuals.     

Marinobufagenin extraction from Rhinella marina toad glands: Alternative approaches for a systematized strategy

Marinobufagenin is a bufadienolide compound detected mainly in skin and parotoid gland secretions of Rhinella marina (L.) toad. Bufadienolides regulate the Na⁺/K⁺‐ATPase pump by inhibiting the cardiotonic steroid dependent‐site and act as cardiac inotropes with vasoconstrictive properties. Marinobufagenin and other bufadienolides, such as telocinobufagin and bufalin, are thought to be found endogenously in mammals in salt‐sensitive hypertensive states such as essential hypertension, congestive heart‐failure, and preeclampsia. The role of marinobufagenin as antimicrobial agent and its cytotoxic potential have also been recognized. The particular interest around marinobufagenin prompts us to consider the Rhinella marina toad venom as a possible source for molecules with pharmacological and/or diagnostic potential. In this article, two different approaches of extraction and purification of marinobufagenin from Rhinella marina (L.) venom are studied: (i) Preparative thin‐layer chromatography combined to mass spectrometry and/or ultraviolet detection and (ii) solid‐phase extraction coupled with fractionation on high‐performance liquid chromatography. Different chromatographic conditions are tested for each approach. The solid‐phase extraction combined with high‐performance liquid chromatography fractionation approach was preferred as it offered a greater yield, was less time‐consuming and allowed us to selectively isolate marinobufagenin. Both protocols aim to provide efficient and convenient methods for toad venom extraction, based on an easily automatable and systematized strategy.     

Method development and validation for determination of thiosultap sodium, thiocyclam, and nereistoxin in pepper matrix

This work reports a method for extraction and analysis of thiosultap sodium, thiocyclam, and nereistoxin in pepper. Different extraction methods were tested to attain the best recoveries. The final extraction method combines acetonitrile extraction in an acidic medium with ultrasonic extraction followed by a cleanup step with anhydrous MgSO₄. The analyses were performed on a Linear Ion Trap Quadrupole LC-MS/MS in negative mode for thiosultap sodium and in positive mode for thiocyclam and nereistoxin. Recovery studies carried out on peppers spiked at different fortification levels (20 and 200 μg?kg^?1) yielded average recoveries in the range 58–87% with RSD (%) values below 20%. Calibration curves covering two orders of magnitude were performed and they were linear over the concentration range studied (0.001–0.5 mg?l^?1). Instrumental detection limits were in the low μg?kg^?1 range. Stability studies of thiosultap sodium in water were performed by evaluating a 100-μg?l^?1 solution of this compound in water. It was analyzed over 7 days, after which more than 80% degradation of thiosultap sodium could be observed.     

金属离子对蛇毒蛋白生物活性及结构效应的影响

从旅顺产白眉蝮蛇(Gloydius blomhoffii brevicaudus, GBB) 蛇毒中纯化得到了3种电泳和质谱纯蛋白活性组分, 其酶解肽段采用高效液相色谱-电喷雾串联质谱(HPLC-nESI-MS/MS)进行序列测定, 与其它同源性蛇毒蛋白氨基酸序列比对发现, 3种蛋白为新的蛇毒磷脂酶A₂、类凝血酶和金属蛋白酶, 分别将其命名为GBB-bPLA₂, GBB-TLE和GBB-MP. 电感耦合等离子体发射光谱法(ICP-AES) 测得每个GBB-bPLA₂和GBB-MP中含有一个Ca²⁺; 每个GBB-TLE中含有2个Zn²⁺. Ca²⁺可分别使GBB-bPLA₂和GBB-MP的荧光发射波长向短波方向移动2.0和1.6 nm, 使二者的荧光发射强度提高14.0%和11.0%; Ca²⁺的存在可显著提高二者的热稳定性, 使GBB-bPLA₂和GBB-MP的热变性温度分别提高1.5和2.0 ℃. Zn²⁺使GBB-TLE的荧光发射强度增高4.3%, 但对GBB-TLE的酯酶水解活性、荧光发射波长和热变性温度无显著影响. 金属离子的存在能够不同程度地影响蛇毒蛋白结构热稳定性, 但对蛇毒蛋白生物活性的作用则不同.     

Sono-extraction as a pretreatment approach for the screening evaluation of element mobility of sediment samples

Application of economically important and time saving pretreatment for the screening element mobility evaluation of contaminated sediments is presented. Ultrasonically-assisted single-step extraction (USAE) was carried out by EDTA solution. The extraction time of USAE was optimized and obtained results were compared with results estimated by conventional (EDTA extraction) and by sequential extraction (modified BCR protocol). The original three step BCR protocol was modified by addition of the first step (water leaching) and the fifth step, total digestion of sediment residue (acid mixture with HF). Zn, Cu and Pb have been determined in extracts by ICP-OES. Good conformity of the ultrasonically-extracted element contents and sum of contents, extracted during first three steps (water-soluble, acid-extractable, reducible — i.e., the most mobile fractions) of sequential extraction, was found. The sono-extraction reduced operating time of the first three steps of sequential extraction from 48 h to 15 min. Thus, USAE can serve as a rapid screening assessment of the mobile and potentially mobile element portions in sediments and other similar solid state environmental media. Analytical quality control was realized by comparison of the sums of element contents obtained at individual (five) extraction steps. Total element contents were also determined by an independent method (XRF).     

Isolation and Characterization of Wax Esters in Fennel and Caraway Seed Oils by SPE-GC

A rapid method for the isolation and quantitative determination of wax esters in vegetable oils was developed. For the first time wax esters in oils were separated from the triglyceride matrix by means of solid-phase extraction, which allows rapid sample preparation in parallel and therefore a high sample throughput. The thus obtained wax ester fractions of fennel and caraway seed oils were analyzed by high temperature gas chromatography. GC-MS analyses were carried out using electron impact ionization in order to characterize the wax ester fraction. With respect to the results of the GC-MS analyses different isomers of saturated wax esters with the same carbon number were observed. Additional monounsaturated wax esters with an unsaturated fatty acid moiety were identified.     

Effects of propyphenazone and other non-steroidal anti-inflammatory agents on the synthetic and endogenous androgenic anabolic steroids urinary excretion and/or instrumental detection

This paper describes the effects of oral administration of non-steroidal anti-inflammatory drugs on the endogenous and synthetic anabolic androgenic steroids urinary excretion as assessed by gas-chromatography mass-spectrometry. Experiments were carried out on 5 male subjects, with pathologies and/or diseases, treated with non-steroidal anti-inflammatory drugs. To set up the individual baseline variability of testosterone and its main metabolites, urine samples were collected for 3 days, every 2 h prior to the administration of the drug(s); whereas the study of the effects of a single dose of each drug, here considered, on the endogenous androgen steroid urinary concentrations, was assessed by collecting urine samples for 2 days, every 2 h. Data obtained after drugs administration were then evaluated taking into account the individual baseline variability. The results showed that, only in the case of propyphenazone administration, the relative urinary concentrations of some testosterone metabolites were significantly altered. More specifically, the urinary levels of dehydroepiandrosterone, 11keto-etiocholanolone, 11β-hydroxyandrosterone, 11β-hydroxyetiocholanolone, androsterone, etiocholanolone and some metabolite ratios decrease significantly, generally between 2 and 10 h after administration of the drug, whereas no effects were observed on urinary calculated concentrations of testosterone, epitestosterone, 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol and testosterone/epitestosterone ratio. The observed effects do not depend on alterations on pharmacokinetics (excretion/metabolism), but on steroid sample preparation steps (hydrolysis and derivatization) inhibition. More specifically the significant decrease of dehydroepiandrosterone and testosterone metabolites urinary levels was due to a reduced yield of the steroid derivatization step for the presence in urine of the main metabolites of propyphenazone, namely hydroxyl-propyphenazone metabolites.     

Length-scale dependence of variability in epoxy modulus extracted from composite prepreg

Property variability in conjunction with morphological variability are important sources of uncertainty in composite modeling. While image processing of experimental microstructures has enabled accurate quantification of morphological variability, the characterization of material variability is not as well established. In this study, the local material properties of epoxy extracted from a prepreg sheet was determined using nanoindentation with a spherical indenter tip with a radius of 50 μm. Indentations were carried out at four different indentation depths to evaluate the change in the variability of epoxy modulus with the sampling volume. For each length scale studied, 40 indentations were carried out to determine the variability in epoxy modulus. A significant decrease was observed in the coefficient of variation as the indentation depth increased. The corresponding modulus distributions were quantified. The results suggest that, similar to morphological variability, material variability is length-scale dependent and the appropriate variability associated with the selected length scale must be considered for stochastic modeling of composite structures.     

Development of a matrix solid-phase dispersion method for the simultaneous determination of pyrethroid and organochlorinated pesticides in cattle feed

A matrix solid-phase dispersion (MSPD) method was developed for the simultaneous extraction of 36 common pesticides and breakdown products (mostly pyrethroids and organochlorines) in cattle feed. Different parameters affecting the extraction efficiency (such as dispersing phase, clean-up adsorbent and elution volume) were investigated. The experimental procedure was optimized using a multivariate statistical approach and the final analyses were carried out by GC-muECD. Several protocols for extract purification were also studied. As far as we know, this is the first application of MSPD for the extraction of most of the target pesticides from animal feed. Using the optimized extraction conditions, the method was validated in terms of accuracy, and precision (within-a-day and among-days), using a certified reference material (CRM 115) as well as spiked cattle feedingstuffs at different concentration levels. A matrix effect study was also carried out using various real samples. The recoveries were satisfactory (>75% in most cases) and the quantification limits, at the sub-ngg(-1) or low-ngg(-1) level, complied with the regulated maximum residue levels (MRLs) in animal feed and in main crops used in the preparation of cattle feeding materials. Finally, the MSPD-GC-muECD methodology was applied to the analysis of real cattle feed samples collected in farms of dairy cattle from NW Spain.     

Mechanisms of Cytochrome C Extraction by Reverse Micelles

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Effect of the carbon dioxide modifier on the lipid composition of wool wax extracted from raw wool

Wool wax or lanolin is a unique substance secreted by sheep and forms a natural protective coating on wool fibres. It is widely used in pharmaceutical and cosmetic formulations. However, different systems of wool wax recovery from scouring liquour provide a dark impurified greasy product. This product has a lipid composition that differs from the wool wax present on wool fibres. The wool wax extraction method from raw wool with pressurised CO₂ and different modifiers at constant pressure and temperature was studied. Thin-layer chromatography coupled to an automated flame ionisation detection system (TLC/FID) was used to analyse the different lipid classes present in the collected extracts. Moreover, a detailed structural comparison of the cholesteryl esters and hydroxycholesteryl esters was carried out by means of sub-ambient pressure chromatography mass spectrometry in the electron impact and in the ammonia positive chemical ionisation modes. For comparison, qualitative and quantitative analyses of the lanolin extracted in Soxhlet with dichloromethane and commercial cosmetic lanolin were carried out. Differences in the quantity of wool wax extraction and in the lipid composition of different wool wax extracts were detected by changing the modifier polarity.     

Matrix-assisted laser desorption ionization/mass spectrometry mapping of human immunodeficiency virus-gp120 epitopes recognized by a limited polyclonal antibody

In this study we have applied epitope excision and epitope extraction strategies, combined with matrix assisted laser desorption/ionization mass spectrometry, to determine the fine structure of epitopes recognized by a polyclonal antibody to human immunodeficiency virus envelope glycoprotein gp120. This is the first application of this approach to epitope mapping on a large, heavily glycosylated protein. In the epitope excision method, gp120 in the native form is first bound to the antibody immobilized on sepharose beads and cleaved with endoproteinase enzymes. In the epitope extraction method, the gp120 was first proteolytically cleaved and then allowed to react with the immobilized antibody. The fragments that remain bound to the antibody, after repeated washing to remove the unbound peptides, contain the antigenic region that is recognized by the antibody, and the bound peptides in both methods can be characterized by direct analysis of the immobilized antibody by matrix assisted laser desorption ionization/mass spectrometry. In this study we have carried out epitope excision and extraction experiments with three different enzymes and have identified residues 472–478 as a major epitope. In addition, antigenic regions containing minor epitopes have also been identified.     

Solvent extraction of Co/III/ by benzoylacetone from acetate solutions

The extraction of Co/III/ by benzoylacetone solutions has been carried out from acetateacetic acid solutions. The effect of different parameters affecting the distribution coefficient of Co/III/ have been determined. Lg D for Co was found to be a third order dependent on extractant concentration and a negative first order with respect to [H⁺]. From the thermodynamic parameters and the data of distribution ratios, the extraction mechanism has been suggested. Addition of some electron donor compounds shows no possibility for the increase of coordination number.     

Determination of chloramphenicol in urine,feed water,milk and honey samples using molecular imprinted polymer clean-up

A method for determination of low concentrations of chloramphenicol in urine, feed water, milk and honey was developed. A comparison was carried out between a routinely used analytical method based on solid phase extraction (SPE-C18) for cleaning the extract and the new procedure for the sample preparation using columns based on the molecular imprinted polymers (MIP) principle. The extracts obtained from the MIP clean-up procedure were clean enough for chromatografic analyses. Confirmatory analyses were conducted using GC/MS-NCI after derivatisation (silylation). The described method was fully validated according to CD 2002/657/EC. This method is considerably robust and allows very dirty samples to be processed. The described MIP procedure is very simple and low-time-consuming, and provides high throughput of the samples examined. This could be used for routine screening and confirmatory analyses as well.     

Variables affecting simulated use determination of residual ethylene oxide in medical devices

In autumn 1993, AAMI/ST/WG 63, Sterilization Residuals Working Group undertook the task of studying factors involved in determining the amount of residual ethylene oxide in medical devices after sterilization and developing a protocol for controlling the relevant variables. The protocol was evaluated by conducting a round robin study consisting of 8 participating laboratories from around the country. Results of this round robin study demonstrated the range over which results may vary despite controls placed on the time and temperature at which determinations were conducted. The data from the study suggest that small, random variations in technique during short sample extraction times can lead to variability in the results. Variables such as initial water temperature, oven temperature, weighing of sample, and length of extraction should be carefully controlled. Inherent variations in the material composition of similar devices are possible contributing factors. The efforts of this working group and the subsequent evaluation and discussion of its findings are presented.     

MSD,ECD and NPD performances compared in the assay of six non steroidal anti-inflammatory drugs in plasma samples

Summary The aim of this work was to compare the performance of the MSD, ECD and NPD systems when used for drug assay in biological fluids. As a practical test, six non steroidal anti-inflammatory drugs added to plasma samples were detected and quantified. The analyses were carried out after solvent extraction from an acidic medium and subsequent methylation. The linearity of response was tested for all the detection systems in the range of 1 to 25 ng/ml. Precision and accuracy were determined at 1, 5 and 10 ng/ml. The minimum quantifiable level for the six drugs was about 1 ng/ml with each of the three detection systems.