Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography

Flavobacterium heparinum is a soil bacterium that produces several mucopolysaccharidases such as heparinase, heparitinases I and II, and chondroitinases AC, B, C and ABC. The purpose of the present study was to optimize the preparation of F. heparinum chondroitinases, which are very useful tools for the identification and structural characterization of chondroitin and dermatan sulfates. We observed that during the routine procedure for cell disruption (ultrasound, 100 kHz, 5 min) some of the chondroitinase B activity was lost. Using milder conditions (2 min), most of the chondroitinase B and AC protein was solubilized and the enzyme activities were preserved. Tryptic soy broth without glucose was the best culture medium both for bacterial growth and enzyme induction. Chondroitinases AC and B were separated from each other and also from glucuronidases and sulfatases by hydrophobic interaction chromatography on HP Phenyl-Sepharose. A rapid method for screening of the column fractions was also developed based on the metachromatic shift of the color of dimethylmethylene blue.

Temperature effects in hydrophobic interaction chromatography.

The effect of temperature from 5 degrees C to 50 degrees C on the retention of dansyl derivatives of amino acids in hydrophobic interaction chromatography (HIC) was investigated by HPLC on three stationary phases. Plots of the logarithmic retention factor against the reciprocal temperature in a wide range were nonlinear, indicative of a large negative heat capacity change associated with retention. By using Kirchoff's relations, the enthalpy, entropy, and heat capacity changes were evaluated from the logarithmic retention factor at various temperatures by fitting the data to a logarithmic equation and a quadratic equation that are based on the invariance and on an inverse square dependence of the heat capacity on temperature, respectively. In the experimental temperature interval, the heat capacity change was found to increase with temperature and could be approximated by the arithmetic average. For HIC retention of a set of dansylamino acids, both enthalpy and entropy changes were positive at low temperatures but negative at high temperatures as described in the literature for other processes based on the hydrophobic effect. The approach presented here shows that chromatographic measurements can be not only a useful adjunct to calorimetry but also an alternative means for the evaluation of thermodynamic parameters.

High-throughput hydrophilic interaction chromatography coupled to tandem mass spectrometry for the optimized quantification of the anti-Gram-negatives antibiotic colistin A/B and its pro-drug colistimethate.

Colistin is a last resort's antibacterial treatment in critically ill patients with multi-drug resistant Gram-negative infections. As appropriate colistin exposure is the key for maximizing efficacy while minimizing toxicity, individualized dosing optimization guided by therapeutic drug monitoring is a top clinical priority. Objective of the present work was to develop a rapid and robust HPLC-MS/MS assay for quantification of colistin plasma concentrations. This novel methodology validated according to international standards simultaneously quantifies the microbiologically active compounds colistin A and B, plus the pro-drug colistin methanesulfonate (colistimethate, CMS). 96-well micro-Elution SPE on Oasis Hydrophilic-Lipophilic-Balanced (HLB) followed by direct analysis by Hydrophilic Interaction Liquid Chromatography (HILIC) with Ethylene Bridged Hybrid - BEH - Amide phase column coupled to tandem mass spectrometry allows a high-throughput with no significant matrix effect. The technique is highly sensitive (limit of quantification 0.014 and 0.006μg/mL for colistin A and B), precise (intra-/inter-assay CV 0.6-8.4%) and accurate (intra-/inter-assay deviation from nominal concentrations -4.4 to +6.3%) over the clinically relevant analytical range 0.05-20μg/mL. Colistin A and B in plasma and whole blood samples are reliably quantified over 48h at room temperature and at +4°C (<6% deviation from nominal values) and after three freeze-thaw cycles. Colistimethate acidic hydrolysis (1M H2SO4) to colistin A and B in plasma was completed in vitro after 15min of sonication while the pro-drug hydrolyzed spontaneously in plasma ex vivo after 4h at room temperature: this information is of utmost importance for interpretation of analytical results. Quantification is precise and accurate when using serum, citrated or EDTA plasma as biological matrix, while use of heparin plasma is not appropriate. This new analytical technique providing optimized quantification in real-life conditions of the microbiologically active compounds colistin A and B offers a highly efficient tool for routine therapeutic drug monitoring aimed at individualizing drug dosing against life-threatening infections.

Determination of nicotine and nicotine metabolites in urine by hydrophilic interaction chromatography-tandem mass spectrometry: Potential use of smokeless tobacco products by ice hockey players.

Consumption of nicotine in the form of smokeless tobacco (snus, snuff, chewing tobacco) or nicotine-containing medication (gum, patch) may benefit sport practice. Indeed, use of snus seems to be a growing trend and investigating nicotine consumption amongst professional athletes is of major interest to sport authorities. Thus, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of nicotine and its principal metabolites cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide in urine was developed. Sample preparation was performed by liquid-liquid extraction followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in electrospray positive ionization (ESI) mode with selective reaction monitoring (SRM) data acquisition. The method was validated and calibration curves were linear over the selected concentration ranges of 10-10,000 ng/mL for nicotine, cotinine, trans-3-hydroxycotinine and 10-5000 ng/mL for nicotine-N'-oxide and cotinine-N-oxide, with calculated coefficients of determination (R(2)) greater than 0.95. The total extraction efficiency (%) was concentration dependent and ranged between 70.4 and 100.4%. The lower limit of quantification (LLOQ) for all analytes was 10 ng/mL. Repeatability and intermediate precision were ?9.4 and ?9.9%, respectively. In order to measure the prevalence of nicotine exposure during the 2009 Ice Hockey World Championships, 72 samples were collected and analyzed after the minimum of 3 months storage period and complete removal of identification means as required by the 2009 International Standards for Laboratories (ISL). Nicotine and/or metabolites were detected in every urine sample, while concentration measurements indicated an exposure within the last 3 days for eight specimens out of ten. Concentrations of nicotine, cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide were found to range between 11 and 19,750, 13 and 10,475, 10 and 8217, 11 and 3396, and 13 and 1640 ng/mL, respectively. When proposing conservative concentration limits for nicotine consumption prior and/or during the games (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N'-oxide and cotinine-N-oxide), about half of the hockey players were qualified as consumers. These findings significantly support the likelihood of extensive smokeless nicotine consumption. However, since such conclusions can only be hypothesized, the potential use of smokeless tobacco as a doping agent in ice hockey requires further investigation.

A fatal overdose of cocaine associated with coingestion of marijuana, buprenorphine, and fluoxetine. Body fluid and tissue distribution of cocaine and its metabolites determined by hydrophilic interaction chromatography-mass spectrometry(HILIC-MS).

Chromatographic separation of highly polar basic drugs with ideal ionspray mass spectrometry volatile mobile phases is a difficult challenge. A new quantification procedure was developed using hydrophilic interaction chromatography-mass spectrometry with turbo-ionspray ionization in the positive mode. After addition of deuterated internal standards and simple clean-up liquid extraction, the dried extracts were reconstituted in 500 microL pure acetonitrile and 5 microL was directly injected onto a Waters Atlantis HILIC 150- x 2.1-mm, 3-microm column. Chromatographic separations of cocaine, seven metabolites, and anhydroecgonine were obtained by linear gradient-elution with decreasing high concentrations of acetonitrile (80-56% in 18 min). This high proportion of organic solvent makes it easier to be coupled with MS. The eluent was buffered with 2 mM ammonium acetate at pH 4.5. Except for m-hydroxy-benzoylecgonine, the within-day and between-day precisions at 20, 100, and 500 ng/mL were below 7 and 19.1%, respectively. Accuracy was also below +/- 13.5% at all tested concentrations. The limit of quantification was 5 ng/mL (%Diff < 16.1, %RSD < 4.3) and the limit of detection below 0.5 ng/mL. This method was successfully applied to a fatal overdose. In Switzerland, cocaine abuse has dramatically increased in the last few years. A 45-year-old man, a known HIV-positive drug user, was found dead at home. According to relatives, cocaine was self-injected about 10 times during the evening before death. A low amount of cocaine (0.45 mg) was detected in the bloody fluid taken from a syringe discovered near the corpse. Besides injection marks, no significant lesions were detected during the forensic autopsy. Toxicological investigations showed high cocaine concentrations in all body fluids and tissues. The peripheral blood concentrations of cocaine, benzoylecgonine, and methylecgonine were 5.0, 10.4, and 4.1 mg/L, respectively. The brain concentrations of cocaine, benzoylecgonine, and methylecgonine were 21.2, 3.8, and 3.3 mg/kg, respectively. The highest concentrations of norcocaine (about 1 mg/L) were measured in bile and urine. Very high levels of cocaine were determined in hair (160 ng/mg), indicating chronic cocaine use. A low concentration of anhydroecgonine methylester was also found in urine (0.65 mg/L) suggesting recent cocaine inhalation. Therapeutic blood concentrations of fluoxetine (0.15 mg/L) and buprenorphine (0.1 microg/L) were also discovered. A relatively high concentration of Delta(9)-THC was measured both in peripheral blood (8.2 microg/L) and brain cortex (13.5 microg/kg), suggesting that the victim was under the influence of cannabis at the time of death. In addition, fluoxetine might have enhanced the toxic effects of cocaine because of its weak pro-arrhythmogenic properties. Likewise, combination of cannabinoids and cocaine might have increase detrimental cardiovascular effects. Altogether, these results indicate a lethal cocaine overdose with a minor contribution of fluoxetine and cannabinoids.

The Hydrophobic interaction: modeling hydrophobic interactions and aggregation of non-polar particles in aqueous solutions

Malgré son importance dans notre vie de tous les jours, certaines propriétés de l?eau restent inexpliquées. L'étude des interactions entre l'eau et les particules organiques occupe des groupes de recherche dans le monde entier et est loin d'être finie. Dans mon travail j'ai essayé de comprendre, au niveau moléculaire, ces interactions importantes pour la vie. J'ai utilisé pour cela un modèle simple de l'eau pour décrire des solutions aqueuses de différentes particules. Récemment, l?eau liquide a été décrite comme une structure formée d?un réseau aléatoire de liaisons hydrogènes. En introduisant une particule hydrophobe dans cette structure à basse température, certaines liaisons hydrogènes sont détruites ce qui est énergétiquement défavorable. Les molécules d?eau s?arrangent alors autour de cette particule en formant une cage qui permet de récupérer des liaisons hydrogènes (entre molécules d?eau) encore plus fortes : les particules sont alors solubles dans l?eau. A des températures plus élevées, l?agitation thermique des molécules devient importante et brise les liaisons hydrogènes. Maintenant, la dissolution des particules devient énergétiquement défavorable, et les particules se séparent de l?eau en formant des agrégats qui minimisent leur surface exposée à l?eau. Pourtant, à très haute température, les effets entropiques deviennent tellement forts que les particules se mélangent de nouveau avec les molécules d?eau. En utilisant un modèle basé sur ces changements de structure formée par des liaisons hydrogènes j?ai pu reproduire les phénomènes principaux liés à l?hydrophobicité. J?ai trouvé une région de coexistence de deux phases entre les températures critiques inférieure et supérieure de solubilité, dans laquelle les particules hydrophobes s?agrègent. En dehors de cette région, les particules sont dissoutes dans l?eau. J?ai démontré que l?interaction hydrophobe est décrite par un modèle qui prend uniquement en compte les changements de structure de l?eau liquide en présence d?une particule hydrophobe, plutôt que les interactions directes entre les particules. Encouragée par ces résultats prometteurs, j?ai étudié des solutions aqueuses de particules hydrophobes en présence de co-solvants cosmotropiques et chaotropiques. Ce sont des substances qui stabilisent ou déstabilisent les agrégats de particules hydrophobes. La présence de ces substances peut être incluse dans le modèle en décrivant leur effet sur la structure de l?eau. J?ai pu reproduire la concentration élevée de co-solvants chaotropiques dans le voisinage immédiat de la particule, et l?effet inverse dans le cas de co-solvants cosmotropiques. Ce changement de concentration du co-solvant à proximité de particules hydrophobes est la cause principale de son effet sur la solubilité des particules hydrophobes. J?ai démontré que le modèle adapté prédit correctement les effets implicites des co-solvants sur les interactions de plusieurs corps entre les particules hydrophobes. En outre, j?ai étendu le modèle à la description de particules amphiphiles comme des lipides. J?ai trouvé la formation de différents types de micelles en fonction de la distribution des regions hydrophobes à la surface des particules. L?hydrophobicité reste également un sujet controversé en science des protéines. J?ai défini une nouvelle échelle d?hydrophobicité pour les acides aminés qui forment des protéines, basée sur leurs surfaces exposées à l?eau dans des protéines natives. Cette échelle permet une comparaison meilleure entre les expériences et les résultats théoriques. Ainsi, le modèle développé dans mon travail contribue à mieux comprendre les solutions aqueuses de particules hydrophobes. Je pense que les résultats analytiques et numériques obtenus éclaircissent en partie les processus physiques qui sont à la base de l?interaction hydrophobe.<br/><br/>Despite the importance of water in our daily lives, some of its properties remain unexplained. Indeed, the interactions of water with organic particles are investigated in research groups all over the world, but controversy still surrounds many aspects of their description. In my work I have tried to understand these interactions on a molecular level using both analytical and numerical methods. Recent investigations describe liquid water as random network formed by hydrogen bonds. The insertion of a hydrophobic particle at low temperature breaks some of the hydrogen bonds, which is energetically unfavorable. The water molecules, however, rearrange in a cage-like structure around the solute particle. Even stronger hydrogen bonds are formed between water molecules, and thus the solute particles are soluble. At higher temperatures, this strict ordering is disrupted by thermal movements, and the solution of particles becomes unfavorable. They minimize their exposed surface to water by aggregating. At even higher temperatures, entropy effects become dominant and water and solute particles mix again. Using a model based on these changes in water structure I have reproduced the essential phenomena connected to hydrophobicity. These include an upper and a lower critical solution temperature, which define temperature and density ranges in which aggregation occurs. Outside of this region the solute particles are soluble in water. Because I was able to demonstrate that the simple mixture model contains implicitly many-body interactions between the solute molecules, I feel that the study contributes to an important advance in the qualitative understanding of the hydrophobic effect. I have also studied the aggregation of hydrophobic particles in aqueous solutions in the presence of cosolvents. Here I have demonstrated that the important features of the destabilizing effect of chaotropic cosolvents on hydrophobic aggregates may be described within the same two-state model, with adaptations to focus on the ability of such substances to alter the structure of water. The relevant phenomena include a significant enhancement of the solubility of non-polar solute particles and preferential binding of chaotropic substances to solute molecules. In a similar fashion, I have analyzed the stabilizing effect of kosmotropic cosolvents in these solutions. Including the ability of kosmotropic substances to enhance the structure of liquid water, leads to reduced solubility, larger aggregation regime and the preferential exclusion of the cosolvent from the hydration shell of hydrophobic solute particles. I have further adapted the MLG model to include the solvation of amphiphilic solute particles in water, by allowing different distributions of hydrophobic regions at the molecular surface, I have found aggregation of the amphiphiles, and formation of various types of micelle as a function of the hydrophobicity pattern. I have demonstrated that certain features of micelle formation may be reproduced by the adapted model to describe alterations of water structure near different surface regions of the dissolved amphiphiles. Hydrophobicity remains a controversial quantity also in protein science. Based on the surface exposure of the 20 amino-acids in native proteins I have defined the a new hydrophobicity scale, which may lead to an improvement in the comparison of experimental data with the results from theoretical HP models. Overall, I have shown that the primary features of the hydrophobic interaction in aqueous solutions may be captured within a model which focuses on alterations in water structure around non-polar solute particles. The results obtained within this model may illuminate the processes underlying the hydrophobic interaction.<br/><br/>La vie sur notre planète a commencé dans l'eau et ne pourrait pas exister en son absence : les cellules des animaux et des plantes contiennent jusqu'à 95% d'eau. Malgré son importance dans notre vie de tous les jours, certaines propriétés de l?eau restent inexpliquées. En particulier, l'étude des interactions entre l'eau et les particules organiques occupe des groupes de recherche dans le monde entier et est loin d'être finie. Dans mon travail j'ai essayé de comprendre, au niveau moléculaire, ces interactions importantes pour la vie. J'ai utilisé pour cela un modèle simple de l'eau pour décrire des solutions aqueuses de différentes particules. Bien que l?eau soit généralement un bon solvant, un grand groupe de molécules, appelées molécules hydrophobes (du grecque "hydro"="eau" et "phobia"="peur"), n'est pas facilement soluble dans l'eau. Ces particules hydrophobes essayent d'éviter le contact avec l'eau, et forment donc un agrégat pour minimiser leur surface exposée à l'eau. Cette force entre les particules est appelée interaction hydrophobe, et les mécanismes physiques qui conduisent à ces interactions ne sont pas bien compris à l'heure actuelle. Dans mon étude j'ai décrit l'effet des particules hydrophobes sur l'eau liquide. L'objectif était d'éclaircir le mécanisme de l'interaction hydrophobe qui est fondamentale pour la formation des membranes et le fonctionnement des processus biologiques dans notre corps. Récemment, l'eau liquide a été décrite comme un réseau aléatoire formé par des liaisons hydrogènes. En introduisant une particule hydrophobe dans cette structure, certaines liaisons hydrogènes sont détruites tandis que les molécules d'eau s'arrangent autour de cette particule en formant une cage qui permet de récupérer des liaisons hydrogènes (entre molécules d?eau) encore plus fortes : les particules sont alors solubles dans l'eau. A des températures plus élevées, l?agitation thermique des molécules devient importante et brise la structure de cage autour des particules hydrophobes. Maintenant, la dissolution des particules devient défavorable, et les particules se séparent de l'eau en formant deux phases. A très haute température, les mouvements thermiques dans le système deviennent tellement forts que les particules se mélangent de nouveau avec les molécules d'eau. A l'aide d'un modèle qui décrit le système en termes de restructuration dans l'eau liquide, j'ai réussi à reproduire les phénomènes physiques liés à l?hydrophobicité. J'ai démontré que les interactions hydrophobes entre plusieurs particules peuvent être exprimées dans un modèle qui prend uniquement en compte les liaisons hydrogènes entre les molécules d'eau. Encouragée par ces résultats prometteurs, j'ai inclus dans mon modèle des substances fréquemment utilisées pour stabiliser ou déstabiliser des solutions aqueuses de particules hydrophobes. J'ai réussi à reproduire les effets dûs à la présence de ces substances. De plus, j'ai pu décrire la formation de micelles par des particules amphiphiles comme des lipides dont la surface est partiellement hydrophobe et partiellement hydrophile ("hydro-phile"="aime l'eau"), ainsi que le repliement des protéines dû à l'hydrophobicité, qui garantit le fonctionnement correct des processus biologiques de notre corps. Dans mes études futures je poursuivrai l'étude des solutions aqueuses de différentes particules en utilisant les techniques acquises pendant mon travail de thèse, et en essayant de comprendre les propriétés physiques du liquide le plus important pour notre vie : l'eau.

Systematic evaluation of matrix effects in hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry.

Reversed phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the gold standard technique in bioanalysis. However, hydrophilic interaction chromatography (HILIC) could represent a viable alternative to RPLC for the analysis of polar and/or ionizable compounds, as it often provides higher MS sensitivity and alternative selectivity. Nevertheless, this technique can be also prone to matrix effects (ME). ME are one of the major issues in quantitative LC-MS bioanalysis. To ensure acceptable method performance (i.e., trueness and precision), a careful evaluation and minimization of ME is required. In the present study, the incidence of ME in HILIC-MS/MS and RPLC-MS/MS was compared for plasma and urine samples using two representative sets of 38 pharmaceutical compounds and 40 doping agents, respectively. The optimal generic chromatographic conditions in terms of selectivity with respect to interfering compounds were established in both chromatographic modes by testing three different stationary phases in each mode with different mobile phase pH. A second step involved the assessment of ME in RPLC and HILIC under the best generic conditions, using the post-extraction addition method. Biological samples were prepared using two different sample pre-treatments, i.e., a non-selective sample clean-up procedure (protein precipitation and simple dilution for plasma and urine samples, respectively) and a selective sample preparation, i.e., solid phase extraction for both matrices. The non-selective pretreatments led to significantly less ME in RPLC vs. HILIC conditions regardless of the matrix. On the contrary, HILIC appeared as a valuable alternative to RPLC for plasma and urine samples treated by a selective sample preparation. Indeed, in the case of selective sample preparation, the compounds influenced by ME were different in HILIC and RPLC, and lower and similar ME occurrence was generally observed in RPLC vs. HILIC for urine and plasma samples, respectively. The complementary of both chromatographic modes was also demonstrated, as ME was observed only scarcely for urine and plasma samples when selecting the most appropriate chromatographic mode.

Quantification of the hydrophobic interaction by simulations of the aggregation of small hydrophobic solutes in water

The hydrophobic interaction, the tendency for nonpolar molecules to aggregate in solution, is a major driving force in biology. In a direct approach to the physical basis of the hydrophobic effect, nanosecond molecular dynamics simulations were performed on increasing numbers of hydrocarbon solute molecules in water-filled boxes of different sizes. The intermittent formation of solute clusters gives a free energy that is proportional to the loss in exposed molecular surface area with a constant of proportionality of 45 ± 6 cal/mol⋅Å2. The molecular surface area is the envelope of the solute cluster that is impenetrable by solvent and is somewhat smaller than the more traditional solvent-accessible surface area, which is the area transcribed by the radius of a solvent molecule rolled over the surface of the cluster. When we apply a factor relating molecular surface area to solvent-accessible surface area, we obtain 24 cal/mol⋅Å2. Ours is the first direct calculation, to our knowledge, of the hydrophobic interaction from molecular dynamics simulations; the excellent qualitative and quantitative agreement with experiment proves that simple van der Waals interactions and atomic point-charge electrostatics account for the most important driving force in biology.

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding similar to42 and 36 mg EPF from 300 ml bacterial and I L Sf9 cultures, respectively. The preparations were highly purified ( greater than or equal to99% purity on SDS-PAGE for the bacterial products and greater than or equal to97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immuno suppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. (C) 2003 Elsevier Inc. All rights reserved.

Heat resistance of Bacillus spores when adhered to stainless steel and its relationship to spore hydrophobicity

Twenty-one strains of Bacillus (10 B. stearothermophilus, 3 B. cereus, and 8 B. licheniformis strains) were assayed for spore surface hydrophobicity on the basis of three measures: contact angle measurement (CAM), microbial adhesion to hydrocarbons (MATH), and hydrophobic interaction chromatography (HIC). On the basis of the spore surface characteristics obtained from these assays, along with data on the heat resistance of these spores in water, eight strains of Bacillus (three B. stearothermophilus, three B. cereus, and two B. licheniformis strains) either suspended in water or adhering to stainless steel were exposed to sublethal heat treatments at 90 to 110degreesC to determine heat resistance (D-value). Significant increases in heat resistance (ranging from 3 to 400%) were observed for the eight strains adhering to stainless steel. No significant correlation was found between these heat resistance increases and spore surface characteristics as determined by the three hydrophobicity assays. There was a significant positive correlation between the hydrophobicity data obtained by the MATH assay and those obtained by the HIC assay, but these data did not correlate with those obtained by the CAM assay.

Different in Vivo Reactivity Profile in Health Care Workers and Patients with Spina Bifida to Internal and External Latex Glove Surface-Derived Allergen Extracts

BACKGROUND: Allergy to natural rubber latex is a well-recognized health problem, especially among health care workers and patients with spina bifida. Despite latex sensitization being acquired in health institutions in both health care workers and patients with spina bifida, differences in allergen sensitization profiles have been described between these two risk groups. OBJECTIVE: To investigate the in vivo reactivity of health care workers and patients with spina bifida to extracts of internal and external surfaces of latex gloves and also to specific extracts enriched in major allergens for these risk groups. METHODS: Gloves from different manufacturers were used for protein extraction, and salt precipitation and hydrophobic interaction chromatography (HIC) were applied to obtain the enriched latex extracts. The major latex allergens were quantified by an enzyme immunoassay. The extracts obtained were tested in 14 volunteers using skin prick tests (SPT). RESULTS: Latex glove extracts enriched in the hydrophobic allergens that are most often seen in patients with spina bifida were obtained by selective precipitation, whereas HIC produced extracts enriched in the hydrophilic allergens commonly found in health care workers. The health care workers had positive SPTs to glove extracts from internal surfaces and to the hydrophilic allergen-enriched extracts. By contrast, patients with spina bifida had larger skin reactions both to external glove extracts and to the extracts enriched with the hydrophobic major allergens for this risk group. Despite the protein concentration of these extracts being less than half the concentration of the commercial extract, the weal-and-flare reactions were of similar magnitude. CONCLUSION: Using novel latex extracts, our study showed a different in vivo reactivity pattern in health care workers and in patients with spina bifida to extracts of the internal and external surfaces of gloves, which suggests that sensitization may occur by different routes of exposure, and that this influences the allergen reactivity profiles of these risk groups

Analysis of toxoplasma gondii proteins after Triton X-114 solubilization and hidropholic chromotograhy

The distribution of the surface proteins of toxoplasma gondii radiodinated were studied using the phase separation technique and ability of binding in the phenyl-Sepharose column. Eight polypeptides with Mr 22 to 180 distributed exclusively in the detergent rich-phase, while six polypeptides with mol. wt. 15,000 to 76,000 distributed exclusively in the detergent poor-phase. Twopolypeptides with 15,000 and 70,000 distributed on both phase. All the polypeptides present in the detergent rich-phase binding in the phenyl-Sepharose column, and can be isolated in two peak according with their relative hydrophobicities.two polypeptides hydrophobic with Mr 60 and 66 recognized by human serum were isolated by the association of the two technique. Our result showed that the surface proteins of t. gondii present different degrees of hydrophobicity and that the use of hydrophobic interaction chromatography after Triton X-114 extraction may be an important isolation method of membrane proteins.

Purification and Partial Characterization of Trypanosoma cruzi Triosephosphate Isomerase

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins

Partial purification of tightly bound mitochondrial hexokinase from maize (Zea mays L.) root membranes

In mammals, hexokinase (HK) is strategically located at the outer membrane of mitochondria bound to the porin protein. The mitochondrial HK is a crucial modulator of apoptosis and reactive oxygen species generation. In plants, these properties related to HK are unknown. In order to better understand the physiological role of non-cytosolic hexokinase (NC-HK) in plants, we developed a purification strategy here described. Crude extract of 400 g of maize roots (230 mg protein) contained a specific activity of 0.042 µmol G6P min-1 mg PTN-1. After solubilization with detergent two fractions were obtained by DEAE column chromatography, NC-HK 1 (specific activity = 3.6 µmol G6P min-1 mg PTN-1 and protein recovered = 0.7 mg) and NC-HK 2. A major purification (yield = 500-fold) was obtained after passage of NC-HK 1 through the hydrophobic phenyl-Sepharose column. The total amount of protein and activity recovered were 0.04 and 18%, respectively. The NC-HK 1 binds to the hydrophobic phenyl-Sepharose matrix, as observed for rat brain HK. Mild chymotrypsin digestion did not affect adsorption of NC-HK 1 to the hydrophobic column as it does for rat HK I. In contrast to mammal mitochondrial HK, glucose-6-phosphate, clotrimazole or thiopental did not dissociate NC-HK from maize (Zea mays) or rice (Oryza sativa) mitochondrial membranes. These data show that the interaction between maize or rice NC-HK to mitochondria differs from that reported in mammals, where the mitochondrial enzyme can be displaced by modulators or pharmacological agents known to interfere with the enzyme binding properties with the mitochondrial porin protein.