Tuotanto- ja pakkausmateriaalien varastojen nykytila ja kehittäminen lääkeannostelijoita valmistavassa tehtaassa

Diplomityön tarkoituksena oli selvittää lääkeannostelijoita valmistavan tehtaan materiaalivarastojen nykytila. Varaston nykytila-analyysi jaettiin pääoma- ja operatiivisen tehokkuuden arviointiin. Työssä haluttiin kartoittaa toimenpide-ehdotuksia varastonohjauksen tehostamiseksi. Diplomityön tarkoituksena oli myös lisätä avainhenkilöiden ymmärrystä koskien varastojen pidon vaikutuksia yrityksen liiketoimintaan. Varastojen nykytila voitiin kiteyttää suureen pääoman sitoutumiseen, hitaisiin kiertoaikoihin sekä tehokkaan työskentelyn kannalta liian pieniin ja ahtaisiin varastoihin. Kustannuksia ei ole otettu kokonaisuudessaan huomioon tilattaessa ja varastoitaessa materiaalia. Varaston hitaita kiertonopeuksia ei ole tunnistettu vaikuttajina yrityksen tulokseen ja kannattavuuteen. Jotta varastonohjaus- ja hankintaprosesseja voitaisiin tehostaa, pitäisi ostotoimintoihin tehdä muutoksia. Tilausrytmiä ja –määriä täytyisi harkita uudelleen, jotta voitaisiin vähentää varastoihin sitoutuvan pääoman määrää. Informaationvirtaa tehtaalla sekä organisaatiorajojen yli pitäisi parantaa, jotta voitaisiin vähentää kysynnän heilahteluja sekä siihen varautumista. Varmuusvarastojen pidosta saatavien korvausten olisi katettava koko materiaalin kiertoajan aikana syntyvät varastoinnin kustannukset.

Electroactive ion exchange membranes based on conducting polymers

Ion exchange membranes are indispensable for the separation of ionic species. They can discriminate between anions and cations depending on the type of fixed ionic group present in the membrane. These conventional ion exchange membranes (CIX) have exceptional ionic conductivity, which is advantageous in various electromembrane separation processes such as electrodialysis, electrodeionisation and electrochemical ion exchange. The main disadvantage of CIX membranes is their high electrical resistance owing to the fact that the membranes are electronically non conductive. An alternative can be electroactive ion exchange membranes, which are ionically and electronically conducting. Polypyrrole (PPy) is a type of electroactive ion exchange material as well as a commonly known conducting polymer. When PPy membranes are repeatedly reduced and oxidised, ions are pumped through the membrane. The main aim of this thesis was to develop electroactive cation transport membranes based on PPy for the selective transport of divalent cations. Membranes developed composed of PPy films deposited on commercially available support materials. To carry out this study, cation exchange membranes based on PPy doped with immobile anions were prepared. Two types of dopant anions known to interact with divalent metal ions were considered, namely 4-sulphonic calix[6]arene (C6S) and carboxylated multiwalled carbon nanotubes (CNT). The transport of ions across membranes containing PPy doped with polystyrene sulphonate (PSS) and PPy doped with para-toluene sulphonate (pTS) was also studied in order to understand the nature of ion transport and permeability across PPy(CNT) and PPy(C6S) membranes. In the course of these studies, membrane characterisation was performed using electrochemical quartz crystal microbalance (EQCM) and scanning electron microscopy (SEM). Permeability of the membranes towards divalent cations was explored using a two compartment transport cell. EQCM results demonstrated that the ion exchange behaviour of polypyrrole is dependent on a number of factors including the type of dopant anion present, the type of ions present in the surrounding medium, the scan rate used during the experiment and the previous history of the polymer film. The morphology of PPy films was found to change when the dopant anion was varied and even when the thickness of the film was altered in some cases. In nearly all cases the permeability of the membranes towards metal ions followed the order K+ > Ca2+ > Mn2+. The one exception was PPy(C6S), for which the permeability followed the order Ca2+ ≥ K+ > Mn2+ > Co2+ > Cr3+. The above permeability sequences show a strong dependence on the size of the metal ions with metal ions having the smallest hydrated radii exhibiting the highest flux. Another factor that affected the permeability towards metal ions was the thickness of the PPy films. Films with the least thickness showed higher metal ion fluxes. Electrochemical control over ion transport across PPy(CNT) membrane was obtained when films composed of the latter were deposited on track-etched Nucleopore® membranes as support material. In contrast, the flux of ions across the same film was concentration gradient dependent when the polymer was deposited on polyvinylidene difluoride membranes as support material. However, electrochemical control over metal ion transport was achieved with a bilayer type of PPy film consisting of PPy(pTS)/PPy(CNT), irrespective of the type of support material. In the course of studying macroscopic charge balance during transport experiments performed using a two compartment transport cell, it was observed that PPy films were non-permselective. A clear correlation between the change in pH in the receiving solution and the ions transported across the membrane was observed. A decrease in solution pH was detected when the polymer membrane acted primarily as an anion exchanger, while an increase in pH occurred when it functioned as a cation exchanger. When there was an approximately equal flux of anions and cations across the polymer membrane, the pH in the receiving solution was in the range 6 - 8. These observations suggest that macroscopic charge balance during the transport of cations and anions across polypyrrole membranes was maintained by introduction of anions (OH-) and cations (H+) produced via electrolysis of water.

AMB system for high-speed motors using automatic commissioning

The general trend towards increasing e ciency and energy density drives the industry to high-speed technologies. Active Magnetic Bearings (AMBs) are one of the technologies that allow contactless support of a rotating body. Theoretically, there are no limitations on the rotational speed. The absence of friction, low maintenance cost, micrometer precision, and programmable sti ness have made AMBs a viable choice for highdemanding applications. Along with the advances in power electronics, such as signi cantly improved reliability and cost, AMB systems have gained a wide adoption in the industry. The AMB system is a complex, open-loop unstable system with multiple inputs and outputs. For normal operation, such a system requires a feedback control. To meet the high demands for performance and robustness, model-based control techniques should be applied. These techniques require an accurate plant model description and uncertainty estimations. The advanced control methods require more e ort at the commissioning stage. In this work, a methodology is developed for an automatic commissioning of a subcritical, rigid gas blower machine. The commissioning process includes open-loop tuning of separate parts such as sensors and actuators. The next step is to apply a system identi cation procedure to obtain a model for the controller synthesis. Finally, a robust model-based controller is synthesized and experimentally evaluated in the full operating range of the system. The commissioning procedure is developed by applying only the system components available and a priori knowledge without any additional hardware. Thus, the work provides an intelligent system with a self-diagnostics feature and an automatic commissioning.

Lignoselluloosapohjaisten väkevähappohydrolysaattien jatkuvatoiminen kromatografinen fraktiointi

Työssä tutkittiin lignoselluloosapohjaisten väkevähappohydrolysaattien (monosakkaridit, rikki- ja etikkahppo) jatkuvatoimista kromatografista fraktiointia käyttäen Japan Organo SMB -prosessia. Adsorbenttinä toimi happomuotoinen (H+) vahva kationinvaihtohartsi. Panostoimista fraktiointia käytettiin vertailukohtana. Jatkuvatoimisen prosessin optimoinnilla saavutettiin monosakkaridien suurimmaksi tuottavuudeksi 283 mol/(m3 h) (panosprosessille 145 mol/(m3 h). JO-prosessilla saavutettiin korkeat rikki- ja etikkahapon saannot: 97,3 % ja 93,5 %. Monosakkaridisaanto jäi hieman alhaisemmaksi (61,7 %) johtuen monosakkaridi- ja rikkihappo-profiilien hankalasta erottamisesta. Ulostulo virtojen puhtaudet olivat korkeat: 89 % monosakkarideille, 89 % rikkihapolle ja 100 % etikkahapolle.

System-level characterization of Th2 cell development and immune cell responses to ZnO and TiO2 nanoparticles

Asthma and allergy are common diseases and their prevalence is increasing. One of the hypotheses that explains this trend is exposure to inhalable chemicals such as traffi c-related air pollution. Epidemiological research supports this theory, as a correlation between environmental chemicals and allergic respiratory diseases has been found. In addition to ambient airborne particles, one may be exposed to engineered nanosized materials that are actively produced due to their favorable physico-chemical properties compared to their bulk size counterparts. On the cellular level, improper activity of T helper (Th) cells has been connected to allergic reactions. Th cells can differentiate into functionally different effector subsets, which are identifi ed according to their characteristic cytokine profi les resulting in specifi c ability to communicate with other cells. Th2 cells activate humoral immunity and stimulate eradication of extracellular pathogens. However, persistent predominance of Th2 cells is involved in a development of number of allergic diseases. The cytokine environment at the time of antigen recognition is the major factor determining the polarization of a naïve Th cell. Th2 cell differentiation is initiated by IL4, which signals via transcription factor STAT6. Although the importance of this pathway has been evaluated in the mouse studies, the signaling components involved have been largely unknown. The aim of this thesis was to identify molecules, which are under the control of IL4 and STAT6 in Th cells. This was done by using system-level analysis of STAT6 target genes at genome, mRNA and protein level resulting in identifi cation of various genes previously not connected to Th2 cell phenotype acquisition. In the study, STAT6-mediated primary and secondary target genes were dissection from each other and a detailed transcriptional kinetics of Th2 cell polarization of naïve human CD4+ T cells was collected. Integration of these data revealed the hierarchy of molecular events that mediates the differentiation towards Th2 cell phenotype. In addition, the results highlighted the importance of exploiting proteomics tools to complement the studies on STAT6 target genes identifi ed through transcriptional profi ling. In the last subproject, the effects of the exposure with ZnO and TiO2 nanoparticles was analyzed in Jurkat T cell line and in primary human monocyte-derived macrophages and dendritic cells to evaluate their toxicity and potential to cause infl ammation. Identifi cation of ZnO-derived gene expression showed that the same nanoparticles may elicit markedly distinctive responses in different cell types, thus underscoring the need for unbiased profi ling of target genes and pathways affected. The results gave additional proof that the cellular response to nanosized ZnO is due to leached Zn2+ ions. The approach used in ZnO and TiO2 nanoparticle study demonstrated the value of assessing nanoparticle responses through a toxicogenomics approach. The increased knowledge of Th2 cell signaling will hopefully reveal new therapeutic nodes and eventually improve our possibilities to prevent and tackle allergic infl ammatory diseases.

Metallien eluointi kelatoivasta ioninvaihtimesta

Kelatoivat ioninvaihtimet ovat yleensä makrohuokoisia hartseja, joiden avulla metalleja poistetaan ja otetaan mahdollisesti talteen teollisuuden puhdistettavista jätevirroista. Ne muodostavat metalli-ionien kanssa komplekseja runkomateriaaliin kovalenttisesti liitettyjen aktiivisten funktionaalisten ryhmiensä välityksellä. Ioninvaihtimen selektiivisyys eri metalli-ioneja kohtaan vaihtelee riippuen siihen liitetystä kelatoivasta ryhmästä. Työssä tutkitaan metallien tarttumista kelatoiviin ioninvaihtimiin, sekä niiden eluoitumista adsorbentistaan. Kelatoivat ioninvaihtimet sitovat metalli-ioneja tehokkaasti ligandiensa monihampaisuudesta johtuen. Metallien tarttuminen kelatoivaan ioninvaihtimeen ei kuitenkaan ole yksiselitteistä, vaan siihen vaikuttaa muun muassa pH yhdessä monen muun tekijän kanssa. Ioninvaihtimien selektiivisyyttä tarkastellaan työssä lähinnä kovien ja pehmeiden happojen ja emästen HSAB -teorian näkökulmasta. Regeneroinnilla ioninvaihdin saadaan jälleen alkuperäiseen muotoonsa, minkä jälkeen se voidaan käyttää uudelleen. Yleensä regenerointi suoritetaan kemiallisesti. Tässä työssä ioninvaihtimien regenerointia ja kohdemetallin eluointia tarkastellaan paitsi teoriassa, myös kokeellisessa osuudessa. Kokeellisessa osuudessa tutkitaan kuparin (Cu2+) eluoitumista kelatoivasta Dowex M-4195 kationinvaihtohartsista. Kokeissa hartsi ladattiin kuparilla erillisessä panoksessa kuparin ollessa syöttöliuoksessa kuparisulfaattina. Eluointiliuoksina käytettiin 2 ja 5 molaarista rikkihappoa, sekä 2 molaarista ammoniumhydroksidia. Eluointi suoritettiin panostoimisena kolonniajona ja eluaatista otetut näytteet analysoitiin atomiadsorptiospektrofotometrillä. Analyysitulokset esitetään läpäisykäyrinä, joiden perusteella 2M ammoniumhydroksidi on kolmesta tutkitusta eluentista tehokkain eluoimaan kuparia Dowex M-4195 hartsista.

Research and development of III-V semiconductor surfaces for improved device interfaces

This thesis is devoted to understanding and improving technologically important III-V compound semiconductor (e.g. GaAs, InAs, and InSb) surfaces and interfaces for devices. The surfaces and interfaces of crystalline III-V materials have a crucial role in the operation of field-effect-transistors (FET) and highefficiency solar-cells, for instance. However, the surfaces are also the most defective part of the semiconductor material and it is essential to decrease the amount of harmful surface or interface defects for the next-generation III-V semiconductor device applications. Any improvement in the crystal ordering at the semiconductor surface reduces the amount of defects and increases the material homogeneity. This is becoming more and more important when the semiconductor device structures decrease to atomic-scale dimensions. Toward that target, the effects of different adsorbates (i.e., Sn, In, and O) on the III-V surface structures and properties have been investigated in this work. Furthermore, novel thin-films have been synthesized, which show beneficial properties regarding the passivation of the reactive III-V surfaces. The work comprises ultra-high-vacuum (UHV) environment for the controlled fabrication of atomically ordered III-V(100) surfaces. The surface sensitive experimental methods [low energy electron diffraction (LEED), scanning tunneling microscopy/spectroscopy (STM/STS), and synchrotron radiation photoelectron spectroscopy (SRPES)] and computational density-functionaltheory (DFT) calculations are utilized for elucidating the atomic and electronic properties of the crucial III-V surfaces. The basic research results are also transferred to actual device tests by fabricating metal-oxide-semiconductor capacitors and utilizing the interface sensitive measurement techniques [capacitance voltage (CV) profiling, and photoluminescence (PL) spectroscopy] for the characterization. This part of the thesis includes the instrumentation of home-made UHV-compatible atomic-layer-deposition (ALD) reactor for growing good quality insulator layers. The results of this thesis elucidate the atomic structures of technologically promising Sn- and In-stabilized III-V compound semiconductor surfaces. It is shown that the Sn adsorbate induces an atomic structure with (1×2)/(1×4) surface symmetry which is characterized by Sn-group III dimers. Furthermore, the stability of peculiar ζa structure is demonstrated for the GaAs(100)-In surface. The beneficial effects of these surface structures regarding the crucial III-V oxide interface are demonstrated. Namely, it is found that it is possible to passivate the III-V surface by a careful atomic-scale engineering of the III-V surface prior to the gate-dielectric deposition. The thin (1×2)/(1×4)-Sn layer is found to catalyze the removal of harmful amorphous III-V oxides. Also, novel crystalline III-V-oxide structures are synthesized and it is shown that these structures improve the device characteristics. The finding of crystalline oxide structures is exploited by solving the atomic structure of InSb(100)(1×2) and elucidating the electronic structure of oxidized InSb(100) for the first time.

Persistence of the biocide activity of atrazine in soils of the southeast of Buenos Aires Province

Atrazine persistence in soils of the southeast of Buenos Aires Province, was studied by an oat bioassay. Atrazine doses of 0.58, 1.16, and 2.32 mg.g-1 dry soil weight (DSW) were applied to pots containing soils from Balcaree, A. Gonzáles Chaves and San Cayetano sites, whose organic matter (OM) content of soils were 5.70, 5.15, and 3,84%, respectively. Avena sativa cv. Millauquén plants were grownth in the pots under greenhouse conditions at different times after atrazine application. Shoots were evenly cut above the soil and dry weight determined as a measure of plant growth. Plants grown in non-sprayed soil were used as controls. Relative dry weight (RDW) of shoots was calculated as percentage of control. Atrazine phytotoxicity was expressed in terms of 50 % plant growth reduction (GR50) in the soils under study. Herbicide persistence was expressed in terms of days after treatment (DAT) needed for the plant to achieve 80% of RDW. Atrazine GR50 values of 0.30, 0.64, and 0.90 mg.g-1 DSW in soils from San Cayetano, Balcare and A.G. Chaves, were respectively obtained at 42 DAT. Herbicide persistences at the recommended dose (1.16 mg.g-1) were 100, 143, and 221 DAT for A.G. Chaves, Balcarce and San Cayetano soils, respectively. San Cayetano soil had both the lowest OM content and cation exchange capacity (CEC), as well as the highest pH, of all the soil studied here. These results were consistent with both the lowest GR50 and the highest persistence abtained for atrazine in this soil.

Chromatographic recovery of chemicals from acidic biomass hydrolysates

Lignocellulosic biomasses (e.g., wood and straws) are a potential renewable source for the production of a wide variety of chemicals that could be used to replace those currently produced by petrochemical industry. This would lead to lower greenhouse gas emissions and waste amounts, and to economical savings. There are many possible pathways available for the manufacturing of chemicals from lignocellulosic biomasses. One option is to hydrolyze the cellulose and hemicelluloses of these biomasses into monosaccharides using concentrated sulfuric acid as catalyst. This process is an efficient method for producing monosaccharides which are valuable platforn chemicals. Also other valuable products are formed in the hydrolysis. Unfortunately, the concentrated acid hydrolysis has been deemed unfeasible mainly due to high chemical consumption resulting from the need to remove sulfuric acid from the obtained hydrolysates prior to the downstream processing of the monosaccharides. Traditionally, this has been done by neutralization with lime. This, however, results in high chemical consumption. In addition, the by-products formed in the hydrolysis are not removed and may, thus, hinder the monosaccharide processing. In order to improve the feasibility of the concentrated acid hydrolysis, the chemical consumption should be decreased by recycling of sulfuric acid without neutralization. Furthermore, the monosaccharides and the other products formed in the hydrolysis should be recovered selectively for efficient downstream processing. The selective recovery of the hydrolysis by-products would have additional economical benefits on the process due to their high value. In this work, the use of chromatographic fractionation for the recycling of sulfuric acid and the selective recovery of the main components from the hydrolysates formed in the concentrated acid hydrolysis was investigated. Chromatographic fractionation based on the electrolyte exclusion with gel type strong acid cation exchange resins in acid (H+) form as a stationary phase was studied. A systematic experimental and model-based study regarding the separation task at hand was conducted. The phenomena affecting the separation were determined and their effects elucidated. Mathematical models that take accurately into account these phenomena were derived and used in the simulation of the fractionation process. The main components of the concentrated acid hydrolysates (sulfuric acid, monosaccharides, and acetic acid) were included into this model. Performance of the fractionation process was investigated experimentally and by simulations. Use of different process options was also studied. Sulfuric acid was found to have a significant co-operative effect on the sorption of the other components. This brings about interesting and beneficial effects in the column operations. It is especially beneficial for the separation of sulfuric acid and the monosaccharides. Two different approaches for the modelling of the sorption equilibria were investigated in this work: a simple empirical approach and a thermodynamically consistent approach (the Adsorbed Solution theory). Accurate modelling of the phenomena observed in this work was found to be possible using the simple empirical models. The use of the Adsorbed Solution theory is complicated by the nature of the theory and the complexity of the studied system. In addition to the sorption models, a dynamic column model that takes into account the volume changes of the gel type resins as changing resin bed porosity was also derived. Using the chromatography, all the main components of the hydrolysates can be recovered selectively, and the sulfuric acid consumption of the hydrolysis process can be lowered considerably. Investigation of the performance of the chromatographic fractionation showed that the highest separation efficiency in this separation task is obtained with a gel type resin with a high crosslinking degree (8 wt. %); especially when the hydrolysates contain high amounts of acetic acid. In addition, the concentrated acid hydrolysis should be done with as low sulfuric acid concentration as possible to obtain good separation performance. The column loading and flow rate also have large effects on the performance. In this work, it was demonstrated that when recycling of the fractions obtained in the chromatographic fractionation are recycled to preceding unit operations these unit operations should included in the performance evaluation of the fractionation. When this was done, the separation performance and the feasibility of the concentrated acid hydrolysis process were found to improve considerably. Use of multi-column chromatographic fractionation processes, the Japan Organo process and the Multi-Column Recycling Chromatography process, was also investigated. In the studied case, neither of these processes could compete with the single-column batch process in the productivity. However, due to internal recycling steps, the Multi-Column Recycling Chromatography was found to be superior to the batch process when the product yield and the eluent consumption were taken into account.

Unexpected effects of pigeon-peas (Cajanus cajan) in the restoration of rupestrian fields

Several degraded areas can be found along the Highway MG-010 that crosses the Espinhaço Mountain Biosphere Reserve in the Brazilian state of Minas Gerais. Restoration by planting the legume Cajanus cajan was implemented in some of these areas. The present study compares plant species richness, diversity, abundance, equitability, similarity, and soil composition between restored and non-restored areas, in an attempt to evaluate the effectiveness of the use of C. cajan in the restoration process in the mountain environment. Each treatment (restored and non-restored) had four sampling areas, each with three 300 m² plots. We counted and identified every individual plant found within these plots. We also collected soil from the superficial layer (0-10 cm) of each sampling area in both treatments. The areas where C. cajan was planted revealed lower species richness, diversity, and plant abundance. The soil of these areas also contained higher levels of Phosphorus and Magnesium. Plant equitability and similarity between plots and other soil components (pH, Nitrogen, Aluminum, Calcium, Potassium, H+Al, sum of bases - SB, cation exchange capacity - CTC, base saturation - V%, aluminum saturation - M%) did not differ between the two treatments. Contrary to the expectations, soil enhancement in the quartzitic soil poor in nutrients in the rupestrian fields can facilitate the invasion by exotic plants, which are not adapted to the lack of nutrients. As it appears, the use of C. cajan in restoration projects represents a mistake and future restoration plans should avoid the use of exotic species, given that they may cause negative effects on the native plant community, as demonstrated here in the rupestrian fields.

A new brain metalloendopeptidase which degrades the Alzheimer ß-amyloid 1-40 peptide producing soluble fragments without neurotoxic effects

A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human brain using successive steps of chromatography on DEAE-Trisacryl, hydroxylapatite and Sephacryl S-200. The purified enzyme cleaved the Gly33-Leu34 bond of the 25-35 neurotoxic sequence of the Alzheimer ß-amyloid 1-40 peptide producing soluble fragments without neurotoxic effects. This enzyme activity was only inhibited by divalent cation chelators such as EDTA, EGTA and o-phenanthroline (1 mM) and was insensitive to phosphoramidon and captopril (1 µM concentration), specific inhibitors of neutral endopeptidase (EC 3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), respectively. The high affinity of this human brain endopeptidase for ß-amyloid 1-40 peptide (Km = 5 µM) suggests that it may play a physiological role in the degradation of this substance produced by normal cellular metabolism. It may also be hypothesized that the abnormal accumulation of the amyloid ß-protein in Alzheimer's disease may be initiated by a defect or an inactivation of this enzyme.

Outward potassium current oscillations in macrophage polykaryons: extracellular calcium entry and calcium-induced calcium release

Outward current oscillations associated with transient membrane hyperpolarizations were induced in murine macrophage polykaryons by membrane depolarization in the absence of external Na+. Oscillations corresponded to a cyclic activation of Ca2+-dependent K+ currents (IKCa) probably correlated with variations in intracellular Ca2+ concentration. Addition of external Na+ (8 mM) immediately abolished the outward current oscillations, suggesting that the absence of the cation is necessary not only for their induction but also for their maintenance. Oscillations were completely blocked by nisoldipine. Ruthenium red and ryanodine reduced the number of outward current cycles in each episode, whereas quercetin prolonged the hyperpolarization 2- to 15-fold. Neither low molecular weight heparin nor the absence of a Na+ gradient across the membrane had any influence on oscillations. The evidence suggests that Ca2+ entry through a pathway sensitive to Ca2+ channel blockers is elicited by membrane depolarization in Na+-free medium and is essential to initiate oscillations, which are also dependent on the cyclic release of Ca2+ from intracellular Ca2+-sensitive stores; Ca2+ ATPase acts by reducing intracellular Ca2+, thus allowing slow deactivation of IKCa. Evidence is presented that neither a Na+/Ca2+ antiporter nor Ca2+ release from IP3-sensitive Ca2+ stores participate directly in the mechanism of oscillation

A single-step purification of bothropstoxin-1

Bothrops venoms are complex mixtures of components with a wide range of biological activities. Among these substances, myotoxins have been investigated by several groups. Bothropstoxin-1 (Bthtx-1) is a phospholipase A2-like basic myotoxin from Bothrops jararacussu. The purification of this component involves two chromatographic steps. Although providing a pure material, the association of these two steps is time consuming and a single-step method using high performance chromatography media would be useful. In the present study, we describe a single-step purification method for Bthtx-1. Bothrops jararacussu venom was dissolved in 1 ml buffer. After centrifugation, the supernatant was injected into a Resource-S cation exchange column connected to an FPLC system and eluted with a linear salt gradient. The complete procedure took 20 min, representing a considerable time gain when compared to a previously described method (Homsi-Brandenburgo MI et al. (1988) Toxicon, 26: 615-627). Bthtx-1 purity and identity, assessed by SDS-PAGE and N-terminal sequencing, resulted in a single band with a molecular mass of about 14 kDa and the expected sequence of the first 5 residues, S-L-F-E-L. Although the amount of protein purified after each run is lower than in the previously described method, we believe that this method may be useful for small-scale purifications.

Erythrocyte glucose-6-phosphate dehydrogenase activity assay and affinity for its substrate under "physiological" conditions

Glucose-6-phosphate dehydrogenase (G6PD) activity and the affinity for its substrate glucose-6-phosphate were investigated under conditions similar to the physiological environment in terms of ionic strength (I: 0.188), cation concentration, pH 7.34, and temperature (37oC). A 12.4, 10.4 and 21.4% decrease was observed in G6PD B, G6PD A+ and G6PD A- activities, respectively. A Km increase of 95.1, 94.4 and 95.4% was observed in G6PD B, G6PD A+ and G6PD A-, respectively, leading to a marked decrease in affinity. In conclusion, the observation of the reduced activity and affinity for its natural substrate reflects the actual pentose pathway rate. It also suggests a much lower NADPH generation, which is crucial mostly in G6PD-deficient individuals, whose NADPH availability is poor.

A liver metalloendopeptidase which degrades the circulating hypotensive peptide hormones bradykinin and atrial natriuretic peptide

A new metalloendopeptidase was purified to apparent homogeneity from a homogenate of normal human liver using successive steps of chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-200. The purified enzyme hydrolyzed the Pro7-Phe8 bond of bradykinin and the Ser25-Tyr26 bond of atrial natriuretic peptide. No cleavage was produced in other peptide hormones such as vasopressin, oxytocin or Met- and Leu-enkephalin. This enzyme activity was inhibited by 1 mM divalent cation chelators such as EDTA, EGTA and o-phenanthroline and was insensitive to 1 µM phosphoramidon and captopril, specific inhibitors of neutral endopeptidase (EC 3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), respectively. With Mr 85 kDa, the enzyme exhibits optimal activity at pH 7.5. The high affinity of this endopeptidase for bradykinin (Km = 10 µM) and for atrial natriuretic peptide (Km = 5 µM) suggests that it may play a physiological role in the inactivation of these circulating hypotensive peptide hormones.