High Throughput Purification of Combinatorial Libraries by Preparative LC/MS

Viime vuosien nopea kehitys on kiihdyttänyt uusien lääkkeiden kehittämisprosessia. Kombinatorinen kemia on tehnyt mahdolliseksi syntetisoida suuria kokoelmia rakenteeltaan toisistaan poikkeavia molekyylejä, nk. kombinatorisia kirjastoja, biologista seulontaa varten. Siinä molekyylien rakenteeseen liittyvä aktiivisuus tutkitaan useilla erilaisilla biologisilla testeillä mahdollisten "osumien" löytämiseksi, joista osasta saatetaan myöhemmin kehittää uusia lääkeaineita. Jotta biologisten tutkimusten tulokset olisivat luotettavia, on syntetisoitujen komponenttien oltava mahdollisimman puhtaita. Tämän vuoksi tarvitaan HTP-puhdistusta korkealaatuisten komponenttien ja luotettavan biologisen tiedon takaamiseksi. Jatkuvasti kasvavat tuotantovaatimukset ovat johtaneet näiden puhdistustekniikoiden automatisointiin ja rinnakkaistamiseen. Preparatiivinen LC/MS soveltuu kombinatoristen kirjastojen nopeaan ja tehokkaaseen puhdistamiseen. Monet tekijät, esimerkiksi erotuskolonnin ominaisuudet sekä virtausgradientti, vaikuttavat preparatiivisen LC/MS puhdistusprosessin tehokkuuteen. Nämä parametrit on optimoitava parhaan tuloksen saamiseksi. Tässä työssä tutkittiin emäksisiä komponentteja erilaisissa virtausolosuhteissa. Menetelmä kombinatoristen kirjastojen puhtaustason määrittämiseksi LC/MS-puhdistuksen jälkeen optimoitiin ja määritettiin puhtaus joillekin komponenteille eri kirjastoista ennen puhdistusta.

STUDY ON THE SUGAR-ACID RATIO AND RELEVANT METABOLIZING ENZYME ACTIVITIES IN NAVEL ORANGE FRUITS FROM DIFFERENT ECO-REGIONS

ABSTRACT The flavor quality of citrus fruits is largely determined by the sugar-acid ratio, but it remains uncertain how sugar- and/or acid-metabolizing enzymes regulate the sugar-acid ratio of navel oranges and further affect the fruit quality. In the present study, Robertson navel oranges (Citrus sinesis Osb.) were collected from six representative habitats in three eco-regions of Sichuan, China. The changes in the sugar-acid ratio and the activities of sucrose phosphate synthase (SPS), sucrose synthase (SS), cytosolic cio-aconitase (ACO), and isocitrate dehydrogenase (IDH) were examined in navel oranges during fruit development. The results indicated that the sugar-acid ratio of fruits in different eco-regions changed significantly from 150 days after full bloom. The SPS and cytosolic ACO fruit activities had minor changes among different ecoregions throughout the experimental periods, whereas the activities of SS and IDH changed significantly in fruits among three eco-regions. Furthermore, the sugar-acid ratio and the activities of SS in the synthetic direction and IDH were the highest in south subtropics and the lowest in north mid-subtropics, probably due to the effects of climate conditions and/or other relevant eco-factors. It demonstrated that SS in the synthetic direction and IDH were of greater importance in regulating the sugar-acid ratio of navel oranges in different eco-regions, which provided new insights into the factors that determine the flavor quality of navel oranges and valuable data for guiding relevant agricultural practices.

Post-mortem determination of insulin using chemiluminescence enzyme immunoassay: preliminary results.

Insulin determination in blood sampled during post-mortem investigation has been repeatedly asserted as being of little diagnostic value due to the rapid occurrence of decompositional changes and blood haemolysis. In this study, we assessed the feasibility of insulin determination in post-mortem serum, vitreous humour, bile, and cerebrospinal and pericardial fluids in one case of fatal insulin self-administration and a series of 40 control cases (diabetics and non-diabetics) using a chemiluminescence enzyme immunoassay. In the case of suicide by insulin self-administration, insulin concentrations in pericardial fluid and bile were higher than blood clinical reference values, though lower than post-mortem serum concentration. Insulin concentrations in vitreous (11.50 mU/L) and cerebrospinal fluid (17.30 mU/L) were lower than blood clinical reference values. Vitreous insulin concentrations in non-diabetic control cases were lower than the estimated detection limit of the method. These preliminary results tend to confirm the usefulness of insulin determination in vitreous humour in situations of suspected fatal insulin administration. Additional findings pertaining to insulin determination in bile, pericardial, and cerebrospinal fluid would suggest that analysis performed in post-mortem serum and injection sites could be complemented, in individual cases, by investigations carried out in alternative biological fluids. Lastly, these results would indicate that analysis with chemiluminescence enzyme immunoassay may provide suitable data, similar to analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoradiometric assay, to support the hypothesis of insulin overdose. Copyright © 2015 John Wiley & Sons, Ltd.

Immunolocalization of glyoxysomal malate synthase from soybean cotyledons (Glycine max. L.)

Malate synthase (MS; EC 4.1.3.2), an enzyme specific to the glyoxylate cycle, was studied in cotyledons of dark-grown soybean (Glycine max L) seedlings with light and electron microscopy techniques. Immunogold localization confirmed biochemical evidence that MS from soybean is a glyoxysomal matrix enzyme.

Glyoxysomal malate-dehydrogenase and malate synthase from Soybean cotyledons (Glycine max. L.): Enzyme association, antibody-production and cDNA cloning

In order to investigate a possible association between soybean malate synthase (MS; L-malate glyoxylate-lyase, CoA-acetylating, EC 4.1.3.2) and glyoxysomal malate dehydrogenase (gMDH; (S)-malate: NAD(+) oxidoreductase, EC 1.1.1.37), two consecutive enzymes in the glyoxylate cycle, their elution profiles were analyzed on Superdex 200 HR fast protein liquid chromatography columns equilibrated in low- and high-ionic-strength buffers. Starting with soluble proteins extracted from the cotyledons of 5-d-old soybean seedlings and a 45% ammonium sulfate precipitation, MS and gMDH coeluted on Superdex 200 HR (low-ionic-strength buffer) as a complex with an approximate relative molecular mass (M(r)) of 670000. Dissociation was achieved in the presence of 50 mM KCl and 5 mM MgCl2, with the elution of MS as an octamer of M, 510 000 and of gMDH as a dimer of M, 73 000. Polyclonal antibodies raised to the native copurified enzymes recognized both denatured MS and gMDH on immunoblots, and their native forms after gel filtration. When these antibodies were used to screen a lambda ZAP II expression library containing cDNA from 3-d-old soybean cotyledons, they identified seven clones encoding gMDH, whereas ten clones encoding MS were identified using an antibody to SDS-PAGE-purified MS. Of these cDNA clones a 1.8 kb clone for MS and a 1.3-kb clone for gMDH were fully sequenced. While 88% identity was found between mature soybean gMDH and watermelon gMDH, the N-terminal transit peptides showed only 37% identity. Despite this low identity, the soybean gMDH transit peptide conserves the consensus R(X(6))HL motif also found in plant and mammalian thiolases.

Lipid mobilization and gluconeogenesis in plants: Do glyoxylate cycle enzyme activities constitute a real cycle? A hypothesis

Glyoxysomes are specialized peroxisomes present in various plant organs such as germinating cotyledons or senescing leaves. They are the site of beta-oxidation and of the glyoxylate cycle. These consecutive pathways are essential to the maintenance of gluconeogenesis initiated by the degradation of reserve or structural lipids. In contrast to mitochondrial beta-oxidation, which is prevalent in animal cells, glyoxysomal beta-oxidation and the glyoxylate cycle have no direct access to the mitochondrial respiratory chain because of the impermeability of the glyoxysomal membrane to the reduced cofactors. The necessity of NAD(+) regeneration can conceivably be fulfilled by membrane redox chains and/or by transmembrane shuttles. Experimental evidence based on the active metabolic roles of higher plant glyoxysomes and yeast peroxisomes suggests the coexistence of two mechanisms, namely a reductase/peroxidase membrane redox chain and a malate/aspartate shuttle susceptible to transfer electrons to the mitochondrial ATP generating system. Such a model interconnects beta-oxidation, the glyoxylate cycle, the respiratory chain and gluconeogenesis in such a way that glyoxysomal malate dehydrogenase is an essential and exclusive component of beta-oxidation (NAD(+) regeneration). Consequently, the classical view of the glyoxylate cycle is superseded by a tentative reactional scheme deprived of cyclic character.

Fast and economic immobilization methods described for non-commercial Pseudomonas lipases

There is an increasing interest to seek new enzyme preparations for the development of new products derived from bioprocesses to obtain alternative bio-based materials. In this context, four non-commercial lipases from Pseudomonas species were prepared, immobilized on different low-cost supports, and examined for potential biotechnological applications. Results: To reduce costs of eventual scaling-up, the new lipases were obtained directly from crude cell extracts or from growth culture supernatants, and immobilized by simple adsorption on Accurel EP100, Accurel MP1000 and Celite (R) 545. The enzymes evaluated were LipA and LipC from Pseudomonas sp. 42A2, a thermostable mutant of LipC, and LipI. 3 from Pseudomonas CR611, which were produced in either homologous or heterologous hosts. Best immobilization results were obtained on Accurel EP100 for LipA and on Accurel MP1000 for LipC and its thermostable variant. Lip I. 3, requiring a refolding step, was poorly immobilized on all supports tested ( best results for Accurel MP1000). To test the behavior of immobilized lipases, they were assayed in triolein transesterification, where the best results were observed for lipases immobilized on Accurel MP1000. Conclusions: The suggested protocol does not require protein purification and uses crude enzymes immobilized by a fast adsorption technique on low-cost supports, which makes the method suitable for an eventual scaling up aimed at biotechnological applications. Therefore, a fast, simple and economic method for lipase preparation and immobilization has been set up. The low price of the supports tested and the simplicity of the procedure, skipping the tedious and expensive purification steps, will contribute to cost reduction in biotechnological lipase-catalyzed processes.

CYP17A1 Enzyme activity is linked to ambulatory blood pressure in a family-based population study.

BACKGROUND: Genome-wide association studies have linked CYP17A1 coding for the steroid hormone synthesizing enzyme 17α-hydroxylase (CYP17A1) to blood pressure (BP). We hypothesized that the genetic signal may translate into a correlation of ambulatory BP (ABP) with apparent CYP17A1 activity in a family-based population study and estimated the heritability of CYP17A1 activity. METHODS: In the Swiss Kidney Project on Genes in Hypertension, day and night urinary excretions of steroid hormone metabolites were measured in 518 participants (220 men, 298 women), randomly selected from the general population. CYP17A1 activity was assessed by 2 ratios of urinary steroid metabolites: one estimating the combined 17α-hydroxylase/17,20-lyase activity (ratio 1) and the other predominantly 17α-hydroxylase activity (ratio 2). A mixed linear model was used to investigate the association of ABP with log-transformed CYP17A1 activities exploring effect modification by urinary sodium excretion. RESULTS: Daytime ABP was positively associated with ratio 1 under conditions of high, but not low urinary sodium excretion (P interaction <0.05). Ratio 2 was not associated with ABP. Heritability estimates (SE) for day and night CYP17A1 activities were 0.39 (0.10) and 0.40 (0.09) for ratio 1, and 0.71 (0.09) and 0.55 (0.09) for ratio 2 (P values <0.001). CYP17A1 activities, assessed with ratio 1, were lower in older participants. CONCLUSIONS: Low apparent CYP17A1 activity (assessed with ratio 1) is associated with elevated daytime ABP when salt intake is high. CYP17A1 activity is heritable and diminished in the elderly. These observations highlight the modifying effect of salt intake on the association of CYP17A1 with BP.

Effects of Cytochrome P450 Enzyme Inhibitors on the Pharmacokinetics of Nonsteroidal Anti-inflammatory Drugs and Venlafaxine

Cytochrome P450 (CYP) enzymes play a pivotal role in the metabolism of many drugs. Inhibition of CYP enzymes usually increases the plasma concentrations of their substrate drugs and can thus alter the safety and efficacy of these drugs. The metabolism of many widely used nonsteroidal antiinflammatory drugs (NSAIDs) as well as the metabolism of the antidepressant venlafaxine is nown to be catalyzed by CYP enzymes. In the present studies, the effect of CYP inhibition on the armacokinetics and pharmacodynamics of NSAIDs and venlafaxine was studied in clinical trials with healthy volunteers and with a crossover design, by using different antifungal agents as CYP inhibitors. The results of these studies demonstrate that the inhibition of CYP enzymes leads to increased concentrations of NSAIDs. In most cases, the exposure to ibuprofen, diclofenac, etoricoxib, and meloxicam was increased 1.5to 2 fold when they were used concomitantly with antifungal agents. CYP2D6 inhibitor, terbinafine, substantially increased the concentration of parent venlafaxine, whereas the concentration of active moiety of venlafaxine (parent drug plus active metabolite) was only slightly increased. Voriconazole, an inhibitor of the minor metabolic pathway of venlafaxine, produced only minor changes in the pharmacokinetics of venlafaxine. These studies show that an evident increase in the concentrations of NSAIDs may be expected, if they are used concomitantly with CYP inhibitors. However, as NSAIDs are generally well tolerated, use of single doses of NSAIDs concomitantly with CYP inhibitors is not likely to adversely affect patient safety, whereas clinical relevance of longterm concomitant use of NSAIDs with CYP inhibitors needs further investigation. CYP2D6 inhibitors considerably affect the pharmacokinetics of venlafaxine, but the clinical significance of this interaction remains unclear.

Synthesis of angiotensin-converting enzyme (ACE) inhibitors: an important class of antihypertensive drugs

This report outlines the discovery, the design and development of new compounds, and, structure-activity relationships for this drug category. Updated approaches to planned syntheses of new worthy ACE-inhibitors are also exploited.