Kinetic and thermodynamic investigation on clavulanic acid formation and degradation during glycerol fermentation by Streptomyces DAUFPE 3060

Clavulanic acid (CA) is a potent inhibitor of beta-lactamases, produced by some resistant pathogenic microorganisms, which allows efficient treatment of infectious diseases. The kinetic and thermodynamic parameters of CA production by a new isolate of Streptomyces DAUFPE 3060 and its degradation were evaluated. The effect of temperature on the system was investigated in the range 24-40 degrees C adopting an overall model accounting for (a) the Arrhenius-type formation of CA by fermentation, (b) the hypothetical reversible unfolding of the enzyme limiting the overall metabolism, and (c) the irreversible first-order degradation of CA. The higher rates of CA formation (k(CA) = 0,107 h(-1)) and degradation (k(d) = 0.062 h(-1)) were observed at 32 and 40 degrees C, respectively. The main thermodynamic parameters of the three above hypothesized events were estimated. In particular, the activation parameters of degradation (activation energy = 39.0 kJ/mol; Delta H(d)* = 36.5 kJ/mol; Delta S(d)* = -219.7 J/(mol K); Delta G(d)* = 103.5 kJ/mol) compare reasonably well with those reported in the literature for similar system without taking into account the other two events. (C) 2009 Elsevier Inc. All rights reserved.

Investigation of charged polymer influence on green fluorescent protein thermal stability

Methods of stabilization and formulation of proteins are important in both biopharmaceutical and biocatalysis industries. Polymers are often used as modifiers of characteristics of biological macromolecules to improve the biochemical activity and stability of proteins or drug bioavailability. Green fluorescent protein (GFP) shows remarkable structural stability and high fluorescence; its stability can be directly related to its fluorescence output, among other characteristics. GFP is stable under increasing temperatures, and its thermal denaturation is highly reproducible. Relative thermal stability was undertaken by incubation of GFP at varying temperatures and GFP fluorescence was used as a reporter for unfolding. At 80 degrees C, DEAE-dextran did not have any effect on GFP fluorescence, indicating that it does not confer stability.

Stereoselective analysis of thioridazine-2-sulfoxide and thioridazine-5-sulfoxide: An investigation of rac-thioridazine biotransformation by some endophytic fungi

The purpose of this study was to develop a method for the stereoselective analysis of thioridazine-2-sulfoxide (THD-2-SO) and thioridazine-5-sulfoxide (THD-5-SO) in culture medium and to study the biotransformation of rac-thioridazine (THD) by some endophytic fungi. The simultaneous resolution of THD-2-SO and THD-5-SO diastereoisomers was performed on a CHIRALPAK(R) AS column using a mobile phase of hexane: ethanol: methanol (92:6:2, v/v/v) + 0.5% diethylamine; UV detection was carried out at 262 nm. Diethyl ether was used as extractor solvent. The validated method was used to evaluate the biotransformation of THD by 12 endophytic fungi isolated from Tithonia diversifolia, Viguiera arenaria and Viguiera robusta. Among the 12 fungi evaluated, 4 of them deserve prominence for presenting an evidenced stereoselective biotransformation potential: Phomopsis sp. (TD2) presented greater mono-2-sulfoxidation to the form (S)-(SE) (12.1%); Glomerella cingulata (VA1) presented greater mono-5-sulfoxidation to the forms (S)-(SE) + (R)-(FE) (10.5%); Diaporthe phaseolorum (VR4) presented greater mono-2-sulfoxidation to the forms (S)-(SE) and (R)-(FE) (84.4% and 82.5%, respectively) and Aspergillus fumigatus (VR12) presented greater mono-2-sulfoxidation to the forms (S)-(SE) and (R)-(SE) (31.5% and 34.4%, respectively). (C) 2007 Elsevier B.V. All rights reserved.

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm x 4.6 mm, 5 mu m) at 35 degrees C under isocratic conditions using 5 mM KH(2)PO(4) buffer solution pH 6.0: methanol: diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min(-1). UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL, plasma using ethyl acetate and Na(2)HPO(4) pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5,4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether: n-hexane: methanol (50:30:20). In both methods the solvent was evaporated at 40 degrees C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL(-1) plasma (r >= 0.9927), limits of quantification of 50 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL(-1) plasma (r >= 0.9900), limits of quantification of 30 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis. (c) 2008 Elsevier B.V. All rights reserved.

INVESTIGATION OF ACOUSTIC SIGNALS AS A TOOL FOR CHARACTERIZING SPOUTED BED DYNAMICS

The feasibility of characterizing the dynamics of a spouted bed based on acoustic emission (AE) signals is evaluated. Acoustic emission signals were measured in a semi-cylindrical Plexiglas column of diameter 150 mm and height 1000 mm with a conical base of internal angle 60 degrees and 25 mm inlet orifice diameter. Data were obtained for U/U(ms), from 0.3 to 2.0, static bed height from 250 to 500 mm, and glass beads of diameter 1.2 and 2.4 mm. AE signals reflected the effects of particle size and U/U(ms), but in general were insensitive to bed depth, even when there were drastic changes in spouting flow patterns. The results indicate that the AE signals were insensitive to the spouted bed hydrodynamics for the conditions studied. Overall, it appears that the AE analysis is unlikely to be a suitable technique for discriminating spouted bed flow regimes, at least for the range of frequencies and operating conditions investigated.

Morphosyntactic and syntactic priming: an investigation of underlying processing mechanisms and the effects of Parkinson’s disease

There is now considerable evidence to suggest that non-demented people with Parkinson's disease (PD) experience difficulties using the morphosyntactic aspects of language. It remains unclear, however, at precisely which point in the processing of morphosyntax, these difficulties emerge. The major objective of the present study was to examine the impact of PD on the processes involved in accessing morphosyntactic information in the lexicon. Nineteen people with PD and 19 matched control subjects participated in the study which employed on-line word recognition tasks to examine morphosyntactic priming for local grammatical dependencies that occur both within (e.g. is going) and across (e.g. she gives) phrasal boundaries (Experiments 1 and 2, respectively). The control group evidenced robust morphosyntactic priming effects that were consistent with the involvement of both pre- (Experiment 1) and post-lexical (Experiment 2) processing routines. Whilst the participants with PD also recorded priming for dependencies within phrasal boundaries (Experiment 1), priming effects were observed over an abnormally brief time course. Further, in contrast to the controls, the PD group failed to record morphosyntactic priming for constructions that crossed phrasal boundaries (Experiment 2). The results demonstrate that attentionally mediated mechanisms operating at both the pre- and post-lexical stages of processing are able to contribute to morphosyntactic priming effects. In addition, the findings support the notion that, whilst people with PD are able to access morphosyntactic information in a normal manner, the time frame in which this information remains available for processing is altered. Deficits may also be experienced at the post-lexical integrational stage of processing.

Allelic Variation Investigation of the Estrogen Receptor Within an Australian Multiple Sclerosis Population

Multiple Sclerosis (MS) is a central nervous system (CNS) chronic inflammatory demyelinating disease leading to various neurological disabilities. The disorder is more prevalent for women with a ratio of 3:2 female to male. Objectives: To investigate variation within the estrogen receptor 1 (ESR1) polymorphism gene in an Australian MS case-control population using two intragenic restriction fragment length polymorphisms; the G594A located in exon 8 detected with the BtgI restriction enzyme and T938C located in intron 1, detected with PvuII. One hundred and ten Australian MS patients were studied, with patients classified clinically as Relapsing Remitting MS (RR-MS), Secondary Progressive MS (SP-MS) or Primary Progressive MS (PP-MS). Also, 110 age, sex and ethnicity matched controls were investigated as a comparative group. No significant difference in the allelic distribution frequency was found between the case and control groups for the ESR1 PvuII (P = 0.50) and Btg1 (P = 0.45) marker. Our results do not support a role for these two ESR1 markers in multiple sclerosis susceptibility, however other markers within ESR1 should not be excluded for potential involvement in the disorder.

Investigation of the role of cadmium sulfide in the surface passivation of lead sulfide quantum dots

Surface passivation of PbS nanocrystals (NC), resulting in strong photoluminescence, can be achieved by the introduction of CdS precursors. The role of CdS in the surface passivation of PbS NCs is uncertain, as the crystalline structure of CdS and PbS are different, which should impede effective epitaxial overgrowth. Absorption spectroscopy is used to show that the CdS precursors strongly interact with the PbS NC surface. Electron microscopy reveals that the introduction of CdS precursors results in an increased particle size, consistent with overcoating. However, we also find the process to be highly non-uniform. Nevertheless, evidence for epitaxial growth is found, suggesting that effective surface passivation may be possible.

Strategic argumentation: A game theoretical investigation

Argumentation is modelled as a game where the payoffs are measured in terms of the probability that the claimed conclusion is, or is not, defeasibly provable, given a history of arguments that have actually been exchanged, and given the probability of the factual premises. The probability of a conclusion is calculated using a standard variant of Defeasible Logic, in combination with standard probability calculus. It is a new element of the present approach that the exchange of arguments is analysed with game theoretical tools, yielding a prescriptive and to some extent even predictive account of the actual course of play. A brief comparison with existing argument-based dialogue approaches confirms that such a prescriptive account of the actual argumentation has been almost lacking in the approaches proposed so far.

How many ways can a VP split? An investigation of the structure of VP shells in Japanese Causatives

The Japanese inchoative-lexical causative pair poses an interesting problem for the Minimalist Program – how should the lexical causative and the syntactic causative be structurally represented and theoretically accounted for? The lexical causative verb and the syntactic causative verb formed by suffixing the syntactic causative morpheme sase onto the inchoative counterpart are both single causative constructions that are semantically similar. Yet, they differ in some ways, most significantly in their clausality – the lexical causative is monoclausal in nature while the syntactic causative is biclausal, comparable to English biclausal constructions formed with let or force. This paper investigates how this difference can be represented by investigating the possible VP shell structures of different Japanese sentences, and the analysis from the discussion suggests a different structure where a CP is embedded into a higher VP shell as the sister of Agro head.

Molecular cocrystals of carboxylic acids. XXVII - An investigation into the use of triphenylphosphine oxide cocrystals for non-linear optics: the crystal structure of the adduct of triphenylphosphine oxide with N-methylpyrrole-2-carboxylic acid

Four adducts of triphenylphosphine oxide with aromatic carboxylic acids have been synthesized and tested for second-order non-linear optical properties. These were with N-methylpyrrole-2-carboxylic acid (I), indole-2-carboxylic acid (2), 3-dimethylaminobenzoic acid (3), and thiophen-2-carboxylic acid (4). Compound (1) produced clear, colourless crystals (space group P2(1)2(1)2(1) With a 9.892(1), b 14.033(1), c 15.305(1) Angstrom, Z 4) which allowed the structure to be determined by X-ray diffraction.

An investigation of the effect of powder on the impact characteristics between a ball and a plate using free falling experiments

The study of the mechanisms of mechanical alloying requires knowledge of the impact characteristics between the ball and vial in the presence of milling powders. In this paper, foe falling experiments have br cn used to investigate the characteristics of impact events involved in mechanical milling. The effects of milling conditions, including impact velocity, ball size and powder thickness. on the coefficient of restitution and impact force are studied. It is found that the powder has a significant influence on the impact process due to its porous structure. This effect can be demonstrated using a modified Kelvin model. This study also confirms that the impact force is a relevant parameter for characterising the impact event due to its sensitivity to the milling conditions. (C) 1998 Elsevier Science S.A.

Investigation of the antinociceptive efficacy and relative potency of extended duration injectable 3-acylmorphine-6-sulfate prodrugs in rats

This investigation was designed to examine the antinociceptive activity in rats of 3-O-acyl prodrugs of M6S relative to the parent drug, after intravenous and intramuscular injection, using the tail flick latency test of antinociception. M6S, 3-acetylmorphine-6-sulfate (3AcM6S), 3-propionylmorphine-6-sulfate (3PrM6S), 3-butanoylmorphine-6-sulfate (3BuM6S) and 3-heptanoylmorphine-6-sulfate (3HpM6S) were administered by the IV route in a dose of 4.10 mu mol/kg. Relatively high levels of antinociception (>40% Maximum Possible Effect) were achieved following administration of M6S, 3AcM6S and 3PrM6S, whereas insignificant antinociception (<20%MPE) was achieved following administration of 3BuM6S or 3HpM6S. Although the mean duration of action for 3AcM6S (6 h) was longer than for M6S or 3PrM6S (4 h), the mean area (+/- S.E.M.) under the degree of antinociception versus time curve (AUG) for 3AcM6S (151.6 +/- 6.9%MPE h) was not significantly different (p <0.05) from that for M6S (120.8 +/- 32.7%MPE h) or for 3PrM6S (106.0 +/- 21.3%MPE h). The mean ED50 (range) doses for M6S, 3AcM6S and 3PrM6S were calculated to be 4.16 (3.61-4.48), 4.32 (3.55-5.09) and 4.54 (4.21-4.79) mu mol/kg, respectively. Preliminary studies were conducted on potential long-acting formulations containing 8 x ED50 doses of M6S and the 3-acetyl and 3-propionyl esters suspended in soybean oil. These showed that 3PrM6S gave a greater AUC (mean + S.E.M.) (1087.4 +/- 97.4%MPE h) and longer duration of action (20 h) than did M6S (613.1 +/- 155.9%MPE h; 10 h duration) or 3AcM6S (379.3 + 114.2%MPE h: 8 h duration). Further studies are needed to more fully investigate these findings. (C) 1998 Elsevier Science B.V. All rights reserved.

Crystal structures of reduced and oxidized DsbA: investigation of domain motion and thiolate stabilization

Background: The redox proteins that incorporate a thioredoxin fold have diverse properties and functions. The bacterial protein-folding factor DsbA is the most oxidizing of the thioredoxin family. DsbA catalyzes disulfide-bond formation during the folding of secreted proteins, The extremely oxidizing nature of DsbA has been proposed to result from either domain motion or stabilizing active-site interactions in the reduced form. In the domain motion model, hinge bending between the two domains of DsbA occurs as a result of redox-related conformational changes. Results: We have determined the crystal structures of reduced and oxidized DsbA in the same crystal form and at the same pH (5.6). The crystal structure of a lower pH form of oxidized DsbA has also been determined (pH 5.0). These new crystal structures of DsbA, and the previously determined structure of oxidized DsbA at pH 6.5, provide the foundation for analysis of structural changes that occur upon reduction of the active-site disulfide bond. Conclusions: The structures of reduced and oxidized DsbA reveal that hinge bending motions do occur between the two domains. These motions are independent of redox state, however, and therefore do not contribute to the energetic differences between the two redox states, instead, the observed domain motion is proposed to be a consequence of substrate binding. Furthermore, DsbA's highly oxidizing nature is a result of hydrogen bond, electrostatic and helix-dipole interactions that favour the thiolate over the disulfide at the active site.

An investigation of the interrelationship between manipulative therapy-induced hypoalgesia and sympathoexcitation

Objective: To investigate a proposed model in which manipulative therapy produces a treatment-specific initial hypoalgesic and sympathoexcitatory effect by activating a descending pain inhibitory system. The a priori hypothesis tested was that manipulative therapy produces mechanical hypoalgesia and sympatho-excitation beyond that produced by placebo or control. Furthermore, these effects would be correlated, thus supporting the proposed model. Design: A randomized, double-blind, placebo-controlled, repeated-measures study of the initial effect of treatment. Setting: Clinical neurophysiology laboratory. Subjects: Twenty-four subjects (13 women and 11 men; mean age, 49 yr) with chronic lateral epicondylalgia (average duration, 6.2 months). Intervention: Cervical spine lateral glide oscillatory manipulation, placebo and control. Outcome Measures: Pressure pain threshold, thermal pain threshold, pain-free grip strength test, upper limb tension test 2b, skin conductance, pileous and glabrous skin temperature and blood flux. Results: Treatment produced hypoalgesic and sympathoexcitatory changes significantly grater than those of placebo and control (p < .03). Confirmatory factor-analysis modeling, which was performed on the pain-related measures and the indicators of sympathetic nervous system function, demonstrated a significant correlation (r = .82) between the latencies of manipulation-induced hypoalgesia and sympathoexcitation. The Lagrange Multiplier test and Wald test indicated that the two latent factors parsimoniously and appropriately represented their observed variables. Conclusions: Manual therapy produces a treatment-specific initial hypoalgesic and sympathoexcitatory effect beyond that of placebo or control. The strong correlation between hypoalgesic and sympathoexcitatory effects suggests that a central control mechanism might be activated by manipulative therapy.