Browsing by Subject "inhibitio"

MRP2 is an efflux-transporter from the group of ABC-transporters located in the apical side of cell membranes mainly in the liver, intestine, kidneys and lungs. This transporter is associated with multidrug resistance, a phenomenon where the absorption of a drug to the cell is prevented by the transporter as it transports the compound out of the cell. To overcome this phenomenon, inhibitors and substrates for MRP2 are constantly studied. Several flavonoids have been presented of being inhibitors and the research of these compounds continues. Pharmaceutical excipients are also another major group of compounds that possess inhibitory effects towards MRP2. Excipients, as well as flavonoids, are an increasingly studied section of drug interactions and today it may be evaluated that excipients are not thought as inert compounds as has been presented for several years. For now the research of MRP2 interactions focuses mainly on in vitro studies. In the experimental part of this thesis the effects of natural compounds and pharmaceutical excipients are studied towards MRP2 with the vesicular transport assay (VT-assay) with MRP2- Spodoptera frugiperda 9 (Sf9)-membrane vesicles using 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF) as probe. A total of 157 compounds are screened using this in vitro method and hits are further experimented studying IC50 and Ki values. Potential compounds are also tested with two types of particle size measurements (Dynamic light scattering and nephelometer) to evaluate inhibition caused by microaggregates. Some compounds are also studied with liquid chromatograph-mass spectrometry (LC-MS) to determine possible substrates for MRP2. 19 (12%) hits were found from the library of 157 compounds. These hits included 6 stimulators (CDCF transport increased ≥ 150%) and 13 inhibitors (CDCF transport decreased ≤ 50%). IC50 determination was conducted for 12 inhibitors with best-fit values of: Ellagic acid 10.4 µM, gossypin 17.4 µM, morin dihydrate 19.4 µM, myricetin 27.1 µM, nordihydroguaiaretic acid (NDGA) 36.2 µM, octyl gallate 20.3 µM, silybin 52.3 µM, pluronic ®F98 6.9 µM, lutrol F127 ~ 8.2 µM and tannic acid 1.99 µM. Ki determination was conducted for 3 compounds where best-fit values were myricetin 42.9 ± 47.4 µM, gossypin 19.4 ± 12.5 µM and tannic acid 0.0538 ± 0.0398 µM. Ki determination allowed determination of inhibition type: competitive inhibition for tannic acid and gossypin, noncompetitive inhibition for myricetin. Particle sizes studied with dynamic light scattering (DLS) and a nephelometer did not show any significant aggregate formation and inhibition by that mechanism can be ruled out granted that the measurement method should be optimised. Stimulators baicalein, baicalin, digitoxigenin and inhibitors myricetin, gossypin and tannic acid were studied finally with the VT-assay with LC-MS as detector in search of substrates for MRP2. With significant changes in ‚àíATP and +ATP at 50 µM was gossypin. To conclude, gossypin possesses competitive inhibition towards MRP2 and exhibits sings of being a substrate for the transporter as well. Further studies need to be performed to confirm these findings.

Production of biofuels from non-food-based materials, such as lignocellulose, provides a good alternative for the traditional burning of fossil fuels. Some of the researched and existing biofuel applications are based on the utilization of enzymes. There are multiple cellulolytic enzymes required in the efficient hydrolysis of lignocellulose, and one of the key enzyme group is β-glucosidases. These enzymatic systems are mainly adopted from wood-decaying fungi. The overall enzymatic system consists of different types of cellulases that first degrade the crystalline cellulose to oligosaccharides and cellobiose. In the final step, β-glucosidases hydrolyse the oligosaccharides to glucose (a fermentable sugar). In fact, β-glucosidases are one of the limiting enzyme classes in this process, due to phenomena such as end-product inhibition. β-Glucosidases belong to Glycoside hydrolases (GH), that can be classified into different protein families. In an industrial perspective, the main interest resides in GH1 and GH3 family enzymes. Many industrially relevant extracellular β-glucosidases belong to GH3 family. However, intracellular GH1 β-glucosidases often exhibit higher tolerance to harsh conditions such as high substrate and product concentrations, high temperatures and low pH. The goal of this MSc thesis work was to purify and characterize a novel GH1 β-glucosidase, named NBG. Both GH1 and GH3 family enzymes were used as references for the characterization work. The GH3 reference enzyme was a β-glucosidase from Aspergillus niger (An Cel3A), derived from the commercial enzyme preparation Novozym 188. The used GH1 reference was a β-glucosidase from termite Nasutitermes takasagoensis (Nt GH1). The applicability of NBG β-glucosidase in biomass hydrolysis was also examined, together with possible considerations for applicability by other type of applications. The purification of An Cel3 reference enzyme was performed as described previously in literature. A novel protocol combining thermal treatment and low resolution IEX purification was developed for the NBG enzyme in this study. The enzyme’s activity on various pNP-substrates was determined, followed by pH stability, thermostability and inhibition studies. According to the result, NBG is a potential candidate for industrial use. The enzyme was found to be thermostable and active in a wide pH range when compared to the reference enzymes (stable up to 20 h at +60 ˚C and in pH 3.5 – 6.0). NBG also exhibited wider activity on pNP-substrates than the reference enzymes, highest specific activity being on pNPG, followed by moderate activity on pNPFuc and low activities on pNPGal and pNPXyl. Furthermore, NBG exhibited higher tolerance to inhibitors such as glucose and ethanol. Glucose inhibition was not observed until concentration of 200 mM for NBG, while in the same concentration the reference enzymes were almost completely inhibited. A Clear activation (of +16 %) by 100 mM glucose was observed with NBG. This enzyme also outperformed the An Cel3A-reference in ethanol tolerance, retaining activity better in 15 and 20 % ethanol. Activation by ethanol was also observed for both of the fungal enzymes, the most pronounced effect being observed for NBG in 15 % ethanol (+21 % of initial activity). The hydrolysis of insoluble cellulosic substrate (Avicel) was investigated using a commercial cellulase mixture (Celluclast 1.5L), where a semi-pure β-glucosidase preparation was added: novel β-glucosidase preparation (NBG (2-S2)) or the reference preparation An Cel3A (Nz188). According to the results, the NBG (2S-2) was outperformed by An Cel3A (Nz188) in Avicel 4 – 72 h hydrolysis experiments. The amount of reducing sugars released from Avicel was approximately 18–19 % higher with the commercial Nz188 preparation when compared to the 2S-2 preparation. Further analyses of samples revealed accumulation of cello-oligosacchardes, which may accumulate due to two possible reasons: Either the NBG enzyme does not possess high enough cellobiase activity (needed in biomass hydrolysis to glucose), or accumulation of cellobiose is due to transglycosylation activity of NBG. According to activity (and 3D modelling) data, NBG may not be a true β-glucosidase belonging to the EC 3.2.1.21 (and having cellobiase activity). Further investigation of the possible substrate specificity and transglycosylation activity of the NBG will be needed in assessing its applicability in other types of biotechnical applications.

Objectives Executive functions and attentional control are important for development of self-regulation and can be studied in childhood with simple eye tracking tasks. Typically saccades to new targets are slower and easier to inhibit when gaze is fixated on a target. This effect decreases in typical development as eye motor functions become more flexible and controlled. In this study orientation was expected to be faster and maintenance of attention and inhibition of orientation more difficult when the target is removed. The effect and eye motor control were expected to vary, and their relationship to executive functions and general cognitive skills was studied. Methods Study consisted of 52 typically developing 5-7 year old children whose saccadic reaction times and attentional stability and inhibition were evaluated in conditions which differed in terms of fixational targets. Reaction times and proportion of successful inhibition and their differences between the conditions in both tasks were used to predict children’s performance in cognitive tests of executive and attentional control, general cognitive ability and parent rated executive skills. Results and conclusions Fixation to target slowed orientation responses and supported attentional stability and inhibition. Smaller effect of the condition on orienting and better attentional stability were related to better perceptual organization and more parent rated hyperactivity. More stable visual attention predicted better inhibitory control in auditory attention. Results concur with other studies on the relationship between visual fixation and orienting in typical development and suggest simple eye tracking tasks to complement assessment of children’s neurocognitive skills.

Objective Executive function deficits are associated with a risk for psychopathology in childhood, but a consensus on the exact details of the relationship is lacking. This study sought to clarify the relationship by investigating in a child psychiatric sample the association between preschool executive functions and concurrent and school-age 1) psychiatric symptoms and 2) ADHD diagnosis, and 3) the role of age and sex of the child and socioeconomic status of the family in the relationships. Methods The baseline data (n=166) used in this study was recruited in 2015-2017 from child psychiatric outpatient clinics, and the follow-up sample (n=65) was collected by contacting the original sample in 2021. At baseline the children were aged 4 to 7 (70.5% boys), and at follow-up 8 to 13 years (75.4% boys). Executive functions (including inhibition, attention, and execution of action) were measured at baseline with The Attention and Executive Function Rating Inventory – Preschool Version filled in by daycare nurses, and psychiatric symptoms (internalizing, externalizing, attention and total) were measured at both timepoints by age-appropriate versions of Child Behavior Check List filled in by parents. ADHD diagnoses at both timepoints were collected from medical records. Linear and logistic regression analyses were performed to investigate the concurrent and predictive associations. Age and sex of the child, and parental education were controlled in the analyses. Results Preschool executive functions were associated with concurrent psychiatric symptoms to varying degrees. Contrary to previous findings, no concurrent associations with total psychiatric symptoms or predictive associations with any psychiatric symptom categories were found. All preschool executive functions were associated with concurrent ADHD diagnosis, and they continued to predict school-age ADHD diagnosis, even when preschool age diagnosis was controlled for. Conclusion The role of executive function deficits in ADHD is evident, and they may precipitate the disorder. Executive functions are an essential part of ADHD assessment. The association with psychiatric symptoms is more complex, as different components of executive functions are differently associated with internalizing, externalizing and attention symptoms. More research is needed to find out if the results are applicable only to clinical populations.