Organophosphate hydrolase (OPH, EC 3.1.8.1) is a bacterial enzyme with a broad spectrum of potential substrates that include organophosphorus pesticides, herbicides, and chemical warfare agents. OPH has been expressed successfully in bacterial, fungal, and insect cell culture systems; however, none of these systems produces amounts of enzyme suitable for applications outside of the research laboratory. Therefore, a transgenic Zea mays L. (maize) system was developed to express OPH as an alternate to the current OPH expression systems. The bacterial gene encoding the OPH protein was optimized for transcriptional and translational expression in maize. The optimized gene was inserted into the maize genome under the control of embryo specific, endosperm specific, and constitutive plant promoters. Select transformants were analyzed for the expression of OPH. Expression was observed in the seeds of plants transformed with each of the three constructs with the highest expression observed with the embryo specific and constitutive promoter constructs. The highest OPH expressing lines of transgenic maize had expression levels higher than those reported for the E. coli expression system. OPH was purified from transgenic maize seed and analyzed for posttranslational modification and kinetic properties. OPH was observed to undergo a glycosylation event when expressed in maize that yielded at least two forms of OPH homogolous dimer. The glycosylated form of OPH bound tightly to the Concanavalin A sepharose and remained active after months of storage at room temperature. OPH activity was checked against a number of organophosphate herbicides. Enzymatic activity was observed against the herbicide Amiprophos-methyl and kinetic properties were measured. Enzymatic activity was also tested against the organophosphate Haloxon. Transgenic maize callus, leaf, and seed tissue could be screened for the presence of the optimized opd gene by enzymatic activity. Comparison of the growth of transgenic and control callus on media containing organophosphates showed that the transgenic callus was resistant to the herbicidal effects of haloxon. Transgenic plants expressing OPH were also resistant to the herbicide bensulide when compared to control plants. This indicates that OPH can be used as a screenable marker in plant systems and may be a potential scorable marker system as well.

Renewable fibers produced by forest trees provide excellentraw material of high economic value for industrialapplications. Despite this, the genes and corresponding enzymesinvolved in wood fiber biosynthesis in trees are poorlycharacterized. This thesis describes a functional genomicsapproach for the identification of carbohydrate-active enzymesinvolved in secondary cell wall (wood) formation in hybridaspen. First, a 3' target amplification method was developed toenable microarray-based gene expression analysis on minuteamounts of RNA. The amplification method was evaluated usingboth a smaller microarray containing 192 cDNA clones and alarger microarray containing 2995 cDNA clones that werehybridized with targets isolated from xylem and phloem.Moreover, a gene expression study of phloem differentiation wasperformed to show the usefulness of the amplificationmethod. A microarray containing 2995 cDNA clones representing aunigene set of a cambial region EST library was used to studygene expression during wood formation. Transcript populationsfrom thin tissue sections representing different stages ofxylem development were hybridized onto the microarrays. It wasdemonstrated that genes encoding lignin and cellulosebiosynthetic enzymes, as well as a number of genes withoutassigned function, were differentially expressed across thedevelopmental gradient. Microarrays were also used to track changes in geneexpression in the developing xylem of transgenic, GA-20 oxidaseoverexpressing hybrid aspens that had increased secondarygrowth. The study revealed that a number of genes encoding cellwall related enzymes were upregulated in the transgenic trees.Moreover, most genes with high transcript changes could beassigned a role in the early events of xylogenesis. Ten genes encoding putative cellulose synthases (CesAs) wereidentified in our ownPopulusESTdatabase. Full length cDNA sequences wereobtained for five of them. Expression analyses performed withreal-time PCR and microarrays in normal wood undergoingxylogenesis and in tension wood revealed xylem specificexpression of four putative CesA isoenzymes. Finally, an approach combining expressionprofiling,bioinformatics as well as EST and full length sequencing wasadopted to identify secondary cell wall related genes encodingcarbohydrate-active enzymes, such as glycosyltransferases andglycoside hydrolases. As expected, glycosyltransferasesinvolved in the carbohydrate biosynthesis dominated thecollection of the secondary cell wall related enzymes that wereidentified. Key words:Populus, xylogenesis, secondary cell wall,cellulose, hemicellulose, microarrays, transcript profiling,carbohydrate-active enzyme, glycosyltransferase, glycosidehydrolase

Anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) exert most of their actions by binding to cannabinoid receptors. The effects of the endocannabinoids are short-lived due to rapid cellular accumulation and metabolism, for AEA, primarily by the enzymes fatty acid amide hydrolase (FAAH). This has led to the hypothesis that by inhibition of the cellular processing of AEA, beneficial effects in conditions such as pain and inflammation can be enhanced. The overall aim of the present thesis has been to examine the mechanisms involved in the cellular processing of AEA and how they can be influenced pharmacologically by both synthetic natural compounds. Liposomes, artificial membranes, were used in paper I to study the membrane retention of AEA. The AEA retention mimicked the early properties of AEA accumulation, such as temperature-dependency and saturability. In paper II, FAAH was blocked by a selective inhibitor, URB597, and reduced the accumulation of AEA into RBL2H3 basophilic leukaemia cells by approximately half. Treating intact cells with the tyrosine kinase inhibitor genistein, an isoflavone found in soy plants and known to disrupt caveolae-related endocytosis, reduced the AEA accumulation by half, but in combination with URB597 no further decrease was seen. Further on, the effects of genistein upon uptake were secondary to inhibition of FAAH. The ability to inhibit the accumulation and metabolism of AEA was shared by several flavonoids (shown in paper III). In paper IV, the isoflavone biochanin A and URB597 had effects in vivo, in a model of persistent pain, effects decreased by the cannabinoid receptor 1 antagonist AM251. In paper VI, the cellular processing of the endocannabinoid metabolites following degradation was examined, a mechanism poorly understood. It was found that nitric oxide (NO) donors significantly increased the retention of tritium in cell membranes following incubation with either tritiated AEA or 2-AG. Further experiments revealed that the effect of NO donors mainly involves the arachidonate part of the molecules. Inhibition of FAAH completely reduced the effect of NO donors in cells with a large FAAH component, indicating that the effects were downstream of the enzyme. These results suggest that the cellular processing of endocannabinoids can be affected in a manner of different ways by pharmacological manipulation in vitro and that naturally occurring flavonoid compounds can interact with the endocannabinoid system.

The kinetic mechanism of an α/β hydrolase fold epoxide hydrolase from potato, StEH1, has been studied with the aims of explaining the underlying causes for enantio- and regioselectivity, both being important for product purity. Further effort has been laid upon understanding the causes of a hysteretic behavior discovered in the measurements leading to Paper I. The enantioselectivity was investigated with substrates differing only in substituent size at one carbon of the oxirane ring structure. In catalysis with trans-stilbene oxide and styrene oxide, enantioselectivity is the result of differences in alkylation rates. In pre-steady state measurement with trans-2-methylstyrene oxide (2-MeSO), a rate-limiting step involving slow transitions, referred to as hysteresis, was discovered. With this substrate enantioselectivity is proposed to be a consequence of the catalytic rate of (1R,2R)-enantiomer being more influenced by the hysteretic behavior than was the rate of the other enantiomer. In steady-state measurements with (1R,2R)-2-MeSO, at different temperatures and pH, hysteretic cooperativity was displayed. It can be concluded that this behavior is dependent on the relationship between kcat and the rate of transition between two Michaelis complexes. From the differences in pH dependence of kcat/KM in formation of the two diols resulting from low regioselectivity in catalysis of (1R,2R)-2-MeSO, it is suggested that hysteresis is a result of the substrates placed in different conformational modes within the active site cavity. Regioselectivity is proposed to be the result of specific interactions between the catalytically important Tyr and the substrate, with a link between KM-values and degree of regioselectivity. Furthermore, the hysteretic kinetic model proposed can explain hysteresis, cooperativity and regioselectivity resulting from StEH1 catalyzed hydrolysis of (1R,2R)-2-MeSO.

The work leading to this thesis has provided additional information and novel knowledge concerning structure-function relationship in the potato epoxide hydrolase. Epoxide hydrolases are enzymes catalyzing the hydrolysis of epoxides to yield the corresponding vicinal diols. The reaction mechanism proceeds via a nucleophilic attack resulting in a covalent alkylenzyme intermediate, which in turn is attacked by a base-activated water molecule, followed by product release. Epoxides and diols are precursors in the production of chiral compounds and the use of epoxide hydrolases as biocatalysts is growing. The promising biocatalyst StEH1, a plant epoxide hydrolase from potato, has been investigated in this thesis. In paper I the active site residue Glu35, was established to be important for the formation of the alkylenzyme intermediate, activating the nucleophile for attack by facilitated proton release through a hydrogen bond network. Glu35 is also important during the hydrolytic half reaction by optimally orienting the hydrolytic water molecule, aiding in the important dual function of the histidine base. Glu35 makes it possible for the histidine to work as both an acid and a base. In paper II a putative proton wire composed of five water molecules lining a protein tunnel was proposed to facilitate effective proton transfer from the exterior to the active site, aiding in protonation of the alkylenzyme intermediate. The protein tunnel is also proposed to stabilize plant epoxide hydrolases via hydrogen bonds between water molecules and protein. Enzyme variants with modified enantiospecificity for the substrate (2,3-epoxypropyl)benzene have been constructed by in vitro evolution using the CASTing approach. Residues lining the active site pocket were targeted for mutagenesis. From the second generation libraries a quadruple enzyme variant, W106L/L109Y/V141K/I155V, displayed a radical shift in enantioselectivity. The wild-type enzyme favored the S-enantiomer with a ratio of 2:1, whereas the quadruple variant showed a 15:1 preference for the R-enantiomer.

This thesis documents the subcellular localization and protein-protein interactions of two methyl recycling enzymes. These two enzymes, adenosine kinase (ADK) and S-adenosyl-L-homocysteine hydrolase (SAHH), are essential to sustain the hundreds of S-adenosyl-L-methionine (SAM)-dependent transmethylation reactions in plants. Both ADK and SAHH are involved in the removal of a competitive inhibitor of methyltransferases (MTs), S-adenosyl-L-homocysteine (SAH), that is generated as a by-product of the each transfer of a methyl group from SAM to a substrate. This research focused on understanding how SAH is metabolized in distinct cellular compartments to maintain MT activities required for plant growth and development. Localization studies using green fluorescent protein (GFP) fusions revealed that both ADK and SAHH localize to the cytoplasm and the nucleus, and possibly to the chloroplast, despite the fact that the primary amino acid sequence of neither protein contains detectable targeting signals. This suggested the possibility that these methyl-recycling enzymes may be targeted by specific protein-protein interactions. Moreover, deletion analysis of SAHH1 indicated that the insertion region (IR) of 41 amino acids (Gly150-Lys190), which is present only in plants and parasitic protozoan SAHHs among eukaryotes, is essential for nuclear targeting. This result suggested that the surface-exposed IR loop may serve as a binding domain for interactions with other proteins that may direct SAHH to the nucleus. To investigate protein-protein interactions, several methods were performed including co-immunoprecipitation, bimolecular fluorescence complementation, and pull-down assays. These results not only revealed that ADK and SAHH possibly interact through the IR loop of SAHH in planta, but also suggested that this interaction is either dynamic or indirect, requiring a cofactor/another protein(s) or post-translational modifications. Moreover, possible interactions of both ADK and SAHH with a putative Arabidopsis mRNA cap methyltransferase (CMT), which is localized predominantly in the nucleus, were also confirmed. These results support the hypothesis that the nuclear targeting of both SAHH and ADK can be mediated by the interaction with CMT. In addition, purification of Strep-tagged SAHH1 expressed in Arabidopsis identified a novel interaction between SAHH and aspartate-semialdehyde dehydrogenase (ASDH), an enzyme that catalyzes the second step of the aspartate-derived amino acid biosynthesis pathway. Analysis of ASDH-GFP fusions revealed that ASDH localizes to the chloroplast and the stromule-like structure that emanates from chloroplasts. Moreover the mutation in three amino acids (Pro164-Asp165-Pro166) located within the IR loop of SAHH disrupted its binding to ASDH which affected the plastid localization of SAHH, suggesting that the interaction between SAHH and ASDH is required for plastid-targeting of SAHH. Taken together, this thesis demonstrated that the localization of ADK and SAHH in or between compartments is possibly mediated by specific protein interactions, and that the surface-exposed IR loop of SAHH is crucial for these interactions.

In eukaryotic cells, most proteins in the cytosol and nucleus are degraded via the ubiquitin-proteasome pathway. Ubiquitin is best known for its role in targeting proteins for degradation by the proteasome. Ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is found specifically in central and peripheral neurons, and is responsible for the removal of small peptide fragments from the ubiquitin chain and for co-translational processing of ubiquitin gene products to generate free monomeric ubiquitin. In response to extreme conditions, cells exhibit an up-regulation of heat shock protein (HSP) expression, which contributes to repair and protective mechanisms. Within the HSP family, HSP70 is the major inducible member that protects against cell death. Based on the pharmacologic property of organophosphates as an inhibitor of cholinesterase, it is generally contended that manifestations of organophosphate poisoning, including secretion and muscle fasciculation, stupor, cardiopulmonary collapse, respiratory failure, coma or death, result from accumulation of, and over-stimulation by acetylcholine at peripheral of central synapses. One approach in furthering our understanding on organophosphate poisoning is delineation of its potential protective mechanisms. In this regard, the information on the cellular and molecular mechanisms that underlie organophosphate poisoning has received attention. Our laboratory demonstrated previously that a crucial brain site via which mevinphos (Mev), an organophosphate insecticide of the P=O type, acts is the rostral ventrolateral medulla (RVLM), the medullary origin of premotor sympathetic neurons that are responsible for the maintenance of vasomotor tone. The phasic changes in cardiovascular events over the course of acute Mev intoxication also parallel fluctuations of the ¡§life-and-death¡¨ signals that emanate form the RVLM. Based on a rat model of organophosphate poisoning that provides continuous information on cellular and molecular mechanisms in the RVLM, the present study was undertaken to evaluate whether changes in protein level of UCH-L1 or HSP70 are associated with death arising from Mev intoxication. We also evaluated the efficacy of both of them in the neuroprotection against fatality during Mev intoxication. The first part of this study investigated whether UCH-L1 plays a neuroprotective role at the RVLM, where Mev acts to elicit cardiovascular toxicity. In Sprague-Dawley rats maintained under propofol anesthesia, Mev (960 µg/kg, i.v.) induced a parallel and progressive augmentation in UCH-L1 or ubiquitin expression at the ventrolateral medulla during the course of Mev intoxication. The increase in UCH-L1 level was significantly blunted on pretreatment with microinjection bilaterally into the RVLM of a transcription inhibitor, actinomycin D (5 nmol) or a translation inhibitor, cycloheximide (20 nmol). Compared to artificial cerebrospinal fluid (aCSF) or sense uch-L1 oligonucleotide (100 pmol) pretreatment, microinjection of an antisense uch-L1 oligonucleotide (100 pmol) bilaterally into the RVLM significantly increased mortality, reduced the duration of the phase I (¡§pro- life¡¨ phase), blunted the increase in ubiquitin expression in ventrolateral medulla, and augmented the induced hypotension in rats that received Mev. The second part of this study investigated whether HSP70 plays a neuroprotective role at the RVLM. Intravenous administration of Mev (960

Organophosphate hydrolase (OPH, EC 3.1.8.1) is a bacterial enzyme with a broad spectrum of potential substrates that include organophosphorus pesticides, herbicides, and chemical warfare agents. OPH has been expressed successfully in bacterial, fungal, and insect cell culture systems; however, none of these systems produces amounts of enzyme suitable for applications outside of the research laboratory. Therefore, a transgenic Zea mays L. (maize) system was developed to express OPH as an alternate to the current OPH expression systems. The bacterial gene encoding the OPH protein was optimized for transcriptional and translational expression in maize. The optimized gene was inserted into the maize genome under the control of embryo specific, endosperm specific, and constitutive plant promoters. Select transformants were analyzed for the expression of OPH. Expression was observed in the seeds of plants transformed with each of the three constructs with the highest expression observed with the embryo specific and constitutive promoter constructs. The highest OPH expressing lines of transgenic maize had expression levels higher than those reported for the E. coli expression system. OPH was purified from transgenic maize seed and analyzed for posttranslational modification and kinetic properties. OPH was observed to undergo a glycosylation event when expressed in maize that yielded at least two forms of OPH homogolous dimer. The glycosylated form of OPH bound tightly to the Concanavalin A sepharose and remained active after months of storage at room temperature. OPH activity was checked against a number of organophosphate herbicides. Enzymatic activity was observed against the herbicide Amiprophos-methyl and kinetic properties were measured. Enzymatic activity was also tested against the organophosphate Haloxon. Transgenic maize callus, leaf, and seed tissue could be screened for the presence of the optimized opd gene by enzymatic activity. Comparison of the growth of transgenic and control callus on media containing organophosphates showed that the transgenic callus was resistant to the herbicidal effects of haloxon. Transgenic plants expressing OPH were also resistant to the herbicide bensulide when compared to control plants. This indicates that OPH can be used as a screenable marker in plant systems and may be a potential scorable marker system as well.

<p>Renewable fibers produced by forest trees provide excellentraw material of high economic value for industrialapplications. Despite this, the genes and corresponding enzymesinvolved in wood fiber biosynthesis in trees are poorlycharacterized. This thesis describes a functional genomicsapproach for the identification of carbohydrate-active enzymesinvolved in secondary cell wall (wood) formation in hybridaspen.</p><p>First, a 3' target amplification method was developed toenable microarray-based gene expression analysis on minuteamounts of RNA. The amplification method was evaluated usingboth a smaller microarray containing 192 cDNA clones and alarger microarray containing 2995 cDNA clones that werehybridized with targets isolated from xylem and phloem.Moreover, a gene expression study of phloem differentiation wasperformed to show the usefulness of the amplificationmethod.</p><p>A microarray containing 2995 cDNA clones representing aunigene set of a cambial region EST library was used to studygene expression during wood formation. Transcript populationsfrom thin tissue sections representing different stages ofxylem development were hybridized onto the microarrays. It wasdemonstrated that genes encoding lignin and cellulosebiosynthetic enzymes, as well as a number of genes withoutassigned function, were differentially expressed across thedevelopmental gradient.</p><p>Microarrays were also used to track changes in geneexpression in the developing xylem of transgenic, GA-20 oxidaseoverexpressing hybrid aspens that had increased secondarygrowth. The study revealed that a number of genes encoding cellwall related enzymes were upregulated in the transgenic trees.Moreover, most genes with high transcript changes could beassigned a role in the early events of xylogenesis.</p><p>Ten genes encoding putative cellulose synthases (CesAs) wereidentified in our own<i>Populus</i>ESTdatabase. Full length cDNA sequences wereobtained for five of them. Expression analyses performed withreal-time PCR and microarrays in normal wood undergoingxylogenesis and in tension wood revealed xylem specificexpression of four putative CesA isoenzymes.</p><p>Finally, an approach combining expressionprofiling,bioinformatics as well as EST and full length sequencing wasadopted to identify secondary cell wall related genes encodingcarbohydrate-active enzymes, such as glycosyltransferases andglycoside hydrolases. As expected, glycosyltransferasesinvolved in the carbohydrate biosynthesis dominated thecollection of the secondary cell wall related enzymes that wereidentified.</p><p><b>Key words:</b>Populus, xylogenesis, secondary cell wall,cellulose, hemicellulose, microarrays, transcript profiling,carbohydrate-active enzyme, glycosyltransferase, glycosidehydrolase</p>

Diagnosis of acute pancreatitis (AP) is erroneous in up to one third of patients when based on clinical criteria and elevated serum amylase values. Furthermore, according to autopsy reports fatal pancreatitis remains clinically undiagnosed in 22 to 86 % of hospitalised patients. Consequently, search for better methods for the diagnosis of AP seems not only justified but urgent. The pancreas secretes an nonspecific lipase, the carboxylic ester hydrolase (CEH) with molecular properties different from other pancreatic secretory enzymes. These differences may imply that sites and rates of clearances from blood of pancreatic enzymes differ. Except for the pancreas this enzyme is secreted from the lactating mammary gland with milk. A sensitive and reproducible sandwich-ELISA for quantitative determination of CEH was developed. When establishing referent values it was noted that in individuals aged 20 to 65 years serum concentrations of CEH did not depend on age, gender, the time of the day or duration from food intake to blood sampling, or use of nicotine. The mammary gland did not contribute significantly to basal serum levels of CEH; enzyme levels in lactating women or women with mammary tumours were identical to those of the reference population. Seventy percent of patients with the diagnosis AP, based on elevated serum amylase levels and abdominal pain, had elevated CEH values. Among the patients with elevated amylase alone a probable cause of pancreatitis was lacking in the majority of patients. Contrastingly, a likely cause of AP could be identified in all patients presenting with abdominal pain and elevated CEH levels alone. These findings suggested that an elevated CEH level indicated AP more reliably than an elevated amylase level. In patients with AP diagnosed by contrast enhanced computed tomography (CECT) alone, or combined with histopathological diagnosis, serum CEH levels were elevated on admission in all but one patient, and in all within the next 24 h. Furthermore, in patients with severe pancreatitis CEH levels remained at a raised level from the second to at least the 10:th day following admission, whereas a significant decrease was noted in patients with mild pancreatitis. In contrast, serum amylase values were higher in patients with mild pancreatitis during the observation period than in those with severe pancreatitis. CEH levels were higher in patients with three or more Ranson signs than in those with less than three signs from the first day after admission. CEH levels were within referent range in 164 patients without known pancreatic disease admitted due to abdominal emergency conditions, or due to planned surgery for chronic extrapancreatic gastrointestinal diseases, and 16 patients having CECT without pathological findings in the pancreas. This suggests that AP can be excluded with very high degree of probability in presence of non-elevated CEH levels. A sandwich ELISA for determination of Guinea pig CEH and a model for graded pancreatitis in the same species were developed. CEH levels showed proportional to severity of inflammation, thus confirming previous clinical observations. CEH levels in bile were proportional to inflammation, while it was absent in urine. Amylase levels in urine were identical regardless of severity of inflammation, but low in bile. These results suggested differences in sites and rates of clearance between the two enzymes. Seemingly elevated CEH levels allowed identification of clinically significant pancreatitis following ERCP, which amylase levels did not. The presented studies have shown that quantitative determination in serum of CEH by the described method is a more reliable test for the diagnosis of AP than determination of amylase activity. The differences between CEH and amylase are, at least partly, due to differences in molecular properties determining rates and routes of clearances of the two enzymes from serum. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 5 uppsatser.</p> / digitalisering@umu.se