Metabolism and Analysis of Desomorphine

Desomorphine is a semi-synthetic opioid that is ten times more potent than morphine, with a faster onset but shorter duration of action. It is a major component of the drug referred to as “Krokodil”, which is used as a heroin substitute. Its prevalence is difficult to estimate due to a lack of analytically confirmed cases, which may in part be due to the limited studies regarding its pharmacology or methodology to detect the drug in biological specimens. This research seeks to further the understanding of both desomorphine’s metabolism and its detection in biological specimens, to facilitate its identification in clinical and forensic toxicology laboratories. Six commercially available enzyme-linked immunosorbent assays were evaluated to determine their effectiveness with respect to desomorphine detection. Cross-reactivities were highly variable between assays, ranging from <2.5-77%. Recombinant human cytochrome P450 enzymes (rCYPs) and recombinant uridine 5'-diphospho-glucuronosyltransferases (rUGTs) were used to investigate the biotransformational pathways involved in desomorphine metabolism. Phase I metabolism could be attributed to seven rCYPs (rCYP2B6, rCYP2C8, rCYP2C9, rCYP2C18, rCYP2C19, rCYP2D6 and rCYP3A4), producing a total of nine phase I metabolites (nordesomorphine, desomorphine-N-oxide, two norhydroxydesomorphine isomers, and five hydroxylated isomers). During phase II metabolism, desomorphine-glucuronide was produced by nine rUGTs (rUGT1A1, rUGT1A3, rUGT1A8, rUGT1A9, rUGT1A10, rUGT2B4, rUGT2B7, rUGT2B15, and rUGT2B17). Chemical and enzymatic hydrolysis of conjugated metabolites were investigated using desomorphine-glucuronide generated in situ using rUGT enzyme. Acid hydrolysis and five β-glucuronidase sources (BGTurbo™, IMCSzyme™, Escherichia coli, Helix pomatia and Patella vulgata) were evaluated. Acid hydrolysis produced complete hydrolysis of desomorphine-glucuronide, and under optimal conditions, each enzyme produced complete or near complete hydrolysis (≥96%), with BGTurbo™ and IMCSzyme™ offering the shortest incubation times. Under simulated challenging conditions, P. vulgata was the most effective enzyme evaluated. Desomorphine was analyzed in blood and urine samples using gas chromatography-mass spectrometry (GC-MS), and urine samples were additionally analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole/time of flight-mass spectrometry (LC-Q/TOF-MS). Each method was validated in accordance with published guidelines for forensic use. Additionally, LC-Q/TOF-MS was used to analyze desomorphine metabolites, which in the absence of commercially available reference material or authentic urine specimens, were generated in-vitro

Changing patterns and perceptions of water use in east central texas since the time of anglo settlement

Patterns and perceptions of water use have changed since Anglo settlement in Texas in the early nineteenth century. Change has not been constant, gradual, or linear, but rather has occurred in fits and spurts. This pattern of punctuated equilibrium in water use regimes is the central finding of this dissertation. Water use is examined in terms of built, organizational, and institutional inertias that resist change in the cultural landscape. Change occurs only when forced by crisis and results in water management at an increasing scale. Perception is critical in forcing response to crisis. Four water use regimes are identified. The agrarian regime was characterized by individual family and plantation units that were self-sufficient in their water supply. Water was perceived as abundant, but used sparingly. The agrarian regime began with Texas’s declaration of independence from Mexico in 1836 and lasted for the remainder of the nineteenth century. The waterworks regime was characterized by the introduction of piped water. During this second regime, water was still perceived as abundant, but was also taken for granted. The crisis forcing the waterworks regime was the need for better fire protection in cities. The almost constant threat of flood and drought, underscored by the Drought of the 1950s, in conjunction with a demographic shift, brought about the dam and levee regime. As a consequence of the Drought of the 1950s, water was for the first time perceived as scarce. We have just entered the groundwater regime. Recent water legislation and a state supreme court decision in favor of a bottled water company are putting new emphasis on groundwater sales from rural property owners to municipal water companies. Empirical studies supporting this theoretical framework are drawn from the heretofore unpublished 1868 journal of Pleasant B. Watson, from municipal bond records in the archives of the Texas Comptroller, from the early history of the waterworks at Bryan, Texas, from newly discovered records of a levee along the Brazos River, from an overview of dam and reservoir construction, and from a recent proliferation of groundwater districts

Neuroanastomosis of orthotopically transplanted palmaris longus muscles

Palmaris longus (PML) muscles of rhesus monkeys were transplanted, with or without anastomosis of the median nerve, to the nerve stump of the autograft. Because PML autografts revascularize spontaneously, vascular anastomoses were not performed. Muscle fibers regenerated in all autografts with neuroanastomosis, but in only three of eight autografts without neuroanastomosis. Five autografts without neuroanastomosis were replaced by noncontractile connective tissue. Growth and differentiation of muscle fibers into three fiber types and development of capillarity were analyzed histochemically, and succinate oxidase activity of whole-muscle homogenates was determined. None of these measures reached values for control PML muscles within 100 days of transplantation. In comparison to control muscles, autografts had slower times to peak tension and less absolute tension, but similar tension per square centimeter of muscle fiber cross-sectional area. Monkey PML autografts with neuroanastomosis were similar in structure and function to cat extensor digitorum longus autografts that had not had neuroanastomosis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50127/1/880020107_ftp.pd

Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin‐3

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102210/1/emboj2008289-sup-0001.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102210/2/emboj2008289.pd

Preventing Ataxin-3 protein cleavage mitigates degeneration in a Drosophila model of SCA3

Protein cleavage is a common feature in human neurodegenerative disease. Ataxin-3 protein with an expanded polyglutamine (polyQ) repeat causes spinocerebellar ataxia type-3 (SCA3), also called Machado–Joseph disease, and is cleaved in mammalian cells, transgenic mice and SCA3 patient brain tissue. However, the pathological significance of Ataxin-3 cleavage has not been carefully examined. To gain insight into the significance of Ataxin-3 cleavage, we developed a Drosophila SL2 cell-based model as well as transgenic fly models. Our data indicate that Ataxin-3 protein cleavage is conserved in the fly and may be caspase-dependent as reported previously. Importantly, comparison of flies expressing either wild-type or caspase-site mutant proteins indicates that Ataxin-3 cleavage enhances neuronal loss in vivo. This genetic in vivo confirmation of the pathological role of Ataxin-3 cleavage indicates that therapies targeting Ataxin-3 cleavage might slow disease progression in SCA3 patients

Splice Isoforms of the Polyglutamine Disease Protein Ataxin-3 Exhibit Similar Enzymatic yet Different Aggregation Properties

Protein context clearly influences neurotoxicity in polyglutamine diseases, but the contribution of alternative splicing to this phenomenon has rarely been investigated. Ataxin-3, a deubiquitinating enzyme and the disease protein in SCA3, is alternatively spliced to encode either a C-terminal hydrophobic stretch or a third ubiquitin interacting motif (termed 2UIM and 3UIM isoforms, respectively). In light of emerging insights into ataxin-3 function, we examined the significance of this splice variation. We confirmed neural expression of several minor 5′ variants and both of the known 3′ ataxin-3 splice variants. Regardless of polyglutamine expansion, 3UIM ataxin-3 is the predominant isoform in brain. Although 2UIM and 3UIM ataxin-3 display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and more rapidly degraded by the proteasome. Our data demonstrate how alternative splicing of sequences distinct from the trinucleotide repeat can alter properties of the encoded polyglutamine disease protein and thereby perhaps contribute to selective neurotoxicity

Understanding the Role of the Josephin Domain in the PolyUb Binding and Cleavage Properties of Ataxin-3

Ataxin-3, the disease protein in the neurodegenerative disorder Spinocerebellar Ataxia Type 3 or Machado Joseph disease, is a cysteine protease implicated in the ubiquitin proteasome pathway. It contains multiple ubiquitin binding sites through which it anchors polyubiquitin chains of different linkages that are then cleaved by the N-terminal catalytic (Josephin) domain. The properties of the ubiquitin interacting motifs (UIMs) in the C-terminus of ataxin-3 are well established. Very little is known, however, about how two recently identified ubiquitin-binding sites in the Josephin domain contribute to ubiquitin chain binding and cleavage. In the current study, we sought to define the specific contribution of the Josephin domain to the catalytic properties of ataxin-3 and assess how the topology and affinity of these binding sites modulate ataxin-3 activity. Using NMR we modeled the structure of diUb/Josephin complexes and showed that linkage preferences are imposed by the topology of the two binding sites. Enzymatic studies further helped us to determine a precise hierarchy between the sites. We establish that the structure of Josephin dictates specificity for K48-linked chains. Site 1, which is close to the active site, is indispensable for cleavage. Our studies open the way to understand better the cellular function of ataxin-3 and its link to pathology

Cytokine responses to Schistosoma haematobium in a Zimbabwean population: contrasting profiles for IFN-γ, IL-4, IL-5 and IL-10 with age

<p>Abstract</p> <p>Background</p> <p>The rate of development of parasite-specific immune responses can be studied by following their age profiles in exposed and infected hosts. This study determined the cytokine-age profiles of Zimbabweans resident in a <it>Schistosoma haematobium </it>endemic area and further investigated the relationship between the cytokine responses and infection intensity.</p> <p>Methods</p> <p>Schistosome adult worm antigen-specific IFN-γ, IL-4, IL-5 and IL-10 cytokine responses elicited from whole blood cultures were studied in 190 Zimbabweans exposed to <it>S. haematobium </it>infection (aged 6 to 40 years old). The cytokines were measured using capture ELISAs and the data thus obtained together with <it>S. haematobium </it>egg count data from urine assays were analysed using a combination of parametric and nonparametric statistical approaches.</p> <p>Results</p> <p>Age profiles of schistosome infection in the study population showed that infection rose to peak in childhood (11–12 years) followed by a sharp decline in infection intensity while prevalence fell more gradually. Mean infection intensity was 37 eggs/10 ml urine (SE 6.19 eggs/10 ml urine) while infection prevalence was 54.7%. Measurements of parasite-specific cytokine responses showed that IL-4, IL-5 and IL-10 but not IFN-γ followed distinct age-profiles. Parasite-specific IL-10 production developed early, peaking in the youngest age group and declining thereafter; while IL-4 and IL-5 responses were slower to develop with a later peak. High IL-10 producers were likely to be egg positive with IL-10 production increasing with increasing infection intensity. Furthermore people producing high levels of IL-10 produced little or no IL-5, suggesting that IL-10 may be involved in the regulation of IL-5 levels. IL-4 and IFN-γ did not show a significant relationship with infection status or intensity and were positively associated with each other.</p> <p>Conclusion</p> <p>Taken together, these results show that the IL-10 responses develop early compared to the IL-5 response and may be down-modulating immunopathological responses that occur during the early phase of infection. The results further support current suggestions that the Th1/Th2 dichotomy does not sufficiently explain susceptibility or resistance to schistosome infection.</p