It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and eight psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition, we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (-30%) and middle (-41%) portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.

1.Adult specimens of Ascaris lumbricoides of similar weights were incubated under nitrogen for 24 hours in a synthetic medium with 1 mM 2,2′-dipyridyl.2.Under these conditions, the viability of the parasites was not affected as evidenced by the amount of ATP in the whole sample and the mobility after mechanical stimulus.3.Incorporation of [14C]proline into non-collagenous proteins in the body wall and cuticle was reproducibly higher in 2,2′-dipyridyl-treated specimens than in untreated worms. Synthesis of collagenous hydroxyproline was inhibited in the cuticle and, to a greater extent, in the muscle layer.4.After transferring the specimens into a fresh medium enriched with 0·1 mM ferrous ions and incubated under 70% oxygen, the muscle collagen remained underhydroxylated. The synthesis of hydroxyproline, however, was almost completely normalized in the cuticle collagen.5.We interpret the data as further evidence of the existence of at least two different enzymes hydroxylating collagenous proline, one located in the subcuticle and the other in the muscle layer of Ascaris lumbricoides.

Phox2a and Phox2b are two homeodomain proteins that control the differentiation of noradrenergic neurons during embryogenesis. In the present study, we examined the possible effect of Phox2a/2b on the in vitro expression of the norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH), two important markers of the noradrenergic system. SK-N-BE(2)C cells were transfected with cDNAs or short hairpin RNAs specific to the human Phox2a and Phox2b genes. Transfection of 0.1 to 5 μg of cDNAs of Phox2a or Phox2b significantly increased mRNA and protein levels of NET and DBH in a concentration-dependent manner. As a consequence of the enhanced expression of NET after transfection, there was a parallel increase in the uptake of [ 3H]norepinephrine. Co-transfection of Phox2a and Phox2b did not further increase the expression of noradrenergic markers when compared with transfection of either Phox2a or Phox2b alone. Transfection of shRNAs specific to Phox2a or Phox2b genes significantly reduced mRNA and protein levels of NET and DBH after shutdown of endogenous Phox2, which was accompanied by a decreased [3H]norepinephrine uptake. Furthermore, there was an additive effect after cotransfection with both shRNAs specific to Phox2a or Phox2b genes on NET mRNA levels. Finally, the reduced DBH expression caused by the shRNA specific to Phox2a could be reversed by transfection with Phox2b cDNA and vice versa. The present findings verify the determinant role of Phox2a and Phox2b on the expression and function of NET and DBH in vitro. Further clarifying the regulatory role of these two transcription factors on key proteins of the noradrenergic system may open a new avenue for therapeutics of aging-caused dysfunction of the noradrenergic system.

The development of a biologic protein involves different stages and becomes a highly complex process which can be costly and time consuming to scale up for industrial production. Therefore, optimization is a necessary part of the production process development to lower the production expenses.An on-going project is working on upscaling the production of a protein derived from mussel adhesive proteins (MAPs) which has great properties to be used as a pharmaceutical drug or in medical devices. The protein is expressed in a bacterial host cell and the necessary post translational modifications (PTMs) are done in-vitro using enzymes. The work presented in this report was done to optimize both the protein production in lab scale bioreactors and the enzymatic reaction using an immobilized prolyl-4-hydroxylase (P4H) which does a post translational modification on prolyl-residues. Additionally, an interaction study was conducted to better understand the hydroxylation using the prolyl-4-hydroxylase.For the bioreactor optimization four initial trials were performed testing different growth and induction temperatures and also comparing exponential to linear feeding. From these trials it appeared that having 30 ℃ growth overnight and induction at the same temperature in combination with an exponential feeding rate gave the best results. The modifications done by the prolyl-4-hydroxylase were analysed by LC-MS and suggest that longer incubation time and more immobilized protein gives more modifications in the tested ranges and the possibilities of reusing the immobilized proteins looks promising. No conclusive data was discovered for the optimal substrate concentration. The interaction study revealed the importance of reagents used for catalysis with the enzyme to be present for interaction to occur, however more work needs to be done to discover an accurate KD for the interaction.

The pesticide/neurotoxin, rotenone, has been shown to cause systemic inhibition of mitochondrial complex I activity, with consequent degeneration of the nigrostriatal pathway, as observed in Parkinson’s disease. A novel intrastriatal rotenone model of Parkinson’s disease was used to examine the neuroprotective effects of valproic acid (VPA) and melatonin, both of which are known to induce neurotrophic gene expression in the central nervous system via mechanisms which may involve epigenetic modulation. In these studies, sham or lesioned rats were treated with either vehicle, VPA (4mg/mL), or melatonin (4µg/mL) in drinking water. Results from a forelimb asymmetry test indicated a significant decrease in use of the contralateral forelimb in rotenone-infused animals, in the third week post-surgery, which was abolished by VPA treatment. Apomorphine administration resulted in significantly higher ipsilateral rotation in rotenone-lesioned (12µg) animals, as compared to controls, which was attenuated by melatonin treatment. Subsequent immunohistochemical examination revealed a decrease in tyrosine hydroxylase immunoreactivity within the striatum and substantia nigra of rotenone-infused animals. VPA or melatonin treatment prevented this decrease in tyrosine hydroxylase in the striatum and substantia nigra. Stereological cell counting indicated a significant decrease in dopamine neurons within the substantia nigra of rotenone-treated animals. Importantly, this loss of dopamine neurons in rotenone-infused animals was blocked by chronic VPA or melatonin treatment. A third study explored whether rotenone infusion into the medial forebrain bundle and substantia nigra in mice could provide a model of Parkinson's disease. Densitometric analysis revealed a significant depletion of tyrosine hydroxylase immunofluorescence within the ipsilateral striatum and substantia nigra of lesioned animals, and a significant bilateral overexpression of α-synuclein in the substantia nigra, as compared to control animals. These novel findings support the use of intracranial rotenone as a Parkinsonian model, and provide a solid platform for future combinatorial therapeutic approaches with VPA and melatonin. / Dissertation / Doctor of Philosophy (PhD)

The intestinal epithelium is able to adapt to varying blood flow and, thus, oxygen availability. Still, the adaptation fails under pathologic situations. A better understanding of the mechanisms underlying the epithelial adaptation to hypoxia could help to improve the therapeutic approach. We hypothesized that the short-term adaptation to hypoxia is mediated via AMP-activated protein kinase (AMPK) and that it is coupled to the long-term adaptation by a common regulation mechanism, the HIF-hydroxylase enzymes. Further, we hypothesized the transepithelial transport of glucose to be part of this short-term adaptation. We conducted Ussing chamber studies using isolated lagomorph jejunum epithelium and cell culture experiments with CaCo-2 cells. The epithelia and cells were incubated under 100% and 21% O2, respectively, with the panhydroxylase inhibitor dimethyloxalylglycine (DMOG) or under 1% O2. We showed an activation of AMPK under hypoxia and after incubation with DMOG by Western blot. This could be related to functional effects like an impairment of Na+-coupled glucose transport. Inhibitor studies revealed a recruitment of glucose transporter 1 under hypoxia, but not after incubation with DMOG. Summing up, we showed an influence of hydroxylase enzymes on AMPK activity and similarities between hypoxia and the effects of hydroxylase inhibition on functional changes.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24670号 / 農博第2553号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5451(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 梅澤 俊明, 教授 矢﨑 一史, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The HIF prolyl 4-hydroxylases (HIF-P4H) control hypoxia-inducible factor (HIF), a powerful mechanism regulating cellular adaptation to decreased oxygenation. The gastrointestinal epithelium subsists in “physiological hypoxia” and should therefore have an especially well-designed control over this adaptation. Thus, we assessed the absolute mRNA expression levels of the HIF pathway components, Hif1a, HIF2a, Hif-p4h-1, 2 and 3 and factor inhibiting HIF (Fih1) in murine jejunum, caecum and colon epithelium using droplet digital PCR.We found a higher expression of all these genes towards the distal end of the gastrointestinal tract. We detected mRNA for Hif-p4h-1, 2 and 3 in all parts of the gastrointestinal tract. Hif-p4h-2 had significantly higher expression levels compared to Hif-p4h-1 and 3 in colon and caecum epithelium. To test the roles each HIF-P4H isoform plays in the gut epithelium, we measured the gene expression of classical HIF target genes in Hif-p4h-1/, Hif-p4h-2 hypomorph and Hif-p4h-3/ mice. Only Hif-p4h-2 hypomorphism led to an upregulation of HIF target genes, confirming a predominant role of HIF-P4H-2. However, the abundance of Hif-p4h-1 and 3 expression in the gastrointestinal epithelium implies that these isoforms may have specific functions as well. Thus, the development of selective inhibitors might be useful for diverging therapeutic needs.

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Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.