Synaptic Integration of Adult-Born Hippocampal Neurons Is Locally Controlled by Astrocytes.

Adult neurogenesis is regulated by the neurogenic niche, through mechanisms that remain poorly defined. Here, we investigated whether niche-constituting astrocytes influence the maturation of adult-born hippocampal neurons using two independent transgenic approaches to block vesicular release from astrocytes. In these models, adult-born neurons but not mature neurons showed reduced glutamatergic synaptic input and dendritic spine density that was accompanied with lower functional integration and cell survival. By taking advantage of the mosaic expression of transgenes in astrocytes, we found that spine density was reduced exclusively in segments intersecting blocked astrocytes, revealing an extrinsic, local control of spine formation. Defects in NMDA receptor (NMDAR)-mediated synaptic transmission and dendrite maturation were partially restored by exogenous D-serine, whose extracellular level was decreased in transgenic models. Together, these results reveal a critical role for adult astrocytes in local dendritic spine maturation, which is necessary for the NMDAR-dependent functional integration of newborn neurons.

The paracaspase MALT1: biological function and potential for therapeutic inhibition.

The paracaspase MALT1 has a central role in the activation of lymphocytes and other immune cells including myeloid cells, mast cells and NK cells. MALT1 activity is required not only for the immune response, but also for the development of natural Treg cells that keep the immune response in check. Exaggerated MALT1 activity has been associated with the development of lymphoid malignancies, and recently developed MALT1 inhibitors show promising anti-tumor effects in xenograft models of diffuse large B cell lymphoma. In this review, we provide an overview of the present understanding of MALT1's function, and discuss possibilities for its therapeutic targeting based on recently developed inhibitors and animal models.

A probable dual mode of action for both L- and D-lactate neuroprotection in cerebral ischemia.

Lactate has been shown to offer neuroprotection in several pathologic conditions. This beneficial effect has been attributed to its use as an alternative energy substrate. However, recent description of the expression of the HCA1 receptor for lactate in the central nervous system calls for reassessment of the mechanism by which lactate exerts its neuroprotective effects. Here, we show that HCA1 receptor expression is enhanced 24 hours after reperfusion in an middle cerebral artery occlusion stroke model, in the ischemic cortex. Interestingly, intravenous injection of L-lactate at reperfusion led to further enhancement of HCA1 receptor expression in the cortex and striatum. Using an in vitro oxygen-glucose deprivation model, we show that the HCA1 receptor agonist 3,5-dihydroxybenzoic acid reduces cell death. We also observed that D-lactate, a reputedly non-metabolizable substrate but partial HCA1 receptor agonist, also provided neuroprotection in both in vitro and in vivo ischemia models. Quite unexpectedly, we show D-lactate to be partly extracted and oxidized by the rodent brain. Finally, pyruvate offered neuroprotection in vitro whereas acetate was ineffective. Our data suggest that L- and D-lactate offer neuroprotection in ischemia most likely by acting as both an HCA1 receptor agonist for non-astrocytic (most likely neuronal) cells as well as an energy substrate.

Mammalian Target of Rapamycin Complex 2 Controls CD8 T Cell Memory Differentiation in a Foxo1-Dependent Manner.

Upon infection, antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor, the core component of mammalian target of rapamycin complex 2 (mTORC2), regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover, mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation, repression of T-bet, enhanced mitochondrial spare respiratory capacity, and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore, mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions.

The fusion protein SS18-SSX1 employs core Wnt pathway transcription factors to induce a partial Wnt signature in synovial sarcoma.

Expression of the SS18/SYT-SSX fusion protein is believed to underlie the pathogenesis of synovial sarcoma (SS). Recent evidence suggests that deregulation of the Wnt pathway may play an important role in SS but the mechanisms whereby SS18-SSX might affect Wnt signaling remain to be elucidated. Here, we show that SS18/SSX tightly regulates the elevated expression of the key Wnt target AXIN2 in primary SS. SS18-SSX is shown to interact with TCF/LEF, TLE and HDAC but not β-catenin in vivo and to induce Wnt target gene expression by forming a complex containing promoter-bound TCF/LEF and HDAC but lacking β-catenin. Our observations provide a tumor-specific mechanistic basis for Wnt target gene induction in SS that can occur in the absence of Wnt ligand stimulation.

The influence of skeletal muscle cell volume on the regulation of carbohydrate uptake and muscle metabolism

This study investigated the regulation of carbohydrate metabolism and glucose uptake through changes in skeletal muscle cell volume. Using an established invitro isolated whole muscle model, soleus (SOL) and extensor digitorum longus (EDL) muscles were dissected from male rats and incubated in an organ bath containing Sigma medium-199 with 8 mM D-glucose altered to target osmolality (hypo-osmotic: HYPO, iso-osmotic: ISO, hyper-osmotic: HYPER; 190, 290, 400 mmol/kg). Muscles were divided into two groups; metabolite (MM) and uptake (MU). MM (N=48) were incubated for 60 minutes and were then immediately flash frozen. MU (N=24) were incubated for 30 minutes and then the extracellular fluid was exchanged for media containing ^H-glucose and ^'*C-mannitol and incubated for another 30 minutes. After the incubation, the muscles were freeze clamped. Results demonstrated a relative water decrease and increase in HYPER and HYPO, respectively. EDL and SOL glucose uptakes were found to be significantly greater in HYPER conditions. The HYPER condition resulted in significant alterations in muscle metabolite concentrations (lower glycogen, elevated lactate, and G-6-P) suggesting a catabolic cell state, and an increase in glycogen synthase transformation when compared to the HYPO group. In conclusion, skeletal muscle cell volume alters rates of glucose uptake with further alterations in muscle metabolites and glycogen synthase transformation.

The genetics of glucosamine resistance in yeast /

Thesis (M. Sc.) - Brock University, 1975.

Lifespan, body size and oxygen : aerobic metabolism and oxidative stress in cultured fibroblasts /

The allometric scaling relationship observed between metabolic rate (MR) and species body mass can be partially explained by differences in cellular MR (Porter & Brand, 1995). Here, I studied cultured cell lines derived from ten mammalian species to determine whether cells propagated in an identical environment exhibited MR scaling. Oxidative and anaerobic metabolic parameters did not scale significantly with donor body mass in cultured cells, indicating the absence of an intrinsic MR setpoint. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms (West et al., 2002). As such cells were cultured under a variety of physiologically relevant oxygen tensions to investigate the effect of oxygen on cellular metabolic rates. Exposure to higher medium oxygen tensions resulted in increased metabolic rates in all cells. Higher MRs have the potential to produce more reactive oxygen species (ROS) which could cause genomic instability and thus reduced lifespan. Longer-lived species are more resistant to oxidative stress (Kapahi et al, 1999), which may be due to greater antioxidant and/or DNA repair capacities. This hypothesis was addressed by culturing primary dermal fibroblasts from eight mammalian species ranging in maximum lifespan from 5 to 120 years. Only the antioxidant manganese superoxide dismutases (MnSOD) positively scaled with species lifespan (p<0.01). Oxidative damage to DNA is primarily repaired by the base excision repair (BER) pathway. BER enzyme activities showed either no correlation or as in the case of polymerase p correlated, negatively with donor species (p<0.01 ). Typically, mammalian cells are cultured in a 20% O2 (atmospheric) environment, which is several-fold higher than cells experience in vivo. Therefore, the secondary aim of this study was to determine the effect of culturing mammalian cells at a more physiological oxygen tension (3%) on BER, and antioxidant, enzyme activities. Consistently, standard culture conditions induce higher antioxidant and DNA ba.se excision repair activities than are present under a more physiological oxygen concentration. Therefore, standard culture conditions are inappropriate for studies of oxidative stress-induced activities and species differences in fibroblast DNA BER repair capacities may represent differences in ability to respond to oxidative stress. An interesting outcome firom this study was that some inherent cellular properties are maintained in culture (i.e. stress responses) while others are not (i.e. MR).

The effect of extracellular osmolality on cell volume and resting skeletal muscle metabolism

The purpose of the current investigation was to establish an in-l'itro skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated. whole muscle (SOL and EDL) was dissected from Long Evans rats and incubated for 60 min in Sigma Medium-199 (resting tension (lg). bubbled with 95:5% 02:C02, 30 ± 2°C, and pH 7.4). Media osmolality was altered to simulate hypo-osmotic (190 ± 10 Osm) (HYPO) or hyper-osmotic conditions (400 ± 10 Osm) (HYPER) while an iso-osmotic condition (290± 1 0 Osm) (CON) served as a control (n= 17.19.17). Following incubation, relative muscle water content decreased with HYPER and increased with HYPO in both muscle types (p<0.05). The cross-sectional area of HYPO SOL type I and type II fibres increased (p<0.05) while the EDL type 11 fibre area decreased in HYPER and increascd from HYPO exposure. Furthermore, HYPER exposure in both muscles lead to decreased ATP and phosphocreatine (p<0.05) and increased creatine and lactate (p<0.05) compared to CON. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acutc alterations in muscle water content and resting muscle metabolism.

The influence of skeletal muscle cell volume on carbohydrate metabolism in contracting skeletal muscle

This study investigated the regulation of carbohydrate metabolism through changes in skeletal muscle cell volume immediately post contraction and during recovery. Using an established in vitro isolated muscle strip model, soleus (SOL) and extensor digitorum longus (EDL) were dissected from male rats and incubated in an organ bath (perfused with 95% O2; 5% CO2, pH 7.4, temperature 25°C) containing medium- 199 altered to a target osmotic condition (iso-, hypo- or hyper-osmotic; 290, 1 80, 400 mmol/kg). Muscles were stimulated for 10 minutes (40 Hz SOL; 30 Hz EDL) and then either immediately flash frozen or allowed to recover for 20 minutes before subsequent metabolite and enzyme analysis. Results demonstrated a relative water decrease in HYPER vs. HYPOosmotic condition (n=8/group; p<0.05) regardless of muscle type. Specifically, the SOL HYPER condition had elevated metabolite concentrations after 10 minutes of stimulation in comparison to both HYPO and ISO (p<0.05), while EDL muscle did not show any significant difTerences between the HYPER or HYPO conditions. After 20 minutes of recovery, metabolic changes occurred in both SOL and EDL with the SOL HYPER condition showing greater relative changes in metabolite concentrations versus HYPO. The results of the current study have demonstrated that osmotic imbalance induces metabolic change within the skeletal muscle cell and muscle type may influence the mechanisms utilized for cell volume regulation.

An investigation into fungal metabolism of halogenated and related steroids

Fungal metabolism of halogenated and related steroids was investigated. The fungi Aspergillus niger ATCC 9142, Curvularia lunata NRRL 2380 and Rhizopus stolonifer ATCC6227b were studied in this regard. 2l-Fluoro-, 2l-chloro, 2l-bromo- and 2l-methyl-pregn-4-ene-3,20diones were prepared and incubated with ~ niger (a C-2l-hydroxylator) in order to observe the effect of the C-2l substituent on the metabolism of these substrates. In all four cases, the C-2l substituent prevented any significant metabolism of these substrates. llB-Fluoropregn-4-ene-3,20-dione was prepared and incubated with C. lunata (an llB-hydroxylator) and ~ stolonifer (an lla-hydroxylator). With ~ lunata, the ll-fluoro- substituent prevent hydroxylation at the 11 position, but diverted it to a site remote from the fluorine atom. In contrast, with ~ stolonifer the llB-fluoro- substituent, although slowing the apparent rate of hydroxylation, did not prevent its occurrence at the 11a- position. llB-Hydroxypregn-4-ene-3,20-dione was also incubated with R. stolonifer. The llB-hydroxy-;group did not appear to have any significant effect on hydroxylation at the lla- position. The incubation of a substrate, unsaturated at a favoured site of hydroxylation with Rhizopus arrhizus ATCC 11145 provided a complex mixture of products; among them were both the a and S epoxides. The formation of these products is rationalized as arising because of the lack of regio- and stereospecificity of the hydroxylase enzyme(s) involved.

The genetics of glucosamine in yeast : cytoplasmic determinants conferring resistance

Two cytoplasmic, glucosamine resistant mutants of Saccharomyces cerevisiae, GR6 and GR10, were examined to determine whether or not the lesions involved were located on mitochondrial DNA. Detailed investigation of crosses of GR6 and GR10 or their derivatives to strains bearing known mitochondrial markers demonstrated that: 1. the frequency of glucos~~ine resistance in diploids was independent of factors influencing mitochondrial marker output. 2. upon tetrad analysis a variety of tetrad ratios was observed for glucosamine resistance whereas mitochondrial markers segregated 4:0 or 0:4 (resistant:sensitive). 3. glucosamine resistance and mitochondrial markers segregated differentially with time. 4. glucosamine resistance persisted following treatment of a GRIO derivative with ethidium bromide at concentrations high enough to eliminate all mitochondrial DNA. 5. haploid spore clones displayed two degrees of glucosamine resistance, weak and strong, while growth due to mitochondrial mutations was generally thick and confluent. 6. a number of glucosamine resistant diploids and haploids, which also possessed a mithchondrial resistance mutation, were unable to grow on medium containing both glucosamine and the particular drug involved. 3 These observations 1~ 6 provided strong evidence that the cytoplasmic glucosamine resistant mutations present in GR6 and GRiO were not situated on mitochondrial DNA. Comparison of the glucosamine resistance mutations to some other known cytoplasmic determinants revealed that: 7. glucosamine resistance and the expression of the killer phenotype were separate phenomena. 8. unlike yeast carrying resistance conferring episomes GR6 and GR10 were not resistant to venturicidin or oligomycin and the GR factor exhibited genetic behaviour different from that of the episomal determinants. These results 7--+8 suggested that glucosamine resistance was not associated with the killer determinant nor with alleged yeast episomes. It is therefore proposed that a yeast plasmid(s), previously undescribed, is responsible for glucosamine resistance. The evidence to date is compatible with the hypothesis that GR6 and GR10 carry allelic mutations of the same plasmid which is tentatively designated (GGM).

The influence of carbon dioxide on growth and metabolism of etiolated Avena sativa 1 coleoptiles

The influence of carbon dioxide on growth and protein synthesis of etiolated Avena coleoptiles was investigated. Evidence is presented that 0.03% carbon dioxide stimulated both these processes; and that carbon dioxide stimulated growth depends on carbon dioxide stimulated protein synthesis, In addition the evidence indicates that carbon dioxide stimulated growth is mediated by metabolism, and that carbon dioxide stimulates growth through a dark fixation process. Growth studies also demonstrated that IAA and carbon dioxide stimulated growth in a synergistic manner.

Synthesis of cytochrome P450 inhibitors of vitamin E metabolism

Please consult the paper edition of this thesis to read. It is available on the 5th Floor of the Library at Call Number: Z 9999 C54 O46 2007