Investigation of the Time Dependent Influence of Extracellular Osmotic Stress on Protein Turnover in Skeletal Muscle Cells

Acute alterations in cell volume can substantively modulate subsequent metabolism of substrates. However, how such alterations in skeletal muscle modulate protein metabolism is limited. The purpose of this study was to determine the time dependent influence of extracellular osmotic stress on protein turnover in skeletal muscle cells. L6 cells were incubated in hyperosmotic (HYPER; 425.3 ± 1.8mmol/kg), hypo-osmotic (HYPO; 235.4 ± 1.0mmol/kg) or control (CON; 333.5 ± 1.4mmol/kg) media for 4, 8, 12, or 24hrs. During the final 4hrs, incorporation of L-[ring-3,5-3H]-tyrosine was measured to estimate protein synthesis. Western blotting measured markers of protein synthesis and degradation. No differences were observed in any outcomes except p70S6K phosphorylation whereby HYPO was lower (p<0.05) than CON and HYPER; which remained similar except for a large increase at 8hrs for HYPER. These findings suggest that regardless of duration, extracellular osmotic stress does not significantly affect protein metabolism in L6 cells.

The role of learning and growth hormone-releasing factor in the control of protein intake in rats /

Both learning and basic biological mechanisms have been shown to play a role in the control of protein int^e. It has previously been shown that rats can adapt their dietary selection patterns successfully in the face of changing macronutrient requirements and availability. In particular, it has been demonstrated that when access to dietary protein is restricted for a period of time, rats selectively increase their consumption of a proteincontaining diet when it becomes available. Furthermore, it has been shown that animals are able to associate various orosensory cues with a food's nutrient content. In addition to the role that learning plays in food intake, there are also various biological mechanisms that have been shown to be involved in the control of feeding behaviour. Numerous studies have documented that various hormones and neurotransmitter substances mediate food intake. One such hormone is growth hormone-releasing factor (GRF), a peptide that induces the release of growth hormone (GH) from the anterior pituitary gland. Recent research by Vaccarino and Dickson ( 1 994) suggests that GRF may stimulate food intake by acting as a neurotransmitter in the suprachiasmatic nucleus (SCN) and the adjacent medial preoptic area (MPOA). In particular, when GRF is injected directly into the SCN/MPOA, it has been shown to selectively enhance the intake of protein in both fooddeprived and sated rats. Thus, GRF may play a role in activating protein consumption generally, and when animals have a need for protein, GRF may serve to trigger proteinseeking behaviour. Although researchers have separately examined the role of learning and the central mechanisms involved in the control of protein selection, no one has yet attempted to bring together these two lines of study. Thus, the purpose of this study is to join these two parallel lines of research in order to further our understanding of mechanisms controlling protein selection. In order to ascertain the combined effects that GRF and learning have on protein intake several hypothesis were examined. One major hypothesis was that rats would successfully alter their dietary selection patterns in response to protein restriction. It was speculated that rats kept on a nutritionally complete maintenance diet (NCMD) would consume equal amount of the intermittently presented high protein conditioning diet (HPCD) and protein-free conditioning diet (PFCD). However, it was hypothesized that rats kept on a protein-free maintenance diet (PFMD) would selectively increase their intake of the HPCD. Another hypothesis was that rats would learn to associate a distinct marker flavour with the nutritional content of the diets. If an animal is able to make the association between a marker flavour and the nutrient content of the food, then it is hypothesized that they will consume more of a mixed diet (equal portion HPCD and PFCD) with the marker flavour that was previously paired with the HPCD (Mixednp-f) when kept on the PFMD. In addition, it was hypothesized that intracranial injection of GRF into the SCN/MPOA would result in a selective increase in HPCD as well as Mixednp-t consumption. Results demonstrated that rats did in fact selectively increase their consumption of the flavoured HPCD and Mixednp-f when kept on the NCMD. These findings indicate that the rats successfully learned about the nutrient content of the conditioning diets and were able to associate a distinct marker flavour with the nutrient content of the diets. However, the results failed to support previous findings that GRF increases protein intake. In contrast, the administration of GRF significantly reduced consumption of HPCD during the first hour of testing as compared to the no injection condition. In addition, no differences in the intake of the HPCD were found between the GRF and vehicle condition. Because GRF did not selectively increase HPCD consumption, it was not surprising that GRF also did not increase MixedHP-rintake. What was interesting was that administration of GRF and vehicle did not reduc^Mixednp-f consumption as it had decreased HPCD consumption.

TIME COURSE OF SIGNALING PROCESSES INVOLVED WITH EXTRACELLULAR OSMOTIC - INDUCED GLUCOSE UPTAKE IN MAMMALIAN SKELETAL MUSCLE

Extracellular hyper-osmotic (HYPER) stress increases glucose uptake to defend cell volume, when compared to iso-osmotic (ISO) conditions in skeletal muscle. The purpose of this study was to determine a time course for changes in common signaling proteins involved in glucose uptake during acute hyper-osmotic stress in isolated mammalian skeletal muscle. Rat extensor digitorum longus (EDL) muscles were excised and incubated in a media formulated to mimic ISO (290 ± 10 mmol/kg) or HYPER (400 ± 10 mmol/kg) extracellular condition (Sigma Media-199). Signaling mechanisms were investigated by determining the phosphorylation states of Akt, AMPK, AS160, cPKC and ERK after 30, 45 and 60 minutes of incubation. AS160 was found to be significantly more phosphorylated in HYPER conditions compared to ISO after 30 minutes (p<0.01). It is speculated that AS160 phosphorylation increases glucose transporter 4 (GLUT4) content at the cell surface thereby facilitating an increase in glucose uptake under hyper-osmotic stress.

Potential Urinary Protein Biomarker Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia Patients

Globally, Prostate cancer (PCa) is the most frequently occurring non-cutaneous cancer, and is the second highest cause of cancer mortality in men. Serum prostate specific antigen (PSA) has been the standard in PCa screening since its approval by the American Food & Drug Administration (FDA) in 1994. Currently, PSA is used as an indicator for PCa - patients with a serum PSA level above 4ng/mL will often undergo prostate biopsy to confirm cancer. Unfortunately fewer than similar to 30% of these men will biopsy positive for cancer, meaning that the majority of men undergo invasive biopsy with little benefit. Despite PSA's notoriously poor specificity (33%), there is still a significant lack of credible alternatives. Therefore an ideal biomarker that can specifically detect PCa at an early stage is urgently required. The aim of this study was to investigate the potential of using deregulation of urinary proteins in order to detect Prostate Cancer (PCa) among Benign Prostatic Hyperplasia (BPH). To identify the protein signatures specific for PCa, protein expression profiling of 8 PCa patients, 12 BPH patients and 10 healthy males was carried out using LC-MS/MS. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR. This approach revealed that significant the down-regulation of Fibronectin and TP53INP2 was a characteristic event among PCa patients. Fibronectin mRNA down-regulation, was identified as offering improved specificity (50%) over PSA, albeit with a slightly lower although still acceptable sensitivity (75%) for detecting PCa. As for TP53INP2 on the other hand, its down-regulation was moderately sensitive (75%), identifying many patients with PCa, but was entirely non-specific (7%), designating many of the benign samples as malignant and being unable to accurately identify more than one negative.

Optimizing Computational Frameworks to Study the Influence of the Protein Environment on the Individual Site Energies of Chromophores in Photosystem II of Photosynthesis

Photosynthesis is a process in which electromagnetic radiation is converted into chemical energy. Photosystems capture photons with chromophores and transfer their energy to reaction centers using chromophores as a medium. In the reaction center, the excitation energy is used to perform chemical reactions. Knowledge of chromophore site energies is crucial to the understanding of excitation energy transfer pathways in photosystems and the ability to compute the site energies in a fast and accurate manner is mandatory for investigating how protein dynamics ef-fect the site energies and ultimately energy pathways with time. In this work we developed two software frameworks designed to optimize the calculations of chro-mophore site energies within a protein environment. The first is for performing quantum mechanical energy optimizations on molecules and the second is for com-puting site energies of chromophores in a fast and accurate manner using the polar-izability embedding method. The two frameworks allow for the fast and accurate calculation of chromophore site energies within proteins, ultimately allowing for the effect of protein dynamics on energy pathways to be studied. We use these frame-works to compute the site energies of the eight chromophores in the reaction center of photosystem II (PSII) using a 1.9 Å resolution x-ray structure of photosystem II. We compare our results to conflicting experimental data obtained from both isolat-ed intact PSII core preparations and the minimal reaction center preparation of PSII, and find our work more supportive of the former.

Role of Phosphorylation of the Oxysterol Binding Protein (OSBP) in (PI(4)P) and Sterol-Containing Membrane Recognition and Binding

Studies have demonstrated that the oxysterol binding protein (OSBP) acts as a phosphatidylinositol phosphate (PIP)-sterol exchanger at membrane contact sites (MCS) of the endoplasmic reticulum (ER) and Golgi. OSBP is known to pick up phosphatidylinositol-4-phosphate (PI(4)P) from the ER, transfer it to the trans-Golgi in exchange for a cholesterol molecule that is then transferred from the trans-Golgi to the ER. Upon further examination of this pathway by Ridgway et al. (1), it appeared that phosphorylation of OSBP played a role in the localization of OSBP. The dephosphorylation state of OSBP was linked to Golgi localization and the depletion of cholesterol at the ER. To mimic the phosphorylated state of OSBP, the mutant OSBP-S5E was designed by Ridgway et al. (1). The lipid and sterol recognition by wt-OSBP and its phosphomimic mutant OSBP-S5E were investigated using immobilized lipid bilayers and dual polarization interferometry (DPI). DPI is a technique in which the protein binding affinity to immobilized lipid bilayers is measured and the binding behavior is examined through real time. Lipid bilayers containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and varying concentrations of PI(4)Ps or sterols (cholesterol or 25-hydroxycholesterol) were immobilized on a silicon nitride chip. It was determined that wt-OSBP binds differently to PI(4)P-containing bilayers compared to OSBP-S5E. The binding behavior suggested that wt-OSBP extracts PI(4)P and the change in the binding behavior, in the case of OSBP-S5E, suggested that the phosphorylation of OSBP may prevent the recognition and/or extraction of PI(4)P. In the presence of sterols, the overall binding behavior of OSBP, regardless of phosphorylation state, was fairly similar. The maximum specific bound mass of OSBP to sterols did not differ as the concentration of sterols increased. However, comparing the maximum specific bound mass of OSBP to cholesterol with oxysterol (25-hydroxycholesterol), OSBP displayed nearly a 2-fold increase in bound mass. With the absence of the wt-OSBP-PI(4)P binding behavior, it can be speculated that the sterols were not extracted. In addition, the binding behavior of OSBP was further tested using a fluorescence based binding assay. Using 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (22-NBD cholesterol), wt-OSBP a one site binding dissociation constant Kd, of 15 ± 1.4 nM was determined. OSBP-S5E did not bind to 22-NBD cholesterol and Kd value was not obtained.