X-ray crystallographic studies on Particulate Methane Monooxygenase, Thioredoxin A and Arginine Decarboxylase

The work presented in this thesis describes the X-ray crystallographic studies of particulate methane monoxygenase (pMMO) from Methylococcus capsulatus (Bath), thioredoxin A (BsTrxA) from Bacillus subtilis and arginine decarboxylase (AdiA) from Escherichia coli. 1. pMMO is a respiratory enzyme that catalyses the first step in the metabolic pathway in methanotrophic bacteria by converting methane to methanol. The crystal structure of this integral membrane protein was determined by molecular replacement to 3.5 Å resolution. The three metal sites in pMMO were confirmed to be a mononuclear copper site, a dinuclear copper site and a mononuclear zinc site. 2. Thioredoxin is a ubiquitous protein present in nearly all known organisms. Its purpose in the cell is to maintain cysteine-containing proteins in the reduced state by converting intramolecular disulfide bonds to dithiols in a redox reaction. The crystal structure of an active site mutant of BsTrxA was determined by molecular replacement to 1.5 Å resolution. The structure shows a homodimer that resembles enzyme-substrate reaction intermediates. 3. AdiA is a vitamin B6-dependent enzyme that catalyses the decarboxylation of arginine into agmatine. It forms a part of an enzymatic system in E. coli that contribute to making this organism acid resistant. The structure of arginine decarboxylase (AdiA) from E. coli was determined by multiple isomorphous replacement and anomalous scattering (MIRAS) methods to 2.4 Å resolution. The structure revealed a ~800 kDa decamer composed as a pentamer of five homodimers. AdiA becomes active as the cellular environment becomes more acidic. The structure of AdiA suggests how functional decamers associate with decreasing pH or disassociates into inactive homodimers with increasing pH. The enzyme mechanism and determinants for substrate specificity are discussed within the framework of the structure and comparisons with related structures are made

Strategies for whole tooth regeneration and possible rejuvenation of adult dental pulp stem cells.

Questa tesi mira ad approfondire la nostra conoscenza delle strategie utilizzate per la rigenerazione del dente intero, che includono la "dental tissue engineering", la stimolazione della formazione della terza dentizione, la ricombinazione cellulo-tissutale e la rigenerazione del dente tramite manipolazione genica. A tal fine, un'attenzione più specifica viene posta sulle cellule staminali della polpa dentale adulta il cui potenziale rigenerativo diminuisce con l'età. Il nostro studio ha ulteriormente indagato se fosse possibile ringiovanire le cellule staminali della polpa dentale umani (hDPSC) invecchiate utilizzando il microambiente delle hDPSC giovani. A questo scopo, le hDPSC di donatori giovani (YDPSC) e anziani (ODPSC) sono state dapprima isolate, coltivate e caratterizzate. Dopo la raccolta dei rispettivi terreni condizionati (CM), le YDPSC sono state trattate con CM derivato da ODPSC (YDPSCs-OCM), ODPSC trattate con CM di YDPSC (ODPSCs-YCM), YDPSC coltivate in terreno basale (YDPSCs-DMEM/F12) e ODPSC coltivate in terreno basale (ODPSCs-DMEM/F12). Ogni gruppo è stato valutato sulla base di cambiamenti morfologici, proliferazione, apoptosi, senescenza e stress ossidativo. I fattori trofici secreti nel giovane CM hanno esercitato il loro effetto ringiovanente attraverso il suo potenziale antiossidante. Il giovane CM ha ridotto significativamente la generazione di ROS e superossido negli ODPSC. Il CM da YDPSC ha anche ridotto la frammentazione del DNA nucleare negli ODPSC invecchiati attraverso un percorso caspasi-indipendente. L'effetto antiproliferativo e anti-senescenza del CM da YDPSC su ODPSC non sono stati evidenziati. I nostri risultati hanno dimostrato che il CM degli YDPSC potrebbe non ringiovanire completamente gli ODPSC e richiederà ulteriori indagini.This thesis aims to deepen our knowledge of the strategies used for whole tooth regeneration, which include dental tissue engineering, stimulation of third dentition formation, cell-tissue recombination, and gene-manipulated tooth regeneration. In doing so, a more specific focus is placed on adult human dental pulp stem cells whose regenerative potential declines with age. Our study investigated if it were possible to rejuvenate aged human DPSCs (hDPSCs) using the microenvironment of young hDPSCs. To accomplish this, hDPSCs from young donors (YDPSCs) and old donors (ODPSCs) were first isolated, cultured and characterized. Following the collection of their respective conditioned medium, YDPSCs were treated with conditioned medium (CM) derived from ODPSCs (YDPSCs-OCM), ODPSCs treated with CM of YDPSCs (ODPSCs-YCM), YDPSCs replenished with basal medium (YDPSCs-DMEM/F12) and ODPSCs replenished with basal medium (ODPSCs-DMEM/F12). Each group was evaluated based on morphologic changes, proliferation, apoptosis, senescence and oxidative stress. The trophic factors secreted by the young CM exerted their rejuvenating effect via its antioxidative potential. Young CM significantly reduced ROS and superoxide generation in aged hDPSCs. Young CM also decreased nuclear DNA fragmentation in aged hDPSCs via a caspase-independent pathway. The antiproliferative and antisenescence effect of the conditioned medium derived from young hDPSCs on aged hDPSCs was not evidenced. Our results demonstrate that the CM of young hDPSCs may not rejuvenate aged hDPSCs fully and will require further investigation

Mechanism of membrane-tethered mitochondrial protein synthesis

Mitochondrial ribosomes (mitoribosomes) are tethered to the mitochondrial inner membrane to facilitate the cotranslational membrane insertion of the synthesized proteins. We report cryo-electron microscopy structures of human mitoribosomes with nascent polypeptide, bound to the insertase oxidase assembly 1-like (OXA1L) through three distinct contact sites. OXA1L binding is correlated with a series of conformational changes in the mitoribosomal large subunit that catalyze the delivery of newly synthesized polypeptides. The mechanism relies on the folding of mL45 inside the exit tunnel, forming two specific constriction sites that would limit helix formation of the nascent chain. A gap is formed between the exit and the membrane, making the newly synthesized proteins accessible. Our data elucidate the basis by which mitoribosomes interact with the OXA1L insertase to couple protein synthesis and membrane delivery.Peer reviewe

Efficacy of an All-Natural Polyherbal Mouthwash in Patients With Periodontitis: A Single-Blind Randomized Controlled Trial

Aim: This study aimed to evaluate the anti-inflammatory effect and the incidence of adverse effects of an all-natural polyherbal mouthwash in patients with periodontitis, after 3 months of use. These aims were accomplished by using full mouth bleeding score (FMBS), full mouth plaque score (FMPS), probing depth (PD) clinical attachment level (CAL) and a questionnaire recording any adverse events.Methods: The present randomized controlled clinical study considered 40 patients with moderate or severe periodontitis, randomized in two groups: a test group (TG) and a control group (CG). TG was instructed to use a polyherbal mouthwash composed of Propolis resin extract, Plantago lanceolata, Salvia officinalis leaves extract, and 1.75% of essential oils and the CG was given a placebo mouthwash. Both groups were instructed to rinse for 2 min, twice daily after their routine oral home care with the different mouthwashes. Clinical measurements of FMBS, FMPS, PD and CAL were recorded at baseline (T0) and after 3 months (T1). The incidence of adverse outcomes was recorded at every follow-up. Mann–Whitney U test and Wilcoxon signed-rank test were used for the statistical analysis (p < 0.05).Results: The final study sample consisted of 34 healthy individuals, 17 individuals in each of the two groups. TG and CG showed a statistically significant reduction in FMBS (p = 0.001 TG; p = 0.002 CG), FMPS (p = 0.001 TG; p = 0.003 CG), PD (p = 0.001 TG; p = 0.011 CG) and CAL (p < 0.001 TG; p = 0.020 CG) values from baseline to 3 months. The TG showed a statistically significant decrease in FMBS and FMPS compared with the CG. No adverse events or side effects were reported or observed in both groups.Conclusion: The use of polyherbal mouthwash in patients with moderate or severe periodontitis has proved safe and effective in reducing bleeding score and plaque accumulation, after 3 months, compared with placebo, although no difference between the two groups were reported on PD and CAL (both improving at T1)

A study to assess changes in myocardial perfusion after treatment with spinal cord stimulation and percutaneous myocardial laser revascularisation; data from a randomised trial

<p>Abstract</p> <p>Background</p> <p>Spinal cord stimulation (SCS) and percutaneous myocardial laser revascularisation (PMR) are treatment modalities used to treat refractory angina pectoris, with the major aim of such treatment being the relief of disabling symptoms. This study compared the change in myocardial perfusion following SCS and PMR treatment.</p> <p>Methods</p> <p>Subjects with Canadian Cardiovascular Society class 3/4 angina and reversible perfusion defects as assessed by single-photon emission computed tomographic myocardial perfusion scintigraphy were randomised to SCS (34) or PMR (34). 28 subjects in each group underwent repeat myocardial perfusion imaging 12 months post intervention. Visual scoring of perfusion images was performed using a 20-segment model and a scale of 0 to 4.</p> <p>Results</p> <p>The mean (standard deviation) baseline summed rest score (SRS) and stress scores (SSS) were 4.6 (5.7) and 13.6 (9.0) in the PMR group and 6.1 (7.4) and 16.8 (11.6) in the SCS group. At 12 months, SRS was 5.5 (6.0) and SSS 15.3 (11.3) in the PMR group and 6.9 (8.2) and 15.1 (10.9) in the SCS group. There was no significant difference between the two treatment groups adjusted for baseline (p = 1.0 for SRS, p = 0.29 for SSS).</p> <p>Conclusion</p> <p>There was no significant difference in myocardial perfusion one year post treatment with SCS or PMR.</p

Hijacked then lost in translation:the plight of the recombinant host cell in membrane protein structural biology projects

Membrane protein structural biology is critically dependent upon the supply of high-quality protein. Over the last few years, the value of crystallising biochemically characterised, recombinant targets that incorporate stabilising mutations has been established. Nonetheless, obtaining sufficient yields of many recombinant membrane proteins is still a major challenge. Solutions are now emerging based on an improved understanding of recombinant host cells; as a 'cell factory' each cell is tasked with managing limited resources to simultaneously balance its own growth demands with those imposed by an expression plasmid. This review examines emerging insights into the role of translation and protein folding in defining high-yielding recombinant membrane protein production in a range of host cells

Spinal cord stimulation in the treatment of refractory angina: systematic review and meta-analysis of randomised controlled trials

<p>Abstract</p> <p>Background</p> <p>The aim of this paper was undertake a systematic review and meta-analysis of the use of spinal cord stimulation (SCS) in the management of refractory angina.</p> <p>Methods</p> <p>We searched a number of electronic databases including Medline, Embase and Cochrane Library up to February 2008 to identify randomised controlled trials (RCTs) reporting exercise capacity, ischemic burden, functional class, quality of life, usage of anti-anginal medication, costs and adverse events including mortality. Results were reported both descriptively for each study and using random effects meta-analysis. Given the variety in outcomes reported, some outcome results were pooled as standardised mean differences (SMD) and reported in standard deviation units.</p> <p>Results</p> <p>Seven RCTs were identified in a total of 270 refractory angina patients. The outcomes of SCS were found to be similar when directly compared to coronary artery bypass grafting (CABG) and percutaneous myocardial laser revascularisation (PMR). Compared to a 'no stimulation' control, there was some evidence of improvement in all outcomes following SCS implantation with significant gains observed in pooled exercise capacity (SMD: 0.76, 0.07 to 1.46, <it>p </it>= 0.03) and health-related quality of life (SMD: 0.83, 95% CI: 0.32 to 1.34, <it>p </it>= 0.001). Trials were small and were judged to range considerably in their quality. The healthcare costs of SCS appeared to be lower than CABG at 2-years follow up.</p> <p>Conclusion</p> <p>SCS appears to be an effective and safe treatment option in the management of refractory angina patients and of similar efficacy and safety to PMR, a potential alternative treatment. Further high quality RCT and cost effectiveness evidence is needed before SCS can be accepted as a routine treatment for refractory angina.</p

Expression and purification of recombinant G protein-coupled receptors: A review

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques