A rapid genome-proteome approach to identify rate-limiting steps in the butyrate production pathway in probiotic Clostridium butyricum, CBM588.

Objectives: Clostridium butyricum ferments non-digested dietary fibre in the colon to produce butyric acid. Butyrate is a four-carbon, short-chain fatty acid (SCFA) which has multiple health benefits. Many microbial products of pharmaceutical or industrial interest, such as butyrate, are produced in low quantities due to rate-limiting steps in their metabolic pathway, including low abundance or low activity of one or more enzymes in the pathway.

Potential role for pyruvate kinase M2 in the regulation of murine cardiac glycolytic flux during in vivo chronic hypoxia.

Carbohydrate metabolism in heart failure shares similarities to that following hypoxic exposure, and is thought to maintain energy homoeostasis in the face of reduced O2 availability. As part of these in vivo adaptations during sustained hypoxia, the heart up-regulates and maintains a high glycolytic flux, but the underlying mechanism is still elusive. We followed the cardiac glycolytic responses to a chronic hypoxic (CH) intervention using [5-3H]-glucose labelling in combination with detailed and extensive enzymatic and metabolomic approaches to provide evidence of the underlying mechanism that allows heart survivability.

Potential Nutraceutical Properties of Leaves from Several Commonly Cultivated Plants.

Chronic dietary ingestion of suitable phytochemicals may assist with limiting or negating neurodegenerative decline. Current therapeutics used to treat Alzheimer disease elicit broad adverse drug reactions, and alternative sources of cholinesterase inhibitors (ChEIs) are required. Herein, we screened methanolic extracts from seven commonly cultivated plants for their nutraceutical potential; ability to inhibit acetylcholinesterase (AChE) and butyryl-cholinesterase (BuChE), and provision of antioxidant activity through their 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) free radical scavenging capabilities.

A pilot study of alterations in oxidized angiotensinogen and antioxidants in pre-eclamptic pregnancy.

The oxidation status of angiotensinogen (AGT) may have a critical role in pre-eclampsia. We used a validated, quantitative, mass spectrometry-based method to measure the oxidized and total AGT levels in plasma of pre-eclamptic women (n = 17), normotensive-matched controls (n = 17), and healthy non-pregnant women (n = 10). Measurements of plasma glutathione peroxidase (GPx) activity and serum selenium concentrations were performed as markers of circulating antioxidant capacity.

Profiling of 54 plasma glycoproteins by label-free targeted LC-MS/MS.

Glycoproteins play a central role in diverse biological processes and are linked with many serious human diseases. In this paper we present a simple, reproducible and cost-effective analytical workflow that enables the reliable quantification of clinically relevant human plasma glycoproteins using label free microflow LC-MS/MS analysis. Plasma N-glycoproteins were selectively extracted via ConA Sepharose lectin affinity chromatography then separated into two fractions using reversed-phase solid phase extraction.

Measurement of the total angiotensinogen and its reduced and oxidised forms in human plasma using targeted LC-MS/MS.

Angiotensinogen (AGT) is a critical protein in the renin-angiotensin-aldosterone system and may have an important role in the pathogenesis of pre-eclampsia. The disulphide linkage between cysteines 18 and 138 has a key role in the redox switch of AGT which modulates the release of angiotensin I with consequential effects on blood pressure. In this paper, we report a quantitative targeted LC-MS/MS method for the reliable measurement of the total AGT and its reduced and oxidised forms in human plasma.

Method for the Purification of Endogenous Unanchored Polyubiquitin Chains.

Unanchored polyubiquitin chains are endogenous non-substrate linked ubiquitin polymers which have emerging roles in the control of cellular physiology. We describe an affinity purification method based on an isolated ubiquitin-binding domain, the ZnF_UBP domain of the deubiquitinating enzyme USP5, which permits the selective purification of mixtures of endogenous unanchored polyubiquitin chains that are amenable to downstream molecular analyses. Further, we present methods for detection of unanchored polyubiquitin chains in purified fractions.

Mass spectrometry insights into a tandem ubiquitin-binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin.

Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate-conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker-optimized ubiquitin-binding domain hybrid (t-UBD) containing two UBDs, a ZnF-UBP domain in tandem with a linkage-selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48-linked polyubiquitin chains.

Isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury.

We had previously shown that alcohol consumption can induce cellular isoaspartate protein damage via an impairment of the activity of protein isoaspartyl methyltransferase (PIMT), an enzyme that triggers repair of isoaspartate protein damage. To further investigate the mechanism of isoaspartate accumulation, hepatocytes cultured from control or 4-week ethanol-fed rats were incubated in vitro with tubercidin or adenosine. Both these agents, known to elevate intracellular S-adenosylhomocysteine levels, increased cellular isoaspartate damage over that recorded following ethanol consumption in vivo.

Alcohol-related brain damage in humans.

Chronic excessive alcohol intoxications evoke cumulative damage to tissues and organs. We examined prefrontal cortex (Brodmann's area (BA) 9) from 20 human alcoholics and 20 age, gender, and postmortem delay matched control subjects. H & E staining and light microscopy of prefrontal cortex tissue revealed a reduction in the levels of cytoskeleton surrounding the nuclei of cortical and subcortical neurons, and a disruption of subcortical neuron patterning in alcoholic subjects.

Characterisation of faecal protease activity in irritable bowel syndrome with diarrhoea: origin and effect of gut transit.

Objectives: Faecal serine proteases (FSPs) may play a role in irritable bowel syndrome with diarrhoea (IBS-D), but their origin is unclear. We aimed to structurally characterise them and define the impact of colonic cleansing and transit time.

Design: Faecal samples were obtained from 30 healthy volunteers (HV) and 79 patients with IBS-D participating in a trial of ondansetron versus placebo.

An economical high-throughput protocol for multidimensional fractionation of proteins.

A sequential protocol of multidimensional fractionation was optimised to enable the comparative profiling of fractions of proteomes from cultured human cells. Differential detergent fractionation was employed as a first step to obtain fractions enriched for cytosolic, membrane/organelle, nuclear, and cytoskeletal proteins. Following buffer exchange using gel-permeation chromatography, cytosolic proteins were further fractionated by 2-dimensional chromatography employing anion-exchange followed by reversed-phase steps.

Insights into the molecular composition of endogenous unanchored polyubiquitin chains.

The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS).

High-level production of animal-free recombinant transferrin from Saccharomyces cerevisiae.

Background: Animal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin.

Results: The first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from Saccharomyces cerevisiae strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin® USP-NF) and albumin fusion proteins (Novozymes' albufuse®).

The production, characterisation and enhanced pharmacokinetics of scFv-albumin fusions expressed in Saccharomyces cerevisiae.

An expression system is described for the production of monomeric scFvs and scFv antibody fragments genetically fused to human albumin (at either the N- or C-terminus or both). Based upon strains of Saccharomyces cerevisiae originally developed for the production of a recombinant human albumin (Recombumin) this system has delivered high levels of secreted product into the supernatant of shake flask and high cell density fed-batch fermentations. Specific binding to the corresponding ligand was demonstrated for each of the scFvs and scFv-albumin fusions and pharmacokinetic studies showed that the fusion products had greatly extended circulatory half-lives.

Framework for a systems approach to proteomic biomarker profiling in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in females of reproductive age, and its prevalence ranges between 6 and 8%. Associated problems include infertility, menstrual disorders, hirsutism and obesity. In addition, individuals with PCOS may be at increased risk of diabetes, endometrial cancer and, possibly, cardiovascular disease and breast cancer in later life.

Methods for the purification of ubiquitinated proteins.

Post-translational protein modification by the covalent conjugation of ubiquitin, originally implicated as a signal for proteolytic degradation by 26S proteasome, has now been realised to play important roles in the regulation of almost all biological processes in eukaryotes. In order to understand these processes in greater detail there is a requirement for techniques that can purify mixtures of ubiquitin-conjugated proteins, as a prerequisite to their identification and characterisation. Here we review the methods that have been applied to the bulk purification of ubiquitinated proteins and discuss their applications in proteomic analyses of the 'ubiquitome'.

Role of Tyr84 in controlling the reactivity of Cys34 of human albumin.

Cys34 in domain I of the three-domain serum protein albumin is the binding site for a wide variety of biologically and clinically important small molecules, provides antioxidant activity, and constitutes the largest portion of free thiol in blood. Analysis of X-ray structures of albumin reveals that the loop containing Tyr84 occurs in multiple conformations. In structures where the loop is well defined, there appears to be an H-bond between the OH of Tyr84 and the sulfur of Cys34.