A dystrophin-immunoreactive protein in mammalian brain.

Dystrophin is expressed only in muscle and brain, but is absent from all tissues of the adult mdx mouse, a mutant with a single base substitution in the dystrophin gene. The brains of both normal and mdx mice contain a protein of approximately 230 kDa that is recognised by anti-dystrophin antibodies raised to the N-terminal region of the rod-like domain. Although the N-terminal and central rod regions of dystrophin share structural homologies with spectrin, the 230-kDa protein represents neither of the presently described forms of brain spectrin by a variety of criteria (molecular weight, cerebellar localisation, and developmental regulation) and is distinct from the product of the dystrophin gene. Studies of mdx and normal mouse brain show different postnatal developmental regulation of the 230-kDa dystrophin-immunoreactive protein.

Multiple antibiotic resistance (mar) locus in Salmonella enterica serovar typhimurium DT104

In order to understand the role of the mar locus in Salmonella with regard to multiple antibiotic resistance, cyclohexane resistance, and outer membrane protein F (OmpF) regulation, a marA::gfp reporter mutant was constructed in an antibiotic-sensitive salmonella enterica serovar Typhimurium DT104 background. Salicylate induced marA, whereas a number of antibiotics, disinfect ants, and various growth conditions did not. Increased antibiotic resistance was observed upon salicylate induction, although this was shown to be by both mar-dependent and mar-independent pathways. Cyclohexane resistance, however, was induced by salicylate by a mar-dependent pathway. Complementation studies with a plasmid that constitutively expressed marA confirmed the involvement of map in Salmonella with low-level antibiotic resistance and cyclohexane resistance, although the involvement of mar in down regulation of OmpF was unclear. However, marA overexpression did increase the expression of a ca. 50-kDa protein, but its identity remains to be elucidated Passage of the marA::gfp reporter mutant with increasing levels of tetracycline, a method reported to select for mar mutants in Escherichia coli, led to both multiple-antibiotic and cyclohexane resistance. Collectively, these data indicate that low-level antibiotic resistance, cyclohexane resistance, and modulation of OMPs in Salmonella, as in E. coli, can occur in both a mar-dependent and mar-independent manner.

Comparison of bioactivities, binding properties and intra-follicular levels of bovine follistatins

Five isoforms of follistatin (FST) (Mr 31, 33, 35, 37, 41kDa) were purified from bovine follicular fluid (bFF). Comparison of their activin- and heparan sulphate proteoglycan (HSP)-binding properties and bio-potencies in neutralization of activin-A action in vitro revealed that all five isoforms bound activin-A, but with different affinities. Only the 31kDa isoform (FST-288) bound to HSP. FST-288 also showed the greatest biopotency with 35 and 41kDa isoforms being least potent. To determine whether bovine follicle development is associated with changing intrafollicular FST and activin profiles, we analyzed bFF from dominant (DF) and subordinate (SF) follicles collected at strategic times during a synchronized estrous cycle. Total FST, activin-A and activin-AB were measured by immunoassay while individual FST isoforms were quantified by immunoblotting. Follicle diameter was positively correlated with estrogen:progesterone ratio (r=0.56) in bFF but negatively correlated with activin-A (r=-0.34), activin-AB (r=-0.80) and ‘total’ FST (r=-0.70) levels. Follicle diameter was positively correlated with abundance of the 41 kDa isoform (r=0.59) but negatively correlated with abundance of 33 and 31 kDa isoforms (r=-0.56, -0.41). Both follicle status (DF vs SF) and cycle stage affected total FST, activin-A, activin-B levels while follicle status, but not cycle stage, affected abundance of 41, 37, 33 and 31kDa FST isoforms. Collectively, these findings indicate that intrafollicular FST isoforms that differ in their ability to bind and neutralise activins and associate with cell-surface proteoglycans, show divergent changes during follicle development. Enhanced FST production may have an important negative role, either directly or via inhibition of the positive effects of activins, on follicle growth and function during follicular waves.

Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

Preliminary X-ray diffraction analysis of YcdB from Escherichia coli: a novel haem-containing and Tat-secreted periplasmic protein with a potential role in iron transport

YcdB is a periplasmic haem-containing protein from Escherichia coli that has a potential role in iron transport. It is currently the only reported haem-containing Tat-secreted substrate. Here, the overexpression, purification, crystallization and structure determination at 2.0 angstrom resolution are reported for the apo form of the protein. The apo-YcdB structure resembles those of members of the haem-dependent peroxidase family and thus confirms that YcdB is also a member of this family. Haem-soaking experiments with preformed apo-YcdB crystals have been optimized to successfully generate haem-containing YcdB crystals that diffract to 2.9 angstrom. Completion of model building and structure refinement are under way.

The M1 matrix protein controls the filamentous phenotype of influenza A virus

We show that most isolates of influenza A induce filamentous changes in infected cells in contrast to A/WSN/33 and A/PR8/34 strains which have undergone extensive laboratory passage and are mouse-adapted. Using reverse genetics, we created recombinant viruses in the naturally filamentous genetic background of A/Victoria/3/75 and established that this property is regulated by the M1 protein sequence, but that the phenotype is complex and several residues are involved. The filamentous phenotype was lost when the amino acid at position 41 was switched from A to V, at the same time, this recombinant virus also became insensitive to the antibody 14C2. On the other hand, the filamentous phenotype could be fully transferred to a virus containing RNA segment 7 of the A/WSN/33 virus by a combination of three mutations in both the amino and carboxy regions of the M1 protein. This observation suggests that an interaction among these regions of M1 may occur during assembly. (C) 2004 Elsevier Inc. All rights reserved.

The relationship between membrane damage, release of protein and loss of viability in Escherichia coli exposed to high hydrostatic pressure

The aim of this work was to examine a possible association between resistance of two Escherichia coli strains to high hydrostatic pressure and the susceptibility of their cell membranes to pressure-induced damage. Cells were exposed to pressures between 100 and 700 MPa at room temperature (~20C) in phosphate-buffered-saline. In the more pressure-sensitive strain E. coli 8164, loss of viability occurred at pressures between 100 MPa and 300 MPa and coincided with irreversible loss of membrane integrity as indicated by uptake of propidium iodide (PI) and leakage of protein of molecular mass between 9 and 78 kDa from the cells. Protein release increased to a maximum at 400 MPa then decreased, possibly due to intracellular aggregation at the higher pressures. In the pressure-resistant strain E. coli J1, PI was taken up during pressure treatment but not after decompression indicating that cells were able to reseal their membranes. Loss of viability in strain J1 coincided with the transient loss of membrane integrity between approximately 200 MPa and 600 MPa. In E. coli J1 leakage of protein occurred before loss of viability and the released protein was of low molecular mass, between 8 and 11 kDa and may have been of periplasmic origin. In these two strains differences in pressure resistance appeared to be related to differences in the ability of their membranes to withstand disruption by pressure. However it appears that transient loss of membrane integrity during pressure can lead to cell death irrespective of whether cells can reseal their membranes afterwards.

The role of 80K/MARCKS, a specific substrate of protein kinase C, in cell growth and tumour progression

Since its discovery more than a decade ago [Wu et al., 1982; Rozengurt et al., 1983], the 80-87 kDa myristoylated a lanine-rich C-kinase substrate (80K/MARCKS) protein has attracted a great deal of attention from researchers interested in cell growth and tumour progression. However, despite its ubiquitous distribution, a definitive functional role for 80K/MARCKS has not been found. The purpose of this review is to describe the properties, distribution and regulation of 80K/MARCKS and to discuss some of the most recent findings, both from our laboratory and from others, that have suggested a functional role for this protein in modulating cell growth and tumour progression. Furthermore, I will present data from our laboratory that implicates 80K/MARCKS as a novel tumour suppressor in cells of melanocyte origin.

Affinity purification of 61- and 65-kDa rat brain corticotropin-releasing factor receptors and receptor-associated G proteins.

Corticotropin-releasing factor (CRF) has been shown to have a central role in physiological adaptation to stress. It is recognized for stimulating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonomic and immunological responses to stress. Much confusion has surrounded the characterization of CRF receptors, with proteins of varying molecular weights having been identified but never purified and characterized. Recently, two CRF receptors have been cloned from brain and pituitary gland, but evidence from in-situ hybridization studies suggests that further CRF receptor types exist. We therefore developed two techniques which enable the isolation of CRF receptors from whole rat brain. The use of a solid-phase CRF analogue affinity column and elution using a competing ligand resulted in the purification of a single protein of 61 kDa. A second technique was devised which allowed the co-isolation of associated signalling proteins and the identification of CRF bound species following purification. CRF was covalently cross-linked to receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximately 65 kDa and associated alpha and beta gamma G protein subunits. This study demonstrates the successful isolation of CRF receptors which are of different molecular weights to those previously observed from affinity cross-linking studies or predicted from cloned genes. In addition, we confirm the involvement of G proteins in CRF stimulated cell signalling by demonstrating their association with purified CRF receptor.

Elevated levels of corticotrophin-releasing factor binding protein in the blood of patients suffering from arthritis and septicaemia and the presence of novel ligands in synovial fluid.

In view of the reported inflammatory effects of corticotrophin-releasing factor (CRF) and the associated regulatory elements in the gene of its binding protein (BP), we postulate that both BP as well as novel BP-ligands other than CRF may be involved in inflammatory disease. We have investigated BP in the blood of patients with arthritis and septicaemia and have attempted to identify CRF and other BP-ligands in synovial fluid. The BP was found to be significantly elevated in the blood of patients with rheumatoid arthritis and septicaemia. There was less BP-ligand and CRF in synovial fluid from patients with rheumatoid arthritis that from those with osteo- or psoriatic arthritis. There was at least 10-fold more BP-ligand than CRF in the fluid of all three groups of patients. A small amount of immunoreactive human (h)CRF, eluting in the expected position of CRF-41, was detected after high-pressure liquid chromatography of arthritic synovial fluid; however, the bulk of material with BP-ligand binding activity eluted earlier, suggesting that synovial fluid contained novel peptides that interacted with the BP. These results would suggest that the BP and its ligands could play an endocrine immunomodulatory role in inflammatory disease.

The ModFOLD4 server for the quality assessment of 3D protein models

Once you have generated a 3D model of a protein, how do you know whether it bears any resemblance to the actual structure? To determine the usefulness of 3D models of proteins, they must be assessed in terms of their quality by methods that predict their similarity to the native structure. The ModFOLD4 server is the latest version of our leading independent server for the estimation of both the global and local (per-residue) quality of 3D protein models. The server produces both machine readable and graphical output, providing users with intuitive visual reports on the quality of predicted protein tertiary structures. The ModFOLD4 server is freely available to all at: http://www.reading.ac.uk/bioinf/ModFOLD/.

The FunFOLD2 server for the prediction of protein-ligand interactions

The FunFOLD2 server is a new independent server that integrates our novel protein–ligand binding site and quality assessment protocols for the prediction of protein function (FN) from sequence via structure. Our guiding principles were, first, to provide a simple unified resource to make our function prediction software easily accessible to all via a simple web interface and, second, to produce integrated output for predictions that can be easily interpreted. The server provides a clean web interface so that results can be viewed on a single page and interpreted by non-experts at a glance. The output for the prediction is an image of the top predicted tertiary structure annotated to indicate putative ligand-binding site residues. The results page also includes a list of the most likely binding site residues and the types of predicted ligands and their frequencies in similar structures. The protein–ligand interactions can also be interactively visualized in 3D using the Jmol plug-in. The raw machine readable data are provided for developers, which comply with the Critical Assessment of Techniques for Protein Structure Prediction data standards for FN predictions. The FunFOLD2 webserver is freely available to all at the following web site: http://www.reading.ac.uk/bioinf/FunFOLD/FunFOLD_form_2_0.html.

The influence of nutritional supplement drinks on providing adequate calorie and protein intake in older adults with dementia

Objectives: Investigate the impact of the provision of ONS on protein and energy intake from food and ability to meet protein and calorie requirements in people with dementia. Design: After consent by proxy was obtained, participants took part in a cross over study comparing oral intake on an intervention day to an adjacent control day. Setting: The study occurred in Nursing homes and hspitalised settings. Participants: Older adults with dementia over the age of 65 were recruited. 26 participants (aged 83.9+/-8.4 years, MMSE 13.08+/-8.13) took part. Intervention (if any): On the intervention day nutritional supplement drinks were provided three times. Each drink provided 283.3+/-41.8 Kcal of energy and 13.8+/-4.7g of protein. Supplements were removed approximately 1 hour before meals were served and weighed waste (g) was obtained. Measurements: Intake of food consumed was determined on intervention and control days using the quartile method (none, quarter, half, three quarters, all) for each meal component. Results: More people achieved their energy and protein requirements with the supplement drink intervention with no sufficient impact on habitual food consumption. Conclusion: Findings from these 26 participants with dementia indicate that supplement drinks may be beneficial in reducing the prevalence of malnutrition within teh group as more people meet their nutritional requirements. As the provision of supplement drinks is also demonstrated to have an additive effect to consumption of habitual foods, these can be used alongside other measures to also improve oral intake.

Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle.

Dystrophin is the product of the Duchenne muscular dystrophy (DMD) gene. Dystrophin-related protein (utrophin), an autosomal homologue of dystrophin, was studied in skeletal muscle from normal fetuses aged 9-26 weeks and one stillbirth of 41 weeks' gestation, and compared with low- and high-risk DMD fetuses aged 9-20 weeks. Utrophin was present at the sarcolemma from before 9 weeks' gestation, although there was variability in intensity both within and between myotubes. Sarcolemmal immunolabelling became more uniform, and levels of utrophin increased to a maximum at approximately 17-18 weeks. Levels then declined, until by 26 weeks sarcolemmal labelling was negligible and levels were similar to adult control muscle. By 41 weeks there was virtually no sarcolemmal labelling, although immunolabelling of capillaries was bright. Similar results were obtained with normal and DMD fetal muscle. Utrophin is therefore expressed in the presence and absence of dystrophin and down-regulated before birth in normal fetal muscle fibres. Samples were not available to determine whether or when, utrophin levels decline in DMD fetal muscle. On Western blots, utrophin was shown to have a smaller relative molecular mass than adult dystrophin, but similar to the fetal isoform. Blood vessels were brightly immunolabelled at all ages, although utrophin immunolabelling of peripheral nerves increased with gestational age.