Detection of elevated protein modification in alcoholic cerebellar degeneration

Modification of proteins by reactive ethanol metabolites has been known, for some time, to occur in the liver, the main site of ethanol metabolism. More recently, similar modifi cation has been detected in organs with lesser ability to metabolise ethanol such as skeletal and cardiac muscle, and brain. Such modifi cation may alter protein function or form a neoantigen, making it a target for immune attack.

A polymorphism in the growth hormone (GH)-releasing hormone (GHRH) receptor gene is associated with elevated response to GHRH by human pituitary somatotrophinomas in vitro

A previous study has suggested that a G to A base change at position 169 of the GHRH-receptor gene in human somatotrophinomas is a mutation and confers hypersensitivity to GHRH. The alternative base converts codon 57 from GCG to AGC, resulting in replacement of alanine (Ala) with threonine (Thr). In the present study, two of five human GH-secreting somatotrophinomas were found to possess the codon 57 AGC sequence. The GCG allele was also detected, indicating heterozygosity. However, the patients' normal blood-derived DNA also yielded the same sequence pattern, indicating that the Ala=> Thr amino acid change is a normal polymorphism, and not a somatic mutation. Nevertheless, in vitro, the tumors possessing the Ala=> Thr amino acid change responded very strongly to GHRH in terms of cAMP formation, being increased 40- and 200-fold, in comparison to the 2-fold increases by tumors without the alternative GHRH-receptor sequence. Likewise, the in vitro response of GH secretion to GHRH was elevated. One of the two tumors with the alternative Thr residue, and the highest responder to GHRH, possessed a gsp muration, despite the fact that these defects are thought to reduce responsiveness to GHRH. These results fail to confirm that the GCG => AGC at codon 57 of the GHRH-receptor gene is a mutation, but do support the concept that the alternative form with Thr confers increased sensitivity to GHRH. (C) 2000 Academic Press.

Transglutaminase 2 null macrophages respond to lipopolysaccharide stimulation by elevated proinflammatory cytokine production due to an enhanced αvβ3 integrin-induced Src tyrosine kinase signaling

Transglutaminase 2 (TG2) is a protein crosslinking enzyme with several additional biochemical functions. Loss of TG2 in vivo results in impaired phagocytosis of apoptotic cells and altered proinflammatory cytokine production by macrophages engulfing apoptotic cells leading to autoimmunity. It has been proposed that TG2 acts as an integrin ß(3) coreceptor in the engulfment process, while altered proinflammatory cytokine production is related to the lack of latent TGFß activation by TG2 null macrophages. Here we report that TG2 null macrophages respond to lipopolysaccharide treatment by elevated IL-6 and TNFa production. Though TGFß has been proposed to act as a feed back regulator of proinflammatory cytokine production in LPS-stimulated macrophages, this phenomenon is not related to the lack of active TGFß production. Instead, in the absence of TG2 integrin ß(3) maintains an elevated basal Src family kinase activity in macrophages, which leads to enhanced phosphorylation and degradation of the I?Ba. Low basal levels of I?Ba explain the enhanced sensitivity of TG2 null macrophages to signals that regulate NF-?B. Our data suggest that TG2 null macrophages bear a proinflammatory phenotype, which might contribute to the enhanced susceptibility of these mice to develop autoimmunity and atherosclerosis.

Decreased osteogenesis, increased cell senescence and elevated Dickkopf-1 secretion in human fracture non union stromal cells

The delicately orchestrated process of bone fracture healing is not always successful and long term non union of fractured bone occurs in 5-20% of all cases. Atrophic fracture non unions have been described as the most difficult to treat and this is thought to arise through a cellular and local failure of osteogenesis. However, little is known about the presence and osteogenic proficiency of cells in the local area of non union tissue. We have examined the growth and differentiation potential of cells isolated from human non union tissues compared with normal human bone marrow mesenchymal stromal cells (BMSC). We report the isolation and culture expansion of a population of non union stromal cells (NUSC) which have a CD profile similar to that of BMSC, i.e. CD34-ve, CD45-ve and CD105+ve. The NUSC demonstrated multipotentiality and differentiated to some extent along chondrogenic, adipogenic and osteogenic lineages. However, and importantly, the NUSC showed significantly reduced osteogenic differentiation and mineralization in vitro compared to BMSC. We also found increased levels of cell senescence in NUSC compared to BMSC based on culture growth kinetics and cell positivity for senescence associated beta galactosidase (SA-beta-Gal) activity. The reduced capacity of NUSC to form osteoblasts was associated with significantly elevated secretion of Dickkopf-1 (Dkk-1) which is an important inhibitor of Wnt signalling during osteogenesis, compared to BMSC. Conversely, treating BMSC with levels of rhDkk-1 that were equivalent to those levels secreted by NUSC inhibited the capacity of BMSC to undergo osteogenesis. Treating BMSC with NUSC conditioned medium also inhibited the capacity of the BMSC to undergo osteogenic differentiation when compared to their treatment with BMSC conditioned medium. Our results suggest that the development of fracture non union is linked with a localised reduced capacity of cells to undergo osteogenesis, which in turn is associated with increased cell senescence and Dkk-1 secretion.

Navigating through the maze: the practical issues of conducting research for novices

New postgraduate students embark on their research journey typically with little or no experience in doing research. Supervisors and other more experienced student researchers might help them to find their feet during the first few weeks of their research by sharing their own experience of how they solved similar problems during their research. In this way each novice researcher can learn and benefit from other researchers´ ways of resolving problems. This paper discusses the real concerns that researchers reflected upon during a two-day research workshop, where researchers share problems, exchange ideas for overcoming them and learn from each other´s experiences of conducting research. The output from the workshop is in the form of hints and tips that can guide novice researchers when faced with initial problems. The paper can also be used by a department to induct a novice researcher into their environment.

Elevated urinary neopterin suggests immune activation in Alzheimer’s disease and Down’s syndrome

Neopterin, an unconjugated pteridine, is secreted in large quantities by activated macrophages and can be used as a clinical marker of activated cellular immunity in a patient. Hence, neopterin levels were measured in urine samples taken from patients with Down’s syndrome (DS), non-hospitalized and hospitalized Alzheimer’s disease (AD) and age and sex matched controls. All subjects and patients were free from infectious and malignant disease. A significant effect of age on urinary neopterin levels was found in control subjects, levels being greater in younger and older subjects. No significant trends with age were found in AD and DS patients. The mean level of neopterin was significantly increased in DS and AD compared with age matched controls suggesting immune activation in these patients. In DS, elevated neopterin levels were present in individuals at least 17yrs old suggesting that immune activation could be associated with the initial deposition of beta/A4 in the brain.

The Mechanics of the drawing of polygonal sections from round at elevated temperature

Economic factors such as the rise in cost of raw materials, labour and power, are compelling manufacturers of cold-drawn polygonal sections, to seek new production routes which will enable the expansion in the varieties of metals used and the inclusion of difficult-to-draw materials. One such method generating considerable industrial interest is the drawing of polygonal sections from round at elevated temperature. The technique of drawing mild steel, medium carbon steel and boron steel wire into octagonal, hexagonal and square sections from round at up to 850 deg C and 50% reduction of area in one pass has been established. The main objective was to provide a basic understanding of the process, with particular emphasis being placed on modelling using both experimental and theoretical considerations. Elevated temperature stress-strain data was obtained using a modified torsion testing machine. Data were used in the upper bound solution derived and solved numerically to predict drawing stress strain, strain-rate, temperature and flow stress distribution in the deforming zone for a range of variables. The success of this warm working process will, of course, depend on the use of a satisfactory elevated temperature lubricant, an efficient cooling system, a suitable tool material having good wear and thermal shock resistance and an efficient die profile design which incorporates the principle of least work. The merits and demerits of die materials such as tungsten carbide, chromium carbide, Syalon and Stellite are discussed, principally from the standpoint of minimising drawing force and die wear. Generally, the experimental and theoretical results were in good agreement, the drawing stress could be predicted within close limits and the process proved to be technically feasible. Finite element analysis has been carried out on the various die geometries and die materials, to gain a greater understanding of the behaviour of these dies under the process of elevated temperature drawing, and to establish the temperature distribution and thermal distortion in the deforming zone, thus establishing the optimum die design and die material for the process. It is now possible to predict, for the materials already tested, (i) the optimum drawing temperature range, (ii) the maximum possible reduction of area per pass, (iii) the optimum drawing die profiles and die materials, (iv) the most efficient lubricant in terms of reducing the drawing force and die wear.

The effect of elevated temperature on the wear of stainless steel in contact with tungsten carbide

The bearings in the air motors of modern jet aircraft engines must operate dry in hostile conditions at temperatures up to 500° C, where the thrust races in the actuators operate at temperatures up to 300° C. One of the few metallurgical combinations which can function efficiently under these conditions is martensitic stainless steel on tungsten carbide. The work described was initiated to isolate the wear mechanisms of two such steels in contact with tungsten carbide at temperatures up to 500° C. Experiments were carried out on angular contact bearings similar to these used in service, where both rolling and sliding is present and also for pure sliding conditions using a pin-on-disc apparatus. Wear measurements of the bearings were obtained with wear rates, friction and surface temperatures from the pin-on-disc machine for a series of loads and speeds. Extensive X-ray diffraction analysis was carried out on the wear debris, with also S.E.M. analysis and hardness tests on the worn surfaces along with profilometry measurements of the disc. The oxidational parameters of the steel were obtained from measurements of oxide growth rates by ellipsometry. Three distinct mechanisms of wear were established and the latter two were found to be present in both configurations. These involve an oxidational-abrasive mechanism at loads below 40 N with pin surface temperatures up to about 300 °C, with the mechanism changing to severe wear for higher loads. As the temperature increases a third wear mechanism appears due to transfer of relatively soft oxide films to the steel surface reducing the wear rate. Theoretical K factors were derived and compared with experimental values which were found to be in good agreement for the severe wear mechanism. The pin-on-disc experiments may be useful as a screening test for material selection, without the considerable cost of producing the angular contact bearings.