Effects of metal ions and hydrogen peroxide on the phenotype of yeast hom6Δ mutant.

UNLABELLED: HOM6 is a major gene in the aspartate pathway which leads to biosynthesis of threonine and methionine. The phenotypes of the gene deletion mutant (hom6∆) in a variety of cultural conditions have previously provided meaningful insights into the biological roles of HOM6 and its upstream intermediate metabolites. Here, we conducted a survey on a spectrum of metal ions for their effect on the aspartate pathway and broader sulphur metabolism. We show that manganese (Mn(2+) ) promoted the growth of hom6∆ under both anaerobic and aerobic conditions. Unexpectedly, 4 mmol l(-1) hydrogen peroxide (H2 O2 ), a dose normally causing temporary cell growth arrest, enhanced the growth of hom6∆ under the anaerobic condition only, while it had no effect on the wild type strain BY4743. We propose that Mn(2+) and H2 O2 promote the growth of hom6∆ by reducing the accumulation of the toxic intermediate metabolite-aspartate ß-semialdehyde, via directing the aspartate pathway to the central sugar metabolism-tricarboxylic acid cycle. SIGNIFICANCE AND IMPACT OF THE STUDY: This study focuses on the yeast strain which lacks homoserine dehydrogenase encoded by HOM6 gene in aspartate metabolism. The HOM6-deletion mutant (hom6Δ) was analysed in the context of varying environmental parameters such as metal ions and oxidants, under anaerobic and aerobic conditions. We demonstrated that both manganese and hydrogen peroxide can promote the growth of hom6Δ, with the latter exerting such effect only under anaerobic condition. The findings are relevant to the research areas of ageing and anti-fungal drug development. It highlights the importance of interactions between gene expression and environmental factors as well as culture conditions.

Achieving cardiac rehabilitation uptake targets: What is the value case for commissioners? A UK case-study.

Cardiac Rehabilitation (CR) has become an established intervention to support patient recovery after a cardiac event, with evidence supporting its effectiveness and cost-effectiveness in improving patient health and reducing future burden on healthcare systems. However, this evidence has focussed on the national value case for CR rather than at the point at which it is commissioned. This analysis uses the UK as a case-study to explore variation in current CR engagement and disassemble the value case from a commissioner perspective. Using data collected by the National Audit of CR (NACR), and an existing model of cost-effectiveness, we present details on the current level of CR uptake by commissioning region (Specialist Clinical Networks) in light of the current UK target of achieving 85% uptake. We then interrogate the value case for achieving the target at a commissioner level, highlighting the expected profile of health benefits and healthcare system costs over the long-term. Importantly we consider where this may differ from the national value case. Each commissioning region has a unique level of CR uptake and sociodemographic profile. Concurrently, the value case for commissioning CR relies on the upfront cost of the service being offset by long-term healthcare savings, and health improvements. The shift in the UK and internationally to more localised commissioning necessitates evidence of cost-effectiveness that better reflects the realities of those decision makers. This paper provides vital additional data to facilitate such commissioners to understand the value case in increasing CR uptake in line with national policy.

Depletion of CD8+ cells in human thymic medulla results in selective immune deficiency.

CD8 molecules expressed on the surface of a subset of T cells participate in the selection of class I MHC antigen-restricted T cells in the thymus, and in MHC-restricted immune responses of mature class I MHC antigen-restricted T cells. Here we describe an immune-deficient patient with lack of CD8+ peripheral blood cells. The patient presented with Pneumocystis carinii pneumonia and was unable to reject an allogeneic skin graft, but had normal primary and secondary antibody responses. Examination of the patient's thymus revealed that the loss of CD8+ cells occurred during intrathymic differentiation: the patient's immature cortical thymocytes included both CD4+ and CD8+ cells while the mature medullary cells expressed the CD4 but not the CD8 protein on their surface. Northern blot and polymerase chain reaction analyses revealed the presence of CD8 alpha and beta mRNA in the patient's thymus but not in the peripheral blood. Both class I MHC antigen expression and the expressed TCR V beta repertoire are normal in this patient. These data are consistent with an impaired selection of CD8+ cells in the patient's thymus and support the role of the CD8 surface protein in thymic selection previously characterized in genetically manipulated and inbred mice.

Friction transfer of polytetrafluoroethylene (PTFE) to produce nanoscale features and influence cellular response in vitro.

A large number of cell types are known to respond to chemical and topographical patterning of substrates. Friction transfer of polytetrafluoroethylene (PTFE) onto substrates has been shown to produce continuous, straight, parallel nanofibres. Ammonia plasma treatment can be used to defluorinate the PTFE, decreasing the dynamic contact angle. Fibroblast and epithelial cells were elongated and oriented with their long axis parallel to the fibres, both individually and in clusters. The fibres restricted cell migration. Cell alignment was slightly reduced on the plasma-treated fibres. These results indicated that although surface topography can affect cellular response, surface chemistry also mediates the extent of this response.

Regulation of the transcript for a lysosomal protein: evidence for a gene program modified by platelet-derived growth factor.

Platelet-derived growth factor (PDGF) stimulates density-arrested BALB/c-3T3 cells to synthesize MEP, a lysosomal protein. This enhanced synthesis appears to be largely regulated by the PDGF-modulated accumulation of MEP mRNA, a 1.8-kilobase species. The increase in the MEP transcript, which is dependent on the PDGF concentration, begins 3 to 4 h after PDGF addition and is maximal at 12 h. The accumulation of the MEP transcript is growth-factor specific: PDGF and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, an agent which acts like PDGF, induce MEP RNA accumulation, whereas epidermal growth factor, somatomedin C, insulin, and whole plasma do not. A spontaneously transformed BALB/c-3T3 cell line (ST2-3T3), which does not require PDGF for growth, optimally expresses MEP RNA in the absence of PDGF. The PDGF-modulated increase in MEP RNA is unlike PDGF-modulated c-myc and c-fos RNA accumulation because it is blocked by cycloheximide, suggesting a requirement for de novo protein synthesis. It appears that PDGF modulates a program of gene expression with the accumulation of some transcripts, typified by MEP, being dependent upon the translation of others.

Malignant transformation and tumor promoter treatment increase levels of a transcript for a secreted glycoprotein.

The major excreted protein of transformed mouse fibroblasts, a secreted, mannose 6-phosphate-containing glycoprotein, is induced in nontransformed cells by a variety of transforming agents, by phorbol esters, and by platelet-derived growth factor. We report here the molecular cloning of the cDNA encoding this protein and demonstrate that its induction is a consequence of enhanced mRNA levels for major excreted protein in both tetradecanoyl phorbol acetate-treated 3T3 cells and 3T3 cells transformed by a variety of retroviruses or retroviral oncogenes. These results indicate that tumor promoters and retroviral transformation might share a common pathway of action in cultured cells and that major excreted protein is a molecular marker for the growth response of cells to these agents.

The effect of silica nanoparticulate coatings on serum protein adsorption and cellular response.

Serum protein adsorption on colloidal silica surfaces was investigated using a quartz crystal microbalance with dissipation (QCM-D) monitoring. The amount of serum proteins adsorbed on colloidal silica-coated surfaces was not significantly different from the control silica surfaces, with the exception of 21nm colloidal silica which experienced significantly less (P<0.05) fibrinogen adsorption compared with control silica. The adhesion and proliferation of human endothelial cells (C11STH) on nano-scale colloidal silica surfaces were significantly reduced compared with control silica surfaces, suggesting that the conformation of adsorbed proteins on the colloidal silica surfaces plays a role in modulating the amount of cell binding. Fibronectin is one of the main extracellular matrix proteins involved in endothelial cell attachment to biomaterial surfaces. There was reduced binding of a monoclonal anti-fibronectin antibody, that reacted specifically with the cell-binding fragment, to fibronectin-coated colloidal silica surfaces compared with control silica surfaces. This suggests that the fibronectin adsorbed on the colloidal silica-coated surfaces was conformationally changed compared with control silica reducing the availability of the cell-binding domain of fibronectin.

The human T cell receptor gamma genes are transcribed from TATA-less promoters containing a conserved heptamer sequence.

We have used the anchored polymerase chain reaction (A-PCR) to clone and compare the 5' upstream regions of the human T cell receptor gamma (TRG) genes. Whereas little homology was found among subgroups I, II, III and IV, sequence alignment of TRG subgroup I members revealed a high degree of homology in the 5' sequences. A conserved heptamer sequence (CTGCAGG), which was found upstream from the translation initiation site of all TRG genes in our analysis. Determination of the transcription initiation site located the conserved heptamer 65 base pairs upstream from the cap sites of V5. No TATA box or other cis-acting promoter sequences could be identified in any of the human TRG upstream sequences.

Expression of the human T cell receptor V beta repertoire.

We have used a sensitive assay, based on amplification of cDNA by the polymerase chain reaction, to determine in a variety of human tissues the relative levels of expression of the genes coding for each of the twenty families of human TcR V beta. We have determined the diversity of the expressed TcR V beta repertoire early in the development of the immune system. We have shown that the full TcR V beta repertoire is expressed early into the second trimester; the expressed repertoire is as diverse at this point, in both fetal thymus and spleen, as it is in mature thymus and peripheral blood lymphocytes. In addition the relative expression in the fetal thymus of each V beta gene is conserved to a large extent in the fetal spleen.

Designing environmental research for impact.

Transdisciplinary research, involving close collaboration between researchers and the users of research, has been a feature of environmental problem solving for several decades, often spurred by the need to find negotiated outcomes to intractable problems. In 2005, the Australian government allocated funding to its environment portfolio for public good research, which resulted in consecutive four-year programmes (Commonwealth Environmental Research Facilities, National Environmental Research Program). In April 2014, representatives of the funders, researchers and research users associated with these programmes met to reflect on eight years of experience with these collaborative research models. This structured reflection concluded that successful multi-institutional transdisciplinary research is necessarily a joint enterprise between funding agencies, researchers and the end users of research. The design and governance of research programmes need to explicitly recognise shared accountabilities among the participants, while respecting the different perspectives of each group. Experience shows that traditional incentive systems for academic researchers, current trends in public sector management, and loose organisation of many end users, work against sustained transdisciplinary research on intractable problems, which require continuity and adaptive learning by all three parties. The likelihood of research influencing and improving environmental policy and management is maximised when researchers, funders and research users have shared goals; there is sufficient continuity of personnel to build trust and sustain dialogue throughout the research process from issue scoping to application of findings; and there is sufficient flexibility in the funding, structure and operation of transdisciplinary research initiatives to enable the enterprise to assimilate and respond to new knowledge and situations.

Lack of effect of vitamin D and its metabolites on intestinal phosphate transport in familial hypophosphatemia of mice.

Intestinal calcium and phosphate transport was studied in normal and hypophosphatemic mice fed a variety of dietary regimens with and without vitamin D. Regardless of dietary phosphorus levels, the genetic hypophosphatemic mice had drastically reduced levels of serum inorganic phosphate and intestinal phosphate transport while showing only slightly reduced serum calcium and intestinal calcium transport levels. The inclusion of vitamin D in the diet did not increase the low serum phosphorus levels and low rates of intestinal phosphate transport in the genetic hypophosphatemic mice, while it did increase serum calcium and intestinal calcium transport levels. The administration of 1,25-dihydroxyvitamin D3 to the hypophosphatemic mice stimulated intestinal calcium transport but had no effect on intestinal phosphate transport. In contrast, the 1,25-dihydroxyvitamin D3 stimulated both phosphate and calcium transport in the intestine of normal mice. The results obtained are consistent with the hypothesis that the primary metabolic disturbance in familial hypophosphatemia involves a defect in phosphate transport mechanisms.

Strong genetic divergence among populations of a marine fish with limited dispersal, Acanthochromis polyacanthus, within the Great Barrier Reef and the Coral Sea.

Acanthochromis polyacanthus is an unusual tropical marine damselfish that uniquely lacks pelagic larvae and has lost the capacity for broad-scale dispersal among coral reefs. On the modern Great Barrier Reef (GBR), three color morphs meet and hydridize at two zones of secondary contact. Allozyme electrophoreses revealed strong differences between morphs from the southern zone but few differences between morphs from the northern counterpart, thus suggesting different contact histories. We explore the phylogeography of Acanthochromis polyacanthus with mitochondrial cytochrome b region sequences (alignment of 565 positions) obtained from 126 individuals representing seven to 12 fish from 13 sites distributed over 12 reefs of the GBR and the Coral Sea. The samples revealed three major clades: (1) black fish collected from the southern GBR; (2) bicolored fish collected from the GBR and one reef (Osprey) from the northern Coral Sea; (3) black and white monomorphs collected from six reefs in the Coral Sea. All three clades were well supported (72-100%) by bootstrap analyses. Sequence divergences were very high between the major clades (mean = 7.6%) as well as within them (2.0-3.6%). Within clades, most reefs segregated as monophyletic assemblages. This was revealed both by phylogenetic analyses and AMOVAs that showed that 72-90% of the variance originated from differences among groups, whereas only 5-13% originated within populations. These patterns are discussed in relation to the known geological history of coral reefs of the GBR and the Coral Sea. Finally, we ask whether the monospecific status of Acanthochromis should be revisited because the sequence divergences found among our samples is substantially greater than those recorded among well-recognized species in other reef fishes.

Localization of alpha-casein gene transcription in sections of epoxy resin-embedded mouse mammary tissues by in situ hybridization.

The objective of our study was to evaluate the suitability of aldehyde-fixed, epoxy resin-embedded tissue for efficient and reproducible detection of casein mRNA in mouse mammary tissue by in situ hybridization. We used mouse alpha-casein-specific, 35S-labeled riboprobes generated from a Gemini-3 vector. Both complementary (anti-sense) and homologous (sense) RNA probes were utilized in our study (specific activity ranged from 5-7 x 10(8) cpm/micrograms). We tested the stability of newly synthesized [3H]-uridine-labeled RNA in tissue sections subjected to epoxy plastic solvents and found that no detectable loss of label occurred during preparation of semi-thin (1-2 micron) plastic sections for situ hybridization. In addition, it was possible to detect alpha-casein mRNA in deplasticized sections of mammary gland tissue taken from normal, pregnant, or lactating mice, pre-neoplastic mammary alveolar hyperplasias, explant cultures, and mammary tumors. A positive hybridization signal was consistently obtained in sections of mammary tissues where the estimated average copy number for total casein mRNA was greater than or equal to 250/cell. In mammary tumors, where the estimated casein mRNA content was much lower (less than 5/cell), our positive hybridization signal occurred in regions of the tumor that, in consecutive sections, stained positive for casein by immunoperoxidase. After formaldehyde-glutaraldehyde fixation, loss of hybridizable RNA from epoxy-embedded tissues and sections appears to be minimal. Image resolution was greatly enhanced over frozen or paraffin sections of mammary tissue. Non-specific binding of the radioactive probes was very low. Protease treatment of the sections was not necessary for detection of hybridizable signal.

Biocompatibility of glass ionomer cements.

Glass ionomer cements (GICs) are widely used in the dental field and are increasingly being considered as materials with potentially good osteogenic properties. This paper details a comprehensive biocompatibility evaluation of a number of GICs. These include conventional, commercially available materials, novel formulations and a new light cured material. The experimental programme entailed in vitro cell culture studies using direct contact and extraction tests and an in vitro rat model in which the GICs were in intimate contact with bone for periods up to 8 wk. The results demonstrate clear differences between the materials and in particular highlight the poor cellular response to the light cured material. Methylthiazolyldiphenyl tetrazolium assay demonstrates stimulation of cell growth by some GIC formulations and indicates that cytotoxic leachable agents can be removed from others. The results obtained following implantation into bone are comprehensively presented using photomicrographs. New bone formation with time is demonstrated with a number of formulations.

Molecular biointeractions of biomedical polymers with extracellular exudate and inflammatory cells and their effects on the biocompatibility, in vivo.

The stability of biomedical polymers in physiological environments is crucial for the normal operation of devices, as well as determining their effect on the tissue response. Degradation is an important factor in polymer biocompatibility, since the environment of the human body can be aggressive to polymers. Most implanted polymers suffer degradation to some extent, and the kinetics and mechanisms of the processes can be affected significantly by various biologically active species, especially enzymes, lipids, peroxides, free radicals and phagocytic cells. The degradation of poly(caprolactone) and poly(DL-lactic acid) under controlled in vivo conditions was studied using a poly(methyl methacrylate) chamber designed to control the exposure of polymers to physiological environments. In particular they may be designed to allow access of extracellular exudate only or access to cells as well as the fluid. The chambers, sealed with filters of pore size either 0.45 micron (impervious to cells) or 3.0 microns (allowing cells to enter the chamber), were implanted subcutaneously into experimental animals for 10, 20 and 30 wk periods. Degradation and molecular interactions of the polymers were characterized by gel permeation chromatography and scanning electron microscopy. The extracellular exudate formed within the implanted chamber is active in promoting the degradation of some biomedical polymers. Inflammatory cells are involved in the biodegradation of implanted polymers by releasing biologically active species such as free radicals into the area surrounding the implant. The data have demonstrated that the hydroxyl radical is likely to be one of the main causes of polymer degradation.

Mechanisms of polymer degradation in implantable devices. I. Poly(caprolactone).

Poly(caprolactone) is a biodegradable aliphatic (poly(alpha-hydroxy acid), with important applications in the field of human therapy, due to its biocompatibility and bioresorbability. The degradation of poly(alpha-hydroxy acids) depends on chemical hydrolysis, but there is much interest in the precise mechanisms, including the role of free radicals, especially oxygen free radicals and their role in human disease. The hydrolytic degradation of poly(caprolactone) in aqueous environments was used as the control in a study of the effects of hydroxyl radicals in aqueous solutions. Different methods (GPC, DSC, SEM) were employed to investigate the mechanism of degradation of this semicrystalline physiologically absorbable polymer. The data indicate that hydroxyl radical is likely to be a major factor in the degradation of this polymer.

Biodegradation of hyaluronic acid derivatives by hyaluronidase.

Hyaluronic acid (salt) (HA) has been chemically modified as a biomaterial for medical applications such as controlled drug release matrices, nerve guides and wound dressings. A series of HA derivatives, which include different ester types and different degrees of esterification, have been used to investigate the stability of these materials in testicular hyaluronidase. Gel permeation chromatography and capillary viscometer have been employed to determine the size of the molecules, the former used for the water insoluble derivatives that dissolve in dimethyl sulphoxide, the latter for the water soluble samples. The preliminary experimental results indicated that the molecular weight of fully esterified hyaluronic acid (both ethyl and benzyl esters) did not decrease after treatment in the enzyme for 7 and 14 days while the water soluble partially esterified HA were degraded by the enzyme producing a sharp reduction of viscosity within minutes. These observations tend to suggest that the carboxylic groups in the beta-glucoronic acid unit are the activation centre of this enzyme and the total blockage of these groups can restrict the cleavage of beta (1-->4) glycoside bonds by this enzyme.

Evaluation of soft tissue response to a poly(urethane urea).

Variations in the performance of vascular prostheses constructed of polyurethanes, and some evidence which suggested that these variations could be due not to the properties of the polymer itself, but to differences in the cellular response to the various microstructures of porous polyurethanes require investigation. Experiments were performed to evaluate quantitatively the extent of the cell behaviour adjacent to a series of polyurethane samples. It was shown that, with Biomer, a polyurethane urea, the profile of cell behaviour as a function of distance from the implant surface and of time following implantation, the response of cells in general and macrophages in particular, varied considerably with different internal microstructure. This supports the suggestion that the cellular response to different structures and susceptibility to degradation are related.

Human neutrophil chemokinesis and polarization induced by hyaluronic acid derivatives.

Neutrophils and macrophages are known to undergo significant modifications in their morphology and basal metabolism in response to chemical factors, in particular changes in the shape, movement, phagocytic activity and degranulation. These phenomena often involve an increase in chemokinesis and cellular secretory activity, usually expressed in antimicrobial activity. Once activated, the cells can move quickly towards the source of the stimulus, where they produce and release great amounts of enzymes (e.g. proteases, hydrolases, lysozyme) and reactive oxygen metabolites (e.g. O2-., H2O2, OH.). This study has examined the ability of surfaces of selected biomaterials to influence neutrophil morphology and locomotion. The surface of two films derived from hyaluronic acid derivatives were compared with that of glass. The two hyaluronic acid derivatives, despite having a similar chemical structure, were shown to interact with human neutrophils in different ways. A hyaluronic acid ethyl ester stimulated the whole population of neutrophils to take up a non-spherical morphology (polarize) and to move with a velocity similar to that of N-formyl-methionine-leucine-phenylalanine-stimulated cells on a glass surface. In contrast, only 44% of the examined cells on the surface of hyaluronic acid benzyl ester were polarized and their mean speed was only slightly higher with respect to that found with non-stimulated cells on glass. Moreover, while on the benzyl ester and on glass a correlation between neutrophil circularity (i.e. the shape of the cell) and cell speed was found, the ethyl ester did not show any correlation.

Quantitative assessment of the tissue response to films of hyaluronan derivatives.

The aim of this study was to evaluate the in vivo response following implantation into a rat model of three innovative hyaluronan derivatives for clinical use: HYAFF 7, HYAFF 11 and HYAFF 11p75 (respectively, the 100% ethyl ester, 100% and 75% benzyl esters). The tissue reaction evoked by films of these new biomaterials implanted into the dorsolumbar musculature of rats was assessed quantitatively using a well established technique based upon an image analysis system. The number of inflammatory cells present and the patterns of cell distribution around the implant up to a distance of 642 microns were examined at different time periods after implantation. Since a well-delineated tissue-material interface was needed for this type of investigation, it was not possible to apply image analysis to sections once dissolution of the implanted materials had begun. Films of both the total esters, HYAFF 7 and HYAFF 11, were found to undergo a slow dissolution process and, after a month, films of these materials were still present at the site of implantation. Differences in response to the two materials were observed only during the first two weeks, particularly with respect to neutrophil distribution and total cellularity. HYAFF 7 was found to be more reactive, with higher numbers of neutrophils near the surface of the implant than HYAFF 11. Thereafter, the differences between the two materials were minimal and owing mainly to a faster dissolution of HYAFF 7 films. After 3 and 5 months, considerable degradation of films of both total esters had occurred. Significant quantities of material appeared inside numerous macrophages with an ED1-positive phenotype. Only a very thin layer of fibrous connective tissue, indicative of low reactivity, was found to surround the site of implantation, separating the dissolved material and the phagocytic cells from healthy muscular tissue. ED2-positive macrophages were primarily confined within the lining connective tissue. The partial benzyl ester, HYAFF 11p75, showed a different behaviour. In fact, evidence of film dissolution was already present a week after the implantation. After two weeks, the implanted films were completely dissolved and numerous ED1-positive macrophages phagocytosing the material were observed at the site of implantation. Therefore, in agreement with previous in vitro studies, which showed a greater susceptibility to degradation of hyaluronan derivatives with lower percentage of esterification, HYAFF 11p75 underwent resorption faster than the corresponding total ester.