Effect of plasma insulin and branched-chain amino acids on skeletal muscle protein synthesis in fasted lambs

The increase in fractional rate of protein synthesis (K-s) in the skeletal muscle of growing rats during the transition from fasted to fed state has been explained by the synergistic action of a rise in plasma insulin and branched-chain amino acids (BCAA). Since growing lambs Also exhibit an increase in K-s with level of feed intake, the objective of the present study was to determine if this synergistic relationship between insulin and BCAA also occurs in ruminant animals. Six 30 kg fasted (72 h) lambs (8 months of age) received each of four treatments, which were based on continuous infusion into the jugular vein for 6 h of: (1) saline (155 mmol NaCl/l); (2) a mixture of BCAA (0.778 mumol leucine, 0.640 mumol isoleucine and 0.693 mumol valine/min.kg); (3) 18.7 mumol glucose/min.kg (to induce endogenous insulin secretion): (4) co-infusion of BCAA and glucose. Within each period all animals received the same isotope of phenylalanine, (Phe) as follows: (1) L-[1-C-13]Phe; (2) L-phenyl-[ring H-2(5)]-alanine; (3) L-[N-15]Phe; (4) L-[ring 2,6-H-3]Phe. Blood was sampled serially during infusions to measure plasma concentrations of insulin, glucose and amino acids, and plasma free Phe isotopic activity; biopsies were taken 6 h after the beginning of infusions to determine K-s in in. longissimus dorsi and vastus muscle. Compared with control (saline-infused) lambs, K-s was increased by an average of 40% at the end of glucose infusion, but this effect was not statistically significant in either of the muscles sampled. BCAA infusion, alone or in combination with glucose, also had no significant effect on K-s compared with control sheep. K-s was approximately 60% greater for vastus muscle than for m. longissimus dorsi (P<0.01), regardless of treatment. It is concluded that there are signals other than insulin and BCAA that are responsible for the feed-induced increase in K-s in muscle of growing ruminant animals.

Short communication: Suppressor of cytokine signaling-2 mRNA increases after parturition in the liver of dairy cows

After parturition, the somatotropic axis of the dairy cow is uncoupled, partly because of reduced concentration of liver-specific GH receptor (GHR) 1A. Estradiol-17 beta (E-2) concentrations increase at parturition and E-2 upregulates suppressors of cytokine signaling-2 (SOCS-2) mRNA expression, potentially inhibiting GH signaling. Therefore, we hypothesized that SOCS-2 mRNA is upregulated after parturition. Multiparous Holstein cows (n = 18) were dried off 45 d before expected parturition and fed diets to meet nutrient requirements at ad libitum or limited dry matter intake during the dry period. All cows were fed the same diet ad libitum from calving until 4 wk after parturition. Blood samples were collected weekly and more frequently near parturition. Liver biopsies obtained at -21, -7, 2, and 28 d relative to parturition were assessed for SOCS-2 and GHR 1A mRNA by quantitative real-time reverse-transcription PCR. The relative amount of SOCS-2 mRNA increased after parturition with both treatments and was greater on d 2 for cows limit-fed during the dry period compared with cows fed at ad libitum dry matter intake. Plasma E2 concentrations increased on d -13, -5 and 1 relative to parturition and the increases were greater in limit-fed cows. Plasma GH concentration was greater for limit-fed cows and increased after parturition in all cows. The amount of GHR 1A mRNA did not differ between diets but decreased on d 2. In addition to reduced GHR 1A, increased SOCS-2 mRNA after parturition, perhaps because of increased E-2, may further uncouple GH signaling in the liver of the transition dairy cow.

Aluminium in human breast tissue

Aluminium is omnipresent in everyday life and increased exposure is resulting in a burgeoning body burden of this non-essential metal. Personal care products are potential contributors to the body burden of aluminium and recent evidence has linked breast cancer with aluminium-based antiperspirants. We have used graphite furnace atomic absorption spectrometry (GFAAS) to measure the aluminium content in breast biopsies obtained following mastectomies. The aluminium content of breast tissue and breast tissue fat were in the range 4-437 nmol/g dry wt. and 3-192 nmol/g oil, respectively. The aluminium content of breast tissue in the outer regions (axilla and lateral) was significantly higher (P = 0.033) than the inner regions (middle and medial) of the breast. Whether differences in the regional distribution of aluminium in the breast are related to the known higher incidence of tumours in the outer upper quadrant of the breast remains to be ascertained. (c) 2007 Elsevier Inc. All rights reserved.

Quantification of mitochondrial DNA mutation load

Mitochondrial DNA (mtDNA) mutations are an important cause of genetic disease and have been proposed to play a role in the ageing process. Quantification of total mtDNA mutation load in ageing tissues is difficult as mutational events are rare in a background of wild-type molecules, and detection of individual mutated molecules is beyond the sensitivity of most sequencing based techniques. The methods currently most commonly used to document the incidence of mtDNA point mutations in ageing include post-PCR cloning, single-molecule PCR and the random mutation capture assay. The mtDNA mutation load obtained by these different techniques varies by orders of magnitude, but direct comparison of the three techniques on the same ageing human tissue has not been performed. We assess the procedures and practicalities involved in each of these three assays and discuss the results obtained by investigation of mutation loads in colonic mucosal biopsies from ten human subjects.

Gene expression profiling of human hibernating myocardium: Increased expression of B-type natriuretic peptide and proenkephalin in hypocontractile vs normally-contracting regions of the heart

A greater understanding of the molecular basis of hibernating myocardium may assist in identifying those patients who would most benefit from revascularization. Paired heart biopsies were taken from hypocontractile and normally-contracting myocardium (identified by cardiovascular magnetic resonance) from 6 patients with chronic stable angina scheduled for bypass grafting. Gene expression profiles of hypocontractile and normally-contracting samples were compared using Affymetrix microarrays. The data for patients with confirmed hibernating myocardium were analysed separately and a different, though overlapping, set (up to 380) of genes was identified which may constitute a molecular fingerprint for hibernating myocardium. The expression of B-type natriuretic peptide (BNP) was increased in hypocontractile relative to normally-contracting myocardium. The expression of BNP correlated most closely with the expression of proenkephalin and follistatin 3, which may constitute additional heart failure markers. Our data illustrate differential gene expression in hypocontractile and/hibernating myocardium relative to normally-contracting myocardium within individual human hearts. Changes in expression of these genes, including increased relative expression of natriuretic and other factors, may constitute a molecular signature for hypocontractile and/or hibernating myocardium.

N-nitroso compound exposure-associated transcriptomic profiles are indicative of an increased risk for colorectal cancer

Endogenous formation of N-nitroso compounds (NOCs), which are known animal carcinogens, could contribute to human carcinogenesis but definitive evidence is still lacking. To investigate the relevance of NOCs in human colorectal cancer (CRC) development, we analyzed whole genome gene expression modifications in human colon biopsies in relation to fecal NOC exposure. We had a particular interest in patients suffering from intestinal inflammation as this may stimulate endogenous NOC formation, and consequently predispose to CRC risk. Inflammatory bowel disease (IBD) patients diagnosed with ulcerative colitis and irritable bowel syndrome patients without inflammation, serving as controls, were therefore recruited. Fecal NOC were demonstrated in the majority of subjects. By associating gene expression levels of all subjects to fecal NOC levels, we identified a NOC exposure-associated transcriptomic response that suggests that physiological NOC concentrations may potentially induce genotoxic responses and chromatin modifications in human colon tissue, both of which are linked to carcinogenicity. In a network analysis, chromatin modifications were linked to 11 significantly modulated histone genes, pointing towards a possible epigenetic mechanism that may be relevant in comprehending NOC-induced carcinogenesis. In addition, pro-inflammatory transcriptomic modifications were identified in visually non-inflamed regions of the IBD colon. However, fecal NOC levels were slightly but not significantly increased in IBD patients, suggesting that inflammation did not strongly stimulate NOC formation. We conclude that NOC exposure is associated with gene expression modifications in the human colon that may suggest a potential role of these compounds in CRC development.

Quantification of mitochondrial DNA mutation load

Mitochondrial DNA (mtDNA) mutations are an important cause of genetic disease and have been proposed to play a role in the ageing process. Quantification of total mtDNA mutation load in ageing tissues is difficult as mutational events are rare in a background of wild-type molecules, and detection of individual mutated molecules is beyond the sensitivity of most sequencing based techniques. The methods currently most commonly used to document the incidence of mtDNA point mutations in ageing include post-PCR cloning, single-molecule PCR and the random mutation capture assay. The mtDNA mutation load obtained by these different techniques varies by orders of magnitude, but direct comparison of the three techniques on the same ageing human tissue has not been performed. We assess the procedures and practicalities involved in each of these three assays and discuss the results obtained by investigation of mutation loads in colonic mucosal biopsies from ten human subjects.

MYOD-1 in normal colonic mucosa - role as a putative biomarker?

Background DNA methylation of promoter-associated CpG islands of certain genes may play a role in the development of colorectal cancer. The MYOD-1 gene which is a muscle differentiation gene has been showed to be significantly methylated in colorectal cancer which, is an age related event. However the role of this gene in the colonic mucosa is not understood and whether methylation occurs in subjects without colon cancer. In this study, we have determined the frequency of methylation of the MYOD-1 gene in normal colonic mucosa and investigated to see if this is associated with established colorectal cancer risk factors primarily ageing. Results We analysed colonic mucosal biopsies in 218 normal individuals and demonstrated that in most individuals promoter hypermethylation was not quantified for MYOD-1. However, promoter hypermethylation increased significantly with age (p < 0.001 using regression analysis) and this was gender independent. We also showed that gene promoter methylation increased positively with an increase in waist to hip (WHR) ratio - the latter is also a known risk factor for colon cancer development. Conclusions Our study suggests that promoter gene hypermethylation of the MYOD-1 gene increases significantly with age in normal individuals and thus may offer potential as a putative biomarker for colorectal cancer.

Nutritional factors and gender influence age-related DNA methylation in the human rectal mucosa

Aberrant methylation of CpG islands (CGI) occurs in many genes expressed in colonic epithelial cells, and may contribute to the dysregulation of signalling pathways associated with carcinogenesis. This cross-sectional study assessed the relative importance of age, nutritional exposures and other environmental factors in the development of CGI methylation. Rectal biopsies were obtained from 185 individuals (84 male, 101 female) shown to be free of colorectal disease, and for whom measurements of age, body size, nutritional status and blood cell counts were available. We used quantitative DNA methylation analysis combined with multivariate modelling to investigate the relationships between nutritional, anthropometric and metabolic factors and the CGI methylation of 11 genes, together with LINE-1 as an index of global DNA methylation. Age was a consistent predictor of CGI methylation for 9/11 genes but significant positive associations with folate status and negative associations with vitamin D and selenium status were also identified for several genes. There was evidence for positive associations with blood monocyte levels and anthropometric factors for some genes. In general, CGI methylation was higher in males than in females and differential effects of age and other factors on methylation in males and females were identified. In conclusion, levels of age-related CGI methylation in the healthy human rectal mucosa are influenced by gender, the availability of folate, vitamin D and selenium, and perhaps by factors related to systemic inflammation

Spirochaetes and other bacterial species associated with bovine digital dermatitis

The 16S rRNA genes from spirochaetes associated with digital dermatitis of British cattle were amplified by polymerase chain reaction from digital dermatitis lesion biopsies using one universal and one treponeme-specific primer. Two treponemal sequences were identified both of which shared a high degree of homology with the oral pathogen Treponema denticola (98%). Two further 16S rRNA gene sequences were obtained and shared similarity to Bacteroides levii (99%) and Mycoplasma hyopharyngis (98%). Polymerase chain reaction with T. denticola-specific primers amplified a potential virulence gene from digital dermatitis lesions which shared a high degree of homology to the 46-kDa haemolysin gene of T. denticola. The significance of the presence of organisms in digital dermatitis lesions of the bovine foot which are closely related to oral pathogens is discussed.

The frequent detection of a treponeme in bovine digital dermatitis by immunocytochemistry and polymerase chain reaction

A study was carried out to determine whether spirochaetes are frequently associated with digital dermatitis in United Kingdom (UK) dairy cattle. Histopathological examination of lesions using a silver stain showed a large number of unidentified spirochaete-like organisms present in digital dermatitis hoof skin tissue in all examined biopsies. Immunocytochemical staining demonstrated that spirochaetes in skin lesions were identified by polyclonal antisera to Borrelia burgdorferi, Treponema denticola and Treponema vincentii (again all biopsies were positively stained), whereas monoclonal antibodies to B. burgdorferi and any Treponema pallidum did not stain any organisms in all biopsies. A PCR of 16S rRNA, previously shown to be specific for a new treponeme, was employed and produced positive results from 82.4% of digital dermatitis tissues. It is concluded that this spirochaete (or related spirochaetes), which is similar to human oral treponemes, is frequently associated with, and may be responsible for, pathological changes in digital dermatitis. (C) 1998 Elsevier Science B.V.

Role for protease activity in visceral pain in irritable bowel syndrome

Mediators involved in the generation of symptoms in patients with irritable bowel syndrome (IBS) are poorly understood. Here we show that colonic biopsy samples from IBS patients release increased levels of proteolytic activity (arginine cleavage) compared to asymptomatic controls. This was dependent on the activation of NF-kappaB. In addition, increased proteolytic activity was measured in vivo, in colonic washes from IBS compared with control patients. Trypsin and tryptase expression and release were increased in colonic biopsies from IBS patients compared with control subjects. Biopsies from IBS patients (but not controls) released mediators that sensitized murine sensory neurons in culture. Sensitization was prevented by a serine protease inhibitor and was absent in neurons lacking functional protease-activated receptor-2 (PAR2). Supernatants from colonic biopsies of IBS patients, but not controls, also caused somatic and visceral hyperalgesia and allodynia in mice, when administered into the colon. These pronociceptive effects were inhibited by serine protease inhibitors and a PAR2 antagonist and were absent in PAR2-deficient mice. Our study establishes that proteases are released in IBS and that they can directly stimulate sensory neurons and generate hypersensitivity symptoms through the activation of PAR2.

The aluminium content of breast tissue taken from women with breast cancer

The aetiology of breast cancer is multifactorial. While there are known genetic predispositions to the disease it is probable that environmental factors are also involved. Recent research has demonstrated a regionally specific distribution of aluminium in breast tissue mastectomies while other work has suggested mechanisms whereby breast tissue aluminium might contribute towards the aetiology of breast cancer. We have looked to develop microwave digestion combined with a new form of graphite furnace atomic absorption spectrometry as a precise, accurate and reproducible method for the measurement of aluminium in breast tissue biopsies. We have used this method to test the thesis that there is a regional distribution of aluminium across the breast in women with breast cancer. Microwave digestion of whole breast tissue samples resulted in clear homogenous digests perfectly suitable for the determination of aluminium by graphite furnace atomic absorption spectrometry. The instrument detection limit for the method was 0.48 μg/L. Method blanks were used to estimate background levels of contamination of 14.80 μg/L. The mean concentration of aluminium across all tissues was 0.39 μg Al/g tissue dry wt. There were no statistically significant regionally specific differences in the content of aluminium. We have developed a robust method for the precise and accurate measurement of aluminium in human breast tissue. There are very few such data currently available in the scientific literature and they will add substantially to our understanding of any putative role of aluminium in breast cancer. While we did not observe any statistically significant differences in aluminium content across the breast it has to be emphasised that herein we measured whole breast tissue and not defatted tissue where such a distribution was previously noted. We are very confident that the method developed herein could now be used to provide accurate and reproducible data on the aluminium content in defatted tissue and oil from such tissues and thereby contribute towards our knowledge on aluminium and any role in breast cancer.

Towards pain-free diagnosis of skin diseases through multiplexed microneedles: biomarker extraction and detection using a highly sensitive blotting method

Immunodiagnostic microneedles provide a novel way to extract protein biomarkers from the skin in a minimally invasive manner for analysis in vitro. The technology could overcome challenges in biomarker analysis specifically in solid tissue, which currently often involves invasive biopsies. This study describes the development of a multiplex immunodiagnostic device incorporating mechanisms to detect multiple antigens simultaneously, as well as internal assay controls for result validation. A novel detection method is also proposed. It enables signal detection specifically at microneedle tips and therefore may aid the construction of depth profiles of skin biomarkers. The detection method can be coupled with computerised densitometry for signal quantitation. The antigen specificity, sensitivity and functional stability of the device were assessed against a number of model biomarkers. Detection and analysis of endogenous antigens (interleukins 1α and 6) from the skin using the device was demonstrated. The results were verified using conventional enzyme-linked immunosorbent assays. The detection limit of the microneedle device, at ≤10 pg/mL, was at least comparable to conventional plate-based solid-phase enzyme immunoassays.

Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin’s substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells’ ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone’s effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.