Intermediates in the oxidative pathway from torulene to torularhodin in the red yeasts Cystofilobasidium infirmominiatum and C. capitatum (Heterobasidiomycetes, Fungi)

Two red Cystofilobasidium spp. isolated from spring sap-flows of Betula pendula were analysed for their carotenoid content. In Cystofilobasidium infirmominiatum, three unusual pigments were detected and identified by structure elucidation as oxidised torulene derivatives. These included 16'-hydroxytorulene and torularhodinaldehyde, two carotenoids known so far only from chemical synthesis or as postulated biosynthetic intermediates en route to torularhodin. Unprecedented formation of beta-apo-2'-carotenal was also observed. The production of these pigments in pure culture was dependent on enhanced oxidative stress caused by cultivation in well-aerated (indented) flasks with or without 2% ethanol (16'-hydroxytorulene), or with 100 microM duroquinone (torularhodinaldehyde and beta-apo-2'-carotenal). Among these three pigments, only 16'-hydroxytorulene was detected in C. capitatum. Torularhodin, a common end product of carotenoid oxidation in red yeasts, was not produced by either species under any incubation conditions. Biosynthetic aspects of incomplete oxidation of torulene by these Cystofilobasidium spp. are discussed.     

The optimization of protocols for proteome difference gel electrophoresis (DiGE) analysis of preneoplastic skin

Difference gel electrophoresis (DiGE) allows the reliable comparison of proteome differences between two or three samples within a single gel, by way of a CyDye fluorescent labeling system. This facilitates identification of protein differences avoiding the difficulties associated with gel-to-gel variation. A drawback of this approach is the necessity for high-purity protein samples, since contaminants can interfere with the labeling process, affecting subsequent analysis. Thus far, DiGE has been applied to the study of various sample types derived from relatively simple starting materials such as serum, cell lines, or primary cells. Herein, we describe optimization of protein extraction and purification from a complex tissue (the murine ear) of which a major component is skin, which is compatible with the CyDye labeling system and DiGE. Protein samples obtained by this method from preneoplastic, transgenic tissue have been effectively compared to normal tissue samples to reveal bona fide differences, verifiable by Western blotting. In total, 41 protein differences (21 up- and 20 down-regulated in the pathological samples) were identified by mass spectrometry (MS). This method can therefore form a guide for those wishing to perform DiGE on complex tissues, and is especially useful for samples with relatively insoluble components such as skin.     

Levothyroxine monotherapy cannot guarantee euthyroidism in all athyreotic patients

Context

Levothyroxine monotherapy is the treatment of choice for hypothyroid patients because peripheral T4 to T3 conversion is believed to account for the overall tissue requirement for thyroid hormones. However, there are indirect evidences that this may not be the case in all patients.

Objective

To evaluate in a large series of athyreotic patients whether levothyroxine monotherapy can normalize serum thyroid hormones and thyroid-pituitary feedback.

Design

Retrospective study.

Setting

Academic hospital.

Patients

1,811 athyreotic patients with normal TSH levels under levothyroxine monotherapy and 3,875 euthyroid controls.

Measurements

TSH, FT4 and FT3 concentrations by immunoassays.

Results

FT4 levels were significantly higher and FT3 levels were significantly lower (p<0.001 in both cases) in levothyroxine-treated athyreotic patients than in matched euthyroid controls. Among the levothyroxine-treated patients 15.2% had lower serum FT3 and 7.2% had higher serum FT4 compared to euthyroid controls. A wide range of FT3/FT4 ratios indicated a major heterogeneity in the peripheral T3 production capacity in different individuals. The correlation between thyroid hormones and serum TSH levels indicated an abnormal feedback mechanism in levothyroxine-treated patients.

Conclusions

Athyreotic patients have a highly heterogeneous T3 production capacity from orally administered levothyroxine. More than 20% of these patients, despite normal TSH levels, do not maintain FT3 or FT4 values in the reference range, reflecting the inadequacy of peripheral deiodination to compensate for the absent T3 secretion. The long-term effects of chronic tissue exposure to abnormal T3/T4 ratio are unknown but a sensitive marker of target organ response to thyroid hormones (serum TSH) suggests that this condition causes an abnormal pituitary response. A more physiological treatment than levothyroxine monotherapy may be required in some hypothyroid patients.     

Regional inequalities in cancer care persist in Italy and can influence survival

BackgroundPopulation-based cancer registry studies of care patterns can help elucidate reasons for the marked geographic variation in cancer survival across Italy. The article provides a snapshot of the care delivered to cancer patients in Italy.MethodsRandom samples of adult patients with skin melanoma, breast, colon and non-small cell lung cancers diagnosed in 2003–2005 were selected from 14 Italian cancer registries. Logistic models estimated odds of receiving standard care (conservative surgery plus radiotherapy for early breast cancer; surgery plus chemotherapy for Dukes C colon cancer; surgery for lung cancer; sentinel node biopsy for >1 mm melanoma, vs. other treatment) in each registry compared to the entire sample (reference).ResultsStage at diagnosis for breast, colon and melanoma was earlier in north/central than southern registries. Odds of receiving standard care were lower than reference in Sassari (0.68, 95%CI 0.51–0.90) and Napoli (0.48, 95%CI 0.35–0.67) for breast cancer; did not differ across registries for Dukes C colon cancer; were higher in Romagna (3.77, 95%CI 1.67–8.50) and lower in Biella (0.38, 95%CI 0.18–0.82) for lung cancer; and were higher in Reggio Emilia (2.37, 95%CI 1.12–5.02) and lower in Ragusa (0.27, 95%CI 0.14–0.54) for melanoma.ConclusionsNotwithstanding limitations due to variations in the availability of clinical information and differences in stage distribution between north/central and southern registries, our study shows that important disparities in cancer care persist across Italy. Thus the public health priority of reducing cancer survival disparities will not be achieved in the immediate future.     

Evaluation of allelic expression of imprinted genes in adult human blood

Background

Imprinted genes are expressed from only one allele in a parent-of-origin dependent manner. Loss of imprinted (LOI) expression can result in a variety of human disorders and is frequently reported in cancer. Biallelic expression of imprinted genes in adult blood has been suggested as a useful biomarker and is currently being investigated in colorectal cancer. In general, the expression profiles of imprinted genes are well characterised during human and mouse fetal development, but not in human adults.

Methodology/Principal Findings

We investigated quantitative expression of 36 imprinted genes in adult human peripheral blood leukocytes obtained from healthy individuals. Allelic expression was also investigated in B and T lymphocytes and myeloid cells. We found that 21 genes were essentially undetectable in adult blood. Only six genes were demonstrably monoallelic, and most importantly, we found that nine genes were either biallelic or showed variable expression in different individuals. Separated leukocyte populations showed the same expression patterns as whole blood. Differential methylation at each of the imprinting control loci analysed was maintained, including regions that contained biallelically expressed genes. This suggests in some cases methylation has become uncoupled from its role in regulating gene expression.

Conclusions/Significance

We conclude that only a limited set of imprinted genes, including IGF2 and SNRPN, may be useful for LOI cancer biomarker studies. In addition, blood is not a good tissue to use for the discovery of new imprinted genes. Finally, lymphocyte DNA methylation status in the adult may not always be a reliable indicator of monoallelic gene expression.     

Prebiotic Potential of a Maize-Based Soluble Fibre and Impact of Dose on the Human Gut Microbiota

Dietary management of the human gut microbiota towards a more beneficial composition is one approach that may improve host health. To date, a large number of human intervention studies have demonstrated that dietary consumption of certain food products can result in significant changes in the composition of the gut microbiota i.e. the prebiotic concept. Thus the prebiotic effect is now established as a dietary approach to increase beneficial gut bacteria and it has been associated with modulation of health biomarkers and modulation of the immune system. Promitor™ Soluble Corn Fibre (SCF) is a well-known maize-derived source of dietary fibre with potential selective fermentation properties. Our aim was to determine the optimum prebiotic dose of tolerance, desired changes to microbiota and fermentation of SCF in healthy adult subjects. A double-blind, randomised, parallel study was completed where volunteers (n = 8/treatment group) consumed 8, 14 or 21 g from SCF (6, 12 and 18 g/fibre delivered respectively) over 14-d. Over the range of doses studied, SCF was well tolerated Numbers of bifidobacteria were significantly higher for the 6 g/fibre/day compared to 12g and 18g/fibre delivered/day (mean 9.25 and 9.73 Log10 cells/g fresh faeces in the pre-treatment and treatment periods respectively). Such a numerical change of 0.5 Log10 bifidobacteria/g fresh faeces is consistent with those changes observed for inulin-type fructans, which are recognised prebiotics. A possible prebiotic effect of SCF was therefore demonstrated by its stimulation of bifidobacteria numbers in the overall gut microbiota during a short-term intervention.     

Palladium(II) derivatives of alkylsulfanyl substituted thiophenes as precursors of inorganic polymers: Spectroscopic, electrochemical investigations and X-ray crystal structure of trans-PdCl2[3-(butylsulfanyl)thiophene]2

Palladium(II) complexes with two thiophene derivatives bearing alkylsulfanyl chains, i.e., 3-(n-butylsulfanyl)thiophene and 4,4′-bis(n-butylsulfanyl)-2,2′-bithiophene, are synthesised and spectroscopically and electrochemically characterised. The molecular structure of PdCl₂[3-(n-butylsulfanyl)thiophene]₂ was determined by X-ray analysis. The properties of the complexes have been compared with those of PdCl₂[3,3′-bis(n-butylsulfanyl)-2,2′-bithiophene] and of a conductive polymer partially coordinated with Pd(II), previously synthesised by us. We found that Pd(II) ions can coordinate sulfanyl sulfur atoms both in cis and trans configuration, leading to a reticulate material, where some kind of interchain bridging may be reasonably supposed to enhance the bulk conductivity.     

Influence of inoculum age on hybridoma culture kinetics

To determine the influence of the inoculum age on the kinetics of hybridoma growth and metabolism, spinner flasks have been inoculated with cells previously propagated in T flasks for 43, 52, 62 and 71 hr respectively. Increasing the age of the inoculum is found to result in a longer lag phase, in a lower maximum specific growth rate and in a reduced maximal cell density. During the growth phase specific rates of glucose and glutamine uptake and of ammonia and lactate production are similar. However, with the older inoculum, much higher metabolic activities are observed during the lag phase. The production of antibodies is delayed with increasing inoculum age, but the final antibody concentrations are similar, which indicates a higher specific antibody production rate when inoculating with older cells.