Prooxidant and antioxidant properties of human serum ultrafiltrates toward LDL: important role of uric acid

Oxidized LDL is present within atherosclerotic lesions, demonstrating a failure of antioxidant protection. A normal human serum ultrafiltrate of M-r below 500 was prepared as a model for the low M-r components of interstitial fluid, and its effects on LDL oxidation were investigated. The ultrafiltrate (0.3%, v/v) was a potent antioxidant for native LDL, but was a strong prooxidant for mildly oxidized LDL when copper, but not a water-soluble azo initiator, was used to oxidize LDL. Adding a lipid hydroperoxide to native LDL induced the antioxidant to prooxidant switch of the ultrafiltrate. Uric acid was identified, using uricase and add-back experiments, as both the major antioxidant and prooxidant within the ultrafiltrate for LDL. The ultrafiltrate or uric acid rapidly reduced Cu2+ to Cu+. The reduction of Cu2+ to Cu+ may help to explain both the antioxidant and prooxidant effects observed. The decreased concentration of Cu2+ would inhibit tocopherol-mediated peroxidation in native LDL, and the generation of Cu+ would promote the rapid breakdown of lipid hydroperoxides in mildly oxidized LDL into lipid radicals. The net effect of the low M-r serum components would therefore depend on the preexisting levels of lipid hydroperoxides in LDL.jlr These findings may help to explain why LDL oxidation occurs in atherosclerotic lesions in the presence of compounds that are usually considered to be antioxidants.

Genetic relatedness and phenotypic characteristics of Treponema associated with human periodontal tissues and ruminant foot disease

Treponema have been implicated recently in the pathogenesis of digital dermatitis (DID) and contagious ovine digital dermatitis (CODD) that are infectious diseases of bovine and ovine foot tissues, respectively. Previous analyses of treponemal 16S rDNA sequences, PCR-amplified directly from DID or CODD lesions, have suggested relatedness of animal Treponema to some human oral Treponema species isolated from periodontal tissues. In this study a range of adhesion and virulence-related properties of three animal Treponema isolates have been compared with representative human oral strains of Treponema denticola and Treponema vincentii. In adhesion assays using biotinylated treponemal cells, T denticola cells bound in consistently higher numbers to fibronectin, laminin, collagen type 1, gelatin, keratin and lactoferrin than did T. vincentii or animal Treponema isolates. However, animal DID strains adhered to fibrinogen at equivalent or greater levels than T denticola. All Treponema strains bound to the amino-terminal heparin l/fibrin I domain of fibronectin. 16S rDNA sequence analyses placed ovine strain UB1090 and bovine strain UB1467 within a cluster that was phylogenetically related to T vincentii, while ovine strain UB1466 appeared more closely related to T denticola. These observations correlated with phenotypic properties. Thus, T denticola ATCC 35405, GM-1, and Treponema UB1466 had similar outer-membrane protein profiles, produced chymotrypsin-like protease (CTLP), trypsin-like protease and high levels of proline iminopeptidase, and co-aggregated with human oral bacteria Porphyromonas gingivalis and Streptococcus crista. Conversely, T vincentii ATCC 35580, D2A-2, and animal strains UB1090 and UB1467 did not express CTLP or trypsin-like protease and did not co-aggregate with P. gingivalis or S. crista. Taken collectively, these results suggest that human oral-related Treponema have broad host specificity and that similar control or preventive strategies might be developed for human and animal Treponema-associated infections.

Localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) in human gastrointestinal tract

Calcitonin gene-related peptide (CGRP) exerts its diverse effects on vasodilation, nociception, secretion, and motor function through a heterodimeric receptor comprising of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). Despite the importance of CLR.RAMP1 in human disease, little is known about its distribution in the human gastrointestinal (GI) tract, where it participates in inflammation and pain. In this study, we determined that CLR and RAMP1 mRNAs are expressed in normal human stomach, ileum and colon by RT-PCR. We next characterized antibodies that we generated to rat CLR and RAMP1 in transfected HEK cells. Having characterized these antibodies in vitro, we then localized CLR-, RAMP1-, CGRP- and intermedin-immunoreactivity (IMD-IR) in various human GI segments. In the stomach, nerve bundles in the myenteric plexus and nerve fibers throughout the circular and longitudinal muscle had prominent CLR-IR. In the proximal colon and ileum, CLR was found in nerve varicosities of the myenteric plexus and surrounding submucosal neurons. Interestingly, CGRP expressing fibers did not co-localize, but were in close proximity to CLR. However, CLR and RAMP1, the two subunits of a functional CGRP receptor were clearly localized in myenteric plexus, where they may form functional cell-surface receptors. IMD, another member of calcitonin peptide family was also found in close proximity to CLR, and like CGRP, did not co-localize with either CLR or RAMP1 receptors. Thus, CGRP and IMD appear to be released locally, where they can mediate their effect on their receptors regulating diverse functions such as inflammation, pain and motility.

Application of auto regressive models of wavelet sub-bands for classifying Terahertz pulse measurements

This paper presents an approach for automatic classification of pulsed Terahertz (THz), or T-ray, signals highlighting their potential in biomedical, pharmaceutical and security applications. T-ray classification systems supply a wealth of information about test samples and make possible the discrimination of heterogeneous layers within an object. In this paper, a novel technique involving the use of Auto Regressive (AR) and Auto Regressive Moving Average (ARMA) models on the wavelet transforms of measured T-ray pulse data is presented. Two example applications are examined - the classi. cation of normal human bone (NHB) osteoblasts against human osteosarcoma (HOS) cells and the identification of six different powder samples. A variety of model types and orders are used to generate descriptive features for subsequent classification. Wavelet-based de-noising with soft threshold shrinkage is applied to the measured T-ray signals prior to modeling. For classi. cation, a simple Mahalanobis distance classi. er is used. After feature extraction, classi. cation accuracy for cancerous and normal cell types is 93%, whereas for powders, it is 98%.

Tissue engineering a fetal membrane

The aim of this study was to construct an artificial fetal membrane (FM) by combination of human amniotic epithelial stem cells (hAESCs) and a mechanically enhanced collagen scaffold containing encapsulated human amniotic stromal fibroblasts (hASFs). Such a tissue-engineered FM may have the potential to plug structural defects in the amniotic sac after antenatal interventions, or to prevent preterm premature rupture of the FM. The hAESCs and hASFs were isolated from human fetal amniotic membrane (AM). Magnetic cell sorting was used to enrich the hAESCs by positive ATP-binding cassette G2 selection. We investigated the use of a laminin/fibronectin (1:1)-coated compressed collagen gel as a novel scaffold to support the growth of hAESCs. A type I collagen gel was dehydrated to form a material mimicking the mechanical properties and ultra-structure of human AM. hAESCs successfully adhered to and formed a monolayer upon the biomimetic collagen scaffold. The resulting artificial membrane shared a high degree of similarity in cell morphology, protein expression profiles, and structure to normal fetal AM. This study provides the first line of evidence that a compacted collagen gel containing hASFs could adequately support hAESCs adhesion and differentiation to a degree that is comparable to the normal human fetal AM in terms of structure and maintenance of cell phenotype.

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.

Within-host evolution of Staphylococcus aureus during asymptomatic carriage

Background Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus. Principal Findings We characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA). Significance This investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease.

Changes in fat, fat-free mass and body water in human normal pregnancy

Measurements of body weight, total body water and total body potassium (40K) were made serially on three occasions during pregnancy and once post partum in 27 normal pregnant women. Skinfold thickness and fat cell diameter were also measured. A model of body composition was formulated to permit the estimation of changes in fat, lean tissue and water content of the maternal body. Total maternal body fat increased during pregnancy, reaching a peak towards the end of the second trimester before diminishing. Serial measurements of fat cell diameter showed poor correlation, whilst total body fat calculated from skinfold thickness correlated well with our estimated values for total body fat in pregnancy.

Dystrophin immunoreactivity in normal and Duchenne human fetal neurons in culture.

Dystrophin, the protein product defective in Duchenne muscular dystrophy (DMD), is present in all types of muscle and in the brain. The function of the protein is unknown and its role in the brain is unclear, although 30% of DMD patients show nonprogressive mental retardation. We have therefore studied the localisation of dystrophin in cultures of normal and DMD human fetal neurons using antibodies raised to different regions of the protein. Dystrophin immunoreactivity was demonstrated in the soma and axon hillock of normal neurons and appeared to be associated with the inner part of the cell membrane, although some intracellular staining was also observed. Positive dystrophin staining was present only in cells with fully developed neuronal features, although not all the neurons were positive. Glial cells were always negative for the antigen. Immunostaining with antibodies to the brain spectrins indicate that the dystrophin antibodies did not crossreact with these proteins. The possibility of cross-reactivity with other proteins is discussed. Studies of cells cultured from a DMD fetus also showed specific dystrophin immunostaining in neurons, although the muscle was generally negative for dystrophin. However, the localisation of dystrophin immunostaining and that of the brain spectrins and neurofilaments appeared abnormal, as did the overall morphology of the cells. This suggests that dystrophin may play a role during brain development and dystrophin deficiency results in abnormal neuronal features. This would be consistent with the nonprogressive nature of the mental retardation observed in DMD patients.

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.

Concentrations of parabens in human breast tumours

Parabens are used as preservatives in many thousands of cosmetic, food and pharmaceutical products to which the human population is exposed. Although recent reports of the oestrogenic properties of parabens have challenged current concepts of their toxicity in these consumer products, the question remains as to whether any of the parabens can accumulate intact in the body from the long-term, low-dose levels to which humans are exposed. Initial studies reported here show that parabens can be extracted from human breast tissue and detected by thin-layer chromatography. More detailed studies enabled identification and measurement of mean concentrations of individual parabens in samples of 20 human breast tumours by high-pressure liquid chromatography followed by tandem mass spectrometry. The mean concentration of parabens in these 20 human breast tumours was found to be 20.6 +/- 4.2 ng g(-1) tissue. Comparison of individual parabens showed that methylparaben was present at the highest level (with a mean value of 12.8 +/- 2.2 ng g(-1) tissue) and represents 62% of the total paraben recovered in the extractions. These studies demonstrate that parabens can be found intact in the human breast and this should open the way technically for more detailed information to be obtained on body burdens of parabens and in particular whether body burdens are different in cancer from those in normal tissues. Copyright (C) 2004 John Wiley Sons, Ltd.