Analysis of the human hephaestin gene and protein: comparative modelling of the N-terminus ecto-domain based upon ceruloplasmin

Hephaestin was implicated in mammalian iron homeostasis following its identification as the defective gene in murine sex-linked anaemia. It is a member of the family of copper oxidases that includes mammalian ceruloplasmin, factors V and VIII, yeast fet3 and fet5 and bacterial ascorbate oxidase. Hephaestin is different from ceruloplasmin, a soluble ferroxidase, in having a membrane-spanning region towards the C-terminus. Here we report the gene structure, spanning approximately 100 kb, of the human homologue of mouse hephaestin. The sequence was assembled from the cDNA clones and the chromosome X genomic sequence data available at the Sanger Centre. It has an open reading frame that encodes a protein of 1158 residues, 85% identical with the murine homologue. A model of the N-terminal ecto-domain has been built based on the known three-dimensional structure of human ceruloplasmin. The overall tertiary structure for the hephaestin and the putative residues involved in binding copper and iron appear to be highly conserved between these proteins, which suggests they share the same fold and a conserved function.     

On the age of Border Cave 5 human mandible

An enamel fragment from the Border Cave 5 specimen was analysed with non-destructive ESR combined with laser ablation ICP-MS for uranium profiling. We obtained an age of 74+/-5 ka which fits exactly into the chronological framework that has been previously established for Border Cave by a variety of dating techniques. The result lays at rest the view that BC5 could be of Iron Age, as was implied by (Journal of Human Evolution, 31 (1996) 499) based on nitrogen contents and infra-red splitting factors.     

Neuromelanin associated redox-active iron is increased in the substantia nigra of patients with Parkinson's disease

Degeneration of dopaminergic neurones during Parkinson's disease is most extensive in the subpopulation of melanized-neurones located in the substantia nigra pars compacta. Neuromelanin is a dark pigment produced in the dopaminergic neurones of the human substantia nigra and has the ability to bind a variety of metal ions, especially iron. Post-mortem analyses of the human brain have established that oxidative stress and iron content are enhanced in association with neuronal death. As redox-active iron (free Fe2+ form) and other transition metals have the ability to generate highly reactive hydroxyl radicals by a catalytic process, we investigated the redox activity of neuromelanin (NM)-aggregates in a group of parkinsonian patients, who presented a statistically significant reduction (- 70%) in the number of melanized-neurones and an increased non-heme (Fe3+) iron content as compared with a group of matched-control subjects. The level of redox activity detected in neuromelanin-aggregates was significantly increased (+ 69%) in parkinsonian patients and was highest in patients with the most severe neuronal loss. This change was not observed in tissue in the immediate vicinity of melanized-neurones. A possible consequence of an overloading of neuromelanin with redox-active elements is an increased contribution to oxidative stress and intraneuronal damage in patients with Parkinson's disease.     

Anti-α-fodrin antibodies do not add much to the diagnosis of Sjögren's syndrome

The presence of anti-α-fodrin autoantibodies has been reported to be a highly specific and sensitive test for the diagnosis of Sjögren's syndrome (SjS). We looked (in Nijmegen) for anti-α-fodrin, anti-Ro60, and anti-La autoantibodies in a cohort of 51 patients with rheumatic diseases (primary SjS [21], secondary SjS [6], rheumatoid arthritis [RA] [12], systemic lupus erythematosus [SLE] [6], and scleroderma [6]) and in 28 healthy subjects, using ELISA, immunoblotting, and immunoprecipitation. The same samples were analyzed with an alternative anti-α-fodrin ELISA in Hanover. The Nijmegen ELISA of the sera from primary SjS showed sensitivities of 43% and 48% for IgA- and IgG-type anti-α-fodrin antibodies, respectively. The Hanover ELISA showed sensitivities of 38% and 10% for IgA- and IgG-type anti-α-fodrin antibodies, respectively. The ELISAs for α-fodrin showed six (Nijmegen) and four (Hanover) anti-α-fodrin-positive RA sera. IgA and IgG anti-fodrin antibodies were also present in four patients with secondary SjS. The sensitivities of Ro60 and La-antibodies in the Nijmegen ELISA were 67% and 62%, respectively. Unlike anti-α-fodrin antibodies, all anti-Ro60 and anti-La positive sera could be confirmed by immunoblotting or RNA immunoprecipitation. Thus, anti-Ro and anti-La autoantibodies were more sensitive than anti-α-fodrin autoantibodies in ELISA and were more frequently confirmed by other techniques. Anti-La antibodies appear to be more disease-specific than anti-α-fodrin antibodies, which are also found in RA sera. Therefore, the measurement of anti-α-fodrin autoantibodies does not add much to the diagnosis of Sjögren's syndrome.     

Bestimmung von Fumonisinen in komplexen Lebensmittelmatrices mittels Accelerated Solvent Extraktion (ASE)

Accelerated solvent extraction (ASE) is an alternative sample extraction procedure for fumonisins in corn and corn products. ASE gave results comparable to that of a draft CEN method, but required less extraction time. Furthermore, ASE gave significantly higher quantitative values than another method reported for extraction of fumonisins (Trucksess et al., 1995).     

Vascular oxidant stress: Molecular mechanisms and pathophysiological implications

The term oxidative stress refers to a situation in which cells are exposed to excessive levels of either molecular oxygen or chemical derivatives of oxygen (ie, reactive oxygen species). Three enzyme systems produce reactive oxygen species in the vascular wall: NADH/NADPH oxidase, xanthine oxidoreductase, and endothelial nitric oxide synthase. Among vascular reactive oxygen species superoxide anion plays a critical role in vascular biology because it is the source for many other reactive oxygen species and various vascular cell functions. It is currently thought that increases in oxidant stress, namely excessive production of superoxide anion, are involved in the pathophysiology of endothelial dysfunction that accompanies a number of cardiovascular risk factors including hypercholesterolemia, hypertension and cigarette smoking. On the other hand, vascular oxidant stress plays a pivotal role in the evolution of clinical conditions such as atherosclerosis, diabetes and heart failure.     

A genome-wide association study identifies 5 loci associated with frozen shoulder and implicates diabetes as a causal risk factor

Frozen shoulder is a painful condition that often requires surgery and affects up to 5% of individuals aged 40–60 years. Little is known about the causes of the condition, but diabetes is a strong risk factor. To begin to understand the biological mechanisms involved, we aimed to identify genetic variants associated with frozen shoulder and to use Mendelian randomization to test the causal role of diabetes. We performed a genome-wide association study (GWAS) of frozen shoulder in the UK Biobank using data from 10,104 cases identified from inpatient, surgical and primary care codes. We used data from FinnGen for replication and meta-analysis. We used one-sample and two-sample Mendelian randomization approaches to test for a causal association of diabetes with frozen shoulder. We identified five genome-wide significant loci. The most significant locus (lead SNP rs28971325; OR = 1.20, [95% CI: 1.16–1.24], p = 5x10^-29) contained WNT7B. This variant was also associated with Dupuytren’s disease (OR = 2.31 [2.24, 2.39], p<1x10^-300) as were a further two of the frozen shoulder associated variants. The Mendelian randomization results provided evidence that type 1 diabetes is a causal risk factor for frozen shoulder (OR = 1.03 [1.02–1.05], p = 3x10^-6). There was no evidence that obesity was causally associated with frozen shoulder, suggesting that diabetes influences risk of the condition through glycemic rather than mechanical effects. We have identified genetic loci associated with frozen shoulder. There is a large overlap with Dupuytren’s disease associated loci. Diabetes is a likely causal risk factor. Our results provide evidence of biological mechanisms involved in this common painful condition.     

GFT projection NMR for efficient 1H/13C sugar spin system identification in nucleic acids

A newly implemented G-matrix Fourier transform (GFT) (4,3)D HC(C)CH experiment is presented in conjunction with (4,3)D HCCH to efficiently identify ¹H/¹³C sugar spin systems in ¹³C labeled nucleic acids. This experiment enables rapid collection of highly resolved relay 4D HC(C)CH spectral information, that is, shift correlations of ¹³C?C¹H groups separated by two carbon bonds. For RNA, (4,3)D HC(C)CH takes advantage of the comparably favorable 1??- and 3??-CH signal dispersion for complete spin system identification including 5??-CH. The (4,3)D HC(C)CH/HCCH based strategy is exemplified for the 30-nucleotide 3??-untranslated region of the pre-mRNA of human U1A protein.     

Individualized,discrete event,simulations provide insight into inter- and intra-subject variability of extended-release,drug products

Objective

Develop and validate particular, concrete, and abstract yet plausible in silico mechanistic explanations for large intra- and interindividual variability observed for eleven bioequivalence study participants. Do so in the face of considerable uncertainty about mechanisms.

Methods

We constructed an object-oriented, discrete event model called subject (we use small caps to distinguish computational objects from their biological counterparts). It maps abstractly to a dissolution test system and study subject to whom product was administered orally. A subject comprises four interconnected grid spaces and event mechanisms that map to different physiological features and processes. Drugs move within and between spaces. We followed an established, Iterative Refinement Protocol. Individualized mechanisms were made sufficiently complicated to achieve prespecified Similarity Criteria, but no more so. Within subjects, the dissolution space is linked to both a product-subject Interaction Space and the GI tract. The GI tract and Interaction Space connect to plasma, from which drug is eliminated.

Results

We discovered parameterizations that enabled the eleven subject simulation results to achieve the most stringent Similarity Criteria. Simulated profiles closely resembled those with normal, odd, and double peaks. We observed important subject-by-formulation interactions within subjects.

Conclusion

We hypothesize that there were interactions within bioequivalence study participants corresponding to the subject-by-formulation interactions within subjects. Further progress requires methods to transition currently abstract subject mechanisms iteratively and parsimoniously to be more physiologically realistic. As that objective is achieved, the approach presented is expected to become beneficial to drug development (e.g., controlled release) and to a reduction in the number of subjects needed per study plus faster regulatory review.