Combined enrichment of neuromelanin granules and synaptosomes from human substantia nigra pars compacta tissue for proteomic analysis.

This article gives a detailed description of a protocol using density gradient centrifugation for the enrichment of neuromelanin granules and synaptosomes from low amounts (≥0.15g) of human substantia nigra pars compacta tissue. This has a great advantage compared to already existing methods as it allows for the first time (i) a combined enrichment of neuromelanin granules and synaptosomes and (ii) just minimal amounts of tissue necessary to enable donor specific analysis. Individual specimens were classified as control or diseased according to clinical evaluation and neuropathological examination. For the enrichment of synaptosomes and neuromelanin granules from the same tissue sample density gradient centrifugations using Percoll® and Iodixanol were performed. The purity of resulting fractions was checked by transmission electron microscopy. We were able to establish a reproducible and easy to handle protocol combining two different density gradient centrifugations: using an Iodixanol gradient neuromelanin granules were enriched and in parallel, from the same sample, a fraction of synaptosomes with high purity using a Percoll® gradient was obtained. Our subfractionation strategy will enable a subsequent in depth proteomic characterization of neurodegenerative processes in the substantia nigra pars compacta in patients with Parkinson's disease and dementia with Lewy bodies compared to appropriate controls. BIOLOGICAL SIGNIFICANCE: Key features of Parkinson's disease are the degeneration of dopaminergic neurons in the substantia nigra pars compacta, an associated loss of the brain pigment neuromelanin and a resulting impairment of the neuronal network. The accumulation of iron binding neuromelanin granules is age- and disease-dependent and disease specific alterations could affect the neuronal iron homeostasis leading to oxidative stress induced cell death. The focus of the described method is the analysis of neuromelanin granules as well as axonal cell-endings of nerve cells (synaptosomes) of individual donors (control and diseased). It is the basis for the identification of disease-relevant changes in the iron homeostasis and the generation of new insight into altered protein compositions or regulations which might lead to disturbed communications between nerve cells resulting in pathogenic processes.

The AICD interacting protein DAB1 is up-regulated in Alzheimer frontal cortex brain samples and causes deregulation of proteins involved in gene expression changes.

AICD is the intracellular subdomain of the amyloid precursor protein thought to play a pivotal role as a potential transcription factor that might be of relevance for the pathophysiology of Alzheimer's disease. For its signal transduction potential AICD requires interacting proteins like FE65 and TIP60. However, many other proteins were described being able to bind to AICD. Here, we studied mRNA levels of AICD interacting proteins and found one of them (DAB1) strongly up-regulated in human post-mortem frontal cortex brain samples of AD patients. Subsequent cell culture experiments revealed that elevated DAB1 level results in the deregulation of the cellular proteome. We found the proliferation associated protein 2G4 as well as the guanine monophosphate synthetase (GMPS) significantly up-regulated in DAB1 over-expressing cells. Both proteins can be involved in cellular transcription processes supporting the hypothesis that DAB1 acts via modification of the AICD-dependent transcriptionally active complex. Of note, expression of the three components of the putative transcription complex (AICD, FE65, and TIP60 (AFT)) also revealed deregulation of the GMPS protein in an opposite fashion. Our results point to a putative relevance of AICD-dependent mechanisms in AD, caused by protein abundance changes of AICD interacting proteins, as shown for DAB1 in this work.

Different mechanical loading protocols influence serum cartilage oligomeric matrix protein levels in young healthy humans.

The purpose of the study was to investigate whether a relationship between the loading mode of physical activity and serum cartilage oligomeric matrix protein (COMP) concentration exists and whether the lymphatic system contributes to COMP release into the serum. Serum COMP levels were determined in healthy male subjects before, after and at 18 further time points within 7 h at four separate experimental days with four different loading interventions. The loading intervention included high impact running exercise, slow but deep knee bends, and lymphatic drainage of 30 min duration, respectively, and a resting protocol. The serum COMP levels were measured using a commercially available quantitative enzyme-linked immunosorbent assay. An increase (p < 0.001) in serum COMP concentration was detected immediately after 30 min running exercise. Slow but deep knee bends did not cause any significant changes in serum COMP levels. Lymphatic drainage also had no effect on the serum COMP concentration. After 30 min of complete rest the serum COMP level was significantly (p = 0.008) reduced. The elevation of COMP serum concentration seems to depend on the loading mode of the physical activity and to reflect the extrusion of COMP fragments from the impact loaded articular cartilage or synovial fluid.

Colour compatibility between teeth and dental shade guides in Quinquagenarians and Septuagenarians.

The aim of this investigation was to determine colour compatibility between dental shade guides, namely, VITA Classical (VC) and VITA 3D-Master (3D), and human teeth in quinquagenarians and septuagenarians. Tooth colour, described in terms of L*a*b* values of the middle third of facial tooth surface of 1391 teeth, was measured using VITA Easyshade in 195 subjects (48% female). These were compared with the colours (L*a*b* values) of the shade tabs of VC and 3D. The mean coverage error and the percentage of tooth colours being within a given colour difference (DeltaE(ab)) from the tabs of VC and 3D were calculated. For comparison, hypothetical, optimized, population-specific shade guides were additionally calculated based on discrete optimization techniques for optimizing coverage. Mean coverage error was DeltaE(ab) = 3.51 for VC and DeltaE(ab) = 2.96 for 3D. Coverage of tooth colours by the tabs of VC and 3D within DeltaE(ab) = 2 was 23% and 24%, respectively, (DeltaE(ab)

Pharmacokinetics of intraarterial mitomycin C in the chemoembolisation treatment of liver metastases.

1. The pharmacokinetics of mitomycin C (MMC) were investigated in 12 colorectal cancer patients with liver metastases undergoing chemoembolisation. Hepatic artery branches were embolized using polyvinylalcohol microspheres (150-250 microns) before applying 20 mg MMC in 4-8 min. 2. Serum MMC concentrations were determined from peripheral venous blood samples by reverse-phase HPLC using ultraviolet detection. Pharmacokinetic parameters were computed assuming an open two-compartment model. 3. Pharmacokinetic parameters were similar to values given in the literature for intravenous (IV) or intraarterial (IA) bolus MMC injections (Tmax = 7.0 min following the beginning of MMC infusion, Vss = 0.57 1/kg, C1 = 8.9 ml/min.kg, T1/2 alpha = 8.3 min, T1/2 beta = 58.6 min). 4. The area under the serum concentration-time-curve (AUC), standardized by the MMC amount injected, was similar to values reported in the literature for IV or IA bolus injections. There is no evidence for reduced systemic MMC exposure following the embolization procedure used.

Mitomycin-C concentration in human ocular aqueous humor after topical administration during trabeculectomy.

UNLABELLED: The concentration of the antitumor antibiotic mitomycin (CAS 50-07-7, mitomycin C, MMC), used in ophthalmic surgery for its antiproliferative effects, was measured in the aqueous humor of 7 glaucoma patients undergoing trabeculectomy. Sponges soaked with MMC-solution (100 microliters of MMC-solution 0.2 mg/ml: 20 micrograms) were applied intraoperatively on the scleral flap for 5 min. 100 to 200 microliters of aqueous humor were drawn with a needle 10 min following the end of topical MMC-treatment. Samples were assayed for MMC using a reverse-phase HPLC-system with ultraviolet detection (C18-column, elution: phosphate buffer (0.01 mol/l, pH: 6.5): methanol, v:v = 70:30, 365 nm). Swabs were extracted in phosphate-buffer (0.1 mol/l, pH: 7.0) before HPLC-analysis. External calibration was used for MMC quantitation. Quantitation limit was 10 ng/ml. In all aqueous humor samples MMC-concentration was below 10 ng/ml. MMC in the swabs amounted to 37% of the MMC amount applied. CONCLUSION: After intraoperative topical application, the MMC concentration in the aqueous humor of patients is very low. The substantial loss of MMC from the swabs used for the topical MMC-treatment suggests 1. rapid systemic absorption of MMC and/or 2. a loss through irrigation of the operative field following topical MMC-application.

Familial Mediterranean fever (FMF) in Moroccan Jews: demonstration of a founder effect by extended haplotype analysis.

Familial Mediterranean fever (FMF) is an autosomal recessive disease causing attacks of fever and serositis. The FMF gene (designated "MEF") is on 16p, with the gene order 16cen-D16S80-MEF-D16S94-D16S283-D16S291-++ +16pter. Here we report the association of FMF susceptibility with alleles as D16S94, D16S283, and D16S291 among 31 non-Ashkenazi Jewish families (14 Moroccan, 17 non-Moroccan). We observed highly significant associations at D16S283 and D16S291 among the Moroccan families. For the non-Moroccans, only the allelic association at D16S94 approached statistical significance. Haplotype analysis showed that 18/25 Moroccan FMF chromosomes, versus 0/21 noncarrier chromosomes, bore a specific haplotype for D16S94-D16S283-D16S291. Among non-Moroccans this haplotype was present in 6/26 FMF chromosomes versus 1/28 controls. Both groups of families are largely descended from Jews who fled the Spanish Inquisition. The strong haplotype association seen among the Moroccans is most likely a founder effect, given the recent origin and genetic isolation of the Moroccan Jewish community. The lower haplotype frequency among non-Moroccan carriers may reflect differences both in history and in population genetics.

Refined mapping of the gene causing familial Mediterranean fever, by linkage and homozygosity studies.

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by attacks of fever and serosal inflammation; the biochemical basis is unknown. We recently reported linkage of the gene causing FMF (designated "MEF") to two markers on chromosome 16p. To map MEF more precisely, we have now tested nine 16p markers. Two-point and multipoint linkage analysis, as well as a study of recombinant haplotypes, placed MEF between D16S94 and D16S80, a genetic interval of about 9 cM. We also examined rates of homozygosity for markers in this region, among offspring of consanguineous marriages. For eight of nine markers, the rate of homozygosity among 26 affected inbred individuals was higher than that among their 20 unaffected sibs. Localizing MEF more precisely on the basis of homozygosity rates alone would be difficult, for two reasons: First, the high FMF carrier frequency increases the chance that inbred offspring could have the disease without being homozygous by descent at MEF. Second, several of the markers in this region are relatively nonpolymorphic, with a high rate of homozygosity, regardless of their chromosomal location.

X-ray detection by the olfactory system: ozone as a masking odorant.

The technique of masking was used to test the hypothesis that x-ray detection is mediated by an odorant produced in irradiated air. Rats conditioned to cease licking during exposure to x-ray (conditioned suppression) did not display this conditioned response in the presence of ozone and strong volatile oxidants.