A novel assay for factor XIII based on cross-linking of synthetic peptides: analysis of different substrates.

A novel assay for factor XIII is described that utilizes exclusively small synthetic peptides as substrates for the cross-linking reaction catalyzed by activated factor XIII (FXIIIa). The acyl donor substrate (selection peptide) is immobilized on a microplate via biotin while the acyl acceptor substrate (detection peptide) is labeled with the fluorochrome Oregon green to allow sensitive detection without the need for secondary enzyme systems for signal amplification. Starting with an amino acid sequence from the fibrin gamma-chain (GQQHHLGGAKQAGDV) as a prototype peptide, the influence of amino acid exchanges were investigated with respect to their impact on the FXIIIa-catalyzed reaction. It was found that FXIIIa readily accepts a broad range of substrate peptides, with a proline neighboring the essential lysine having the most detrimental effect. The assay appears to be valuable for the molecular characterization of factor XIII and may be used for a deeper investigation into the substrate requirements of this final enzyme of wound repair, and eventually also for the characterization of other transglutaminases.     

Freie Gelenkk?rper im Kiefergelenk

Zusammenfassung Die Osteochondrosis dissecans (OD) und die synoviale Chondromatose (SC) des Kiefergelenks sind seltene Erkrankungen, die mit freien Gelenkkörpern einhergehen. Für die OD wird ursächlich eine traumatische Ursache angenommen. Bei der metaplastischen proliferativen SC lassen sich Gendefekte feststellen, die Chondrosarkomen ähnlich sind. Eine Ursache dieser Veränderungen ist bis dato nicht bekannt. Beide Erkrankungen sind in ihren klinischen Symptomen fast identisch. Präaurikuläre Schwellung, Gesichts- oder Kiefergelenkschmerzen und gelegentliche Kiefergelenkblockaden werden häufig von Patienten berichtet. Die bildgebende Diagnostik mittels Magnetresonanztomographie (MRT) bereitet Schwierigkeiten in der Differenzierung von OD und SC untereinander sowie gegenüber proliferativen intra- und periartikulären Veränderungen. Die Kiefergelenkarthroskopie bietet hier die einzige Möglichkeit einer in vivo-Beurteilung der Gelenkverhältnisse mit der Option einer histopathologischen Untersuchung. Therapeutisch kann so über eine Dissekatentfernung und Synovektomie bei SC sowie die ausschließliche Dissekatentfernung bei OD geurteilt werden.     

Uncoupling of neuroinflammation from axonal degeneration in mice lacking the myelin protein tetraspanin‐2

Deficiency of the major constituent of central nervous system (CNS) myelin, proteolipid protein (PLP), causes axonal pathology in spastic paraplegia type‐2 patients and in Plp1^null‐mice but is compatible with almost normal myelination. These observations led us to speculate that PLP's role in myelination may be partly compensated for by other tetraspan proteins. Here, we demonstrate that the abundance of the structurally related tetraspanin‐2 (TSPAN2) is highly increased in CNS myelin of Plp1^null‐mice. Unexpectedly, Tspan2^null‐mutant mice generated by homologous recombination in embryonic stem cells displayed low‐grade activation of astrocytes and microglia in white matter tracts while they were fully myelinated and showed no signs of axonal degeneration. To determine overlapping functions of TSPAN2 and PLP, Tspan2^null*Plp1^null double‐mutant mice were generated. Strikingly, the activation of astrocytes and microglia was strongly enhanced in Tspan2^null*Plp1^null double‐mutants compared with either single‐mutant, but the levels of dysmyelination and axonal degeneration were not increased. In this model, glial activation is thus unlikely to be caused by axonal pathology, and vice versa does not potentiate axonal degeneration. Our results support the concept that multiple myelin proteins have distinct roles in the long‐term preservation of a healthy CNS, rather than in myelination per se. GLIA 2013;61:1832–1847     

Unmodified kraft lignin isolated at room temperature from aqueous solution for preparation of highly flexible transparent polyurethane coatings

Polyurethane (PU) coatings were successfully produced using unmodified kraft lignin (KL) as an environmentally benign component in contents of up to 80 wt%. Lignin samples were precipitated from industrial black liquor in aqueous solution working at room temperature and different pH levels (pH 2 to pH 5). Lignins were characterized by UV-Vis, FTIR, pyrolysis-GC/MS, SEC and ³¹P-NMR. Results show a correlation between pH level, OH number and molecular weight M_w of isolated lignins. Lignin-based polyurethane coatings were prepared in an efficient one step synthesis dissolving lignin in THF and PEG₄₂₅ in an ultrasonic bath followed by addition of 4,4-diphenylmethanediisocyanate (MDI) and triethylamine (TEA). Crosslinking was achieved under very mild conditions (1 hour at room temperature followed by 3 hours at 35 °C). The resulting coatings were characterized regarding their physical properties including ATR-IR, TGA, optical contact angle, light microscopy, REM-EDX and AFM data. Transparent homogeneous films of high flexibility resulted from lignins isolated at pH 4, possessing a temperature resistance up to 160 °C. Swelling tests revealed a resistance against water. Swelling in DMSO depends on index, pH of precipitation and catalyst utilization for PU preparation. According to AFM studies, surface roughness is between 10 and 28 nm.

Polyurethane (PU) coatings were successfully produced using unmodified kraft lignin (KL) as an environmentally benign component in contents of up to 80 wt%.     

Freie Gelenkkörper im Kiefergelenk

Osteochondrosis dissecans (OD) and synovial chondromatosis (SC) are two rare causes for loose bodies in the temporomandibular joint. It is assumed that OD is a reaction of some type of joint trauma. Gene mutations similar to what is known in chondrosarcomas can be found in metaplastic SC. The reasons for mutations are still unknown. Both diseases have very similar symptoms. Patients suffer from preauricular swelling, facial or temporomandibular joint pain, and occasional joint locking. In addition, radiological imaging (MRI) is difficult in evaluating the differential diagnosis of presented intra- or periarticular pathology. To overcome diagnostic problems, we strongly support temporomandibular joint arthroscopy as a diagnostic and therapeutic tool. The decision whether or not total synovectomy is needed or if simple removal of fragments is adequate can be evaluated in vivo including the option of histopathological examination.      

Extension of myocardial necrosis differently affects MIBG retention in heart failure caused by ischaemic heart disease or by dilated cardiomyopathy

Purpose This study aims to investigate the relationship between cardiac sympathetic nervous function (CSNF) and myocardial perfusion/function in patients with heart failure (HF) due to dilated cardiomyopathy (DCM) or ischaemic heart disease (CAD).Methods Twenty patients (10 DCM, 10 CAD, 17 males, age 69±5 years) with NYHA class IIIb HF were studied. CSNF was evaluated by early/delayed ¹²³I-metaiodobenzylguanidine (MIBG) uptake and regional washout (WO). Myocardial perfusion and function were evaluated by ^99mTc-tetrofosmin gated single-photon emission tomography (G-SPECT) using a 20-segment model for 400 segments. In each segment, regional MIBG WO was computed as (count density in early images–count density in delayed images/count density in early images)×100.Results DCM and CAD showed similar summed rest perfusion score (6.7±5 vs 9.5±5, p=NS) and mean ejection fraction values (29±7% vs 30±9%, p=NS). By contrast, the summed thickening score was higher in DCM than in CAD patients (26±7 vs 17±6, p<0.05). QGS analysis identified akinesis/dyskinesis in 129/137 (94%) severely hypoperfused segments which were considered as damaged. According to the underlying aetiology of HF, marked differences in regional MIBG WO were observed. In fact, within the CAD group, regional MIBG WO was lower in reference than in damaged segments (38±21% vs 46±19%, p<0.05). By contrast, in DCM patients, regional MIBG WO was faster in reference than in damaged segments (49±18% vs 41±30%, p<0.05). When the two groups were directly compared, regional MIBG WO from damaged areas was similar irrespective of the underlying disease, while it was faster in DCM than in CAD patients from reference segments.Conclusion These data confirm the hypothesis that the presence of myocardial necrosis in HF due to CAD and the consequent loss of neuronal endings cause alterations in regional MIBG WO different from those observed in DCM.     

Novel TRPM6 mutations in 21 families with primary hypomagnesemia and secondary hypocalcemia

Primary hypomagnesemia with secondary hypocalcemia is a rare autosomal recessive disorder characterized by profound hypomagnesemia associated with hypocalcemia. Pathophysiology is related to impaired intestinal absorption of magnesium accompanied by renal magnesium wasting as a result of a reabsorption defect in the distal convoluted tubule. Recently, mutations in the TRPM6 gene coding for TRPM6, a member of the transient receptor potential (TRP) family of cation channels, were identified as the underlying genetic defect. Here, the results of a TRPM6 mutational analysis of 21 families with 28 affected individuals are presented. In this large patient cohort, a retrospective clinical evaluation based on a standardized questionnaire was also performed. Genotype analysis revealed TRPM6 mutations in 37 of 42 expected mutant alleles. Sixteen new TRPM6 mutations were identified, including stop mutations, frame-shift mutations, splice-site mutations, and deletions of exons. Electrophysiologic analysis of mutated ion channels after heterologous expression in Xenopus oocytes proved complete loss of function of TRPM6. Clinical evaluation revealed a homogeneous clinical picture at manifestation with onset in early infancy with generalized cerebral convulsions. Initial laboratory evaluation yielded extremely low serum magnesium levels, low serum calcium levels, and inadequately low parathyroid hormone levels. Treatment usually consisted of acute intravenous magnesium supplementation leading to relief of clinical symptoms and normocalcemia, followed by lifelong oral magnesium supplementation. Serum magnesium levels remained in the subnormal range despite adequate therapy. This is best explained by a disturbed magnesium conservation in the distal convoluted tubule, which emerged in all patients upon magnesium supplementation. Delay of diagnosis resulted in permanent neurologic damage in three patients.