Localization and characterization of N-ethylmaleimide sensitive inhibitor(s) of thiol cathepsin activity from cultured nil and polyoma virus-transformed nil hamster cells

Exposure of cultured Nil (a stable line of fibroblast cells from Syrian hamsters) or polyoma virus-transformed (PyNil) hamster fibroblasts to 0.5 mM N-ethylmaleimide for 5 minutes resulted in striking increases in thiol cathepsin activity in unfractionated cell-free lysates. The paradoxical increase in activity of the normally N-ethylmaleimide-sensitive cathepsins apparently occurred as the result of the protective compartmentalization of the cathepsins in the lysosomes (20,000 X g sedimented fraction) and the unprotected localization of an inhibitor(s) in the soluble cytoplasm (175,000 X g supernatant fraction). Under continuous exposure of the cells to N-ethylmaleimide, a rapid increase in cathepsin activity (seen in the first 5 minutes) was followed by a steady decrease in activity (half inactivation time, 90 minutes). The relative difference in rates of N-ethylmaleimide inactivation of thiol cathepsins and thiol cathepsin inhibitors provides a means for estimating lysosomal cathepsin activity in whole cell extracts without the need for more time-consuming fractionation procedure. In reciprocal inhibition tests, it was found that, regardless of the source of cathepsins, the Nil and PyNil cathepsin inhibitor(s) inactivated the cathepsins to approximately the same extent. The inhibitors were heat stable (90-100 degrees C for 15 minutes) at pH 4, but were totally inactivated when boiled at pH 8.5. On a calibrated Sephadex G-100 column, the relative molecular weight (Mr) of the inhibitor(s) was 13,000 daltons. On the same column, the Mr of the cathepsins was 24,000 daltons. Compared with the cathepsin activity from Nil cells, there was about five times less cathepsin activity recoverable from the PyNil cells.     

A new chemical synthesis of Ascopyrone P from 1,5-anhydro-D-fructose

The naturally occurring antioxidant Ascopyrone P (1,5-anhydro-4-deoxy-D-glycero-hex-1-en-3-ulose, 1) was prepared from the rare sugar 1,5-anhydro-D-fructose (AF, 3) in three steps in an overall yield of 36%. Thus, acetylation of 3 afforded the enolone 3,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-3-en-2-ulopyranose (4), which could be isomerised to 2,6-di-O-acetyl-1,5-anhydro-4-deoxy-D-glycero-hex-1-ene-3-ulose (6). Deacetylation of 6 under mild conditions gave crystalline Ascopyrone P (1).     

Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin

We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC(50)) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78+/-21 microM. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 microM) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin.     

Prion-Dependent Lethality of sup45 Mutants in Saccharomyces cerevisiae

In yeast Saccharomyces cerevisiae translation termination factors eRF1 (Sup45) and eRF3 (Sup35) are encoded by the essential genes SUP45 and SUP35 respectively. Heritable aggregation of Sup35 results in formation of the yeast prion [PSI⁺]. It is known that combination of [PSI⁺] with some mutant alleles of the SUP35 or SUP45 genes in one and the same haploid yeast cell causes synthetic lethality. In this study, we perform detailed analysis of synthetic lethality between various sup45 nonsense and missense mutations on one hand, and different variants of [PSI⁺] on the other hand. Synthetic lethality with sup45 mutations was detected for [PSI⁺] variants of different stringencies. Moreover, we demonstrate for the first time that in some combinations, synthetic lethality is dominant and occurs at the postzygotic stage after only a few cell divisions. The tRNA suppressor SUQ5 counteracts the prion-dependent lethality of the nonsense alleles but not of the missense alleles of SUP45, indicating that the lethal effect is due to the depletion of Sup45. Synthetic lethality is also suppressed in the presence of the C-proximal fragment of Sup35 (Sup35C) that lacks the prion domain and cannot be included into the prion aggregates. Remarkably, the production of Sup35C in a sup45 mutant strain is also accompanied by an increase in the Sup45 levels, suggesting that translationally active Sup35 up-regulates Sup45 or protects it from degradation.Key Words: Sup45, Sup35, eRF1, eRF3, amyloid, [PSI⁺], translation termination, Saccharomyces cerevisiae     

Muscle Histidine-Containing Dipeptides Are Elevated by Glucose Intolerance in Both Rodents and Men

Objective

Muscle carnosine and its methylated form anserine are histidine-containing dipeptides. Both dipeptides have the ability to quench reactive carbonyl species and previous studies have shown that endogenous tissue levels are decreased in chronic diseases, such as diabetes.

Design and Methods

Rodent study: Skeletal muscles of rats and mice were collected from 4 different diet-intervention studies, aiming to induce various degrees of glucose intolerance: 45% high-fat feeding (male rats), 60% high-fat feeding (male rats), cafeteria feeding (male rats), 70% high-fat feeding (female mice). Body weight, glucose-tolerance and muscle histidine-containing dipeptides were assessed. Human study: Muscle biopsies were taken from m. vastus lateralis in 35 males (9 lean, 8 obese, 9 prediabetic and 9 newly diagnosed type 2 diabetic patients) and muscle carnosine and gene expression of muscle fiber type markers were measured.

Results

Diet interventions in rodents (cafeteria and 70% high-fat feeding) induced increases in body weight, glucose intolerance and levels of histidine-containing dipeptides in muscle. In humans, obese, prediabetic and diabetic men had increased muscle carnosine content compared to the lean (+21% (p>0.1), +30% (p<0.05) and +39% (p<0.05), respectively). The gene expression of fast-oxidative type 2A myosin heavy chain was increased in the prediabetic (1.8-fold, p<0.05) and tended to increase in the diabetic men (1.6-fold, p = 0.07), compared to healthy lean subjects.

Conclusion

Muscle histidine-containing dipeptides increases with progressive glucose intolerance, in male individuals (cross-sectional). In addition, high-fat diet-induced glucose intolerance was associated with increased muscle histidine-containing dipeptides in female mice (interventional). Increased muscle carnosine content might reflect fiber type composition and/or act as a compensatory mechanism aimed at preventing cell damage in states of impaired glucose tolerance.     

Blütenbildung von Pinguicula lusitanica in vitro durch Fütterung mit Pollen

Zusammenfassung Pinguicula lusitanica wurde in vitro auf stickstoff- und phorphorfreiem Mineralsalzagar kultiviert; jede Pflanze stand für sich in einem Erlenmeyerkolben.Als die Pflanzen 8 Wochen alt waren, wurden 20 Exemplare innerhalb von 5 Wochen viermal mit Pinuspollen gefüttet. 20 gleichgroße und gleichalte dienten als Kontrollen.Durch die Fütterung steigerte sich die Blattzahl und der Durchmesser der Blattrosette, die Blätter wurden intensiver grün und alterten langsamer.Vor allem wurde durch die Pollenfütterung die Blütenbildung ausgelöst. Schon nach der 2. Fütterung traten die ersten Knospen auf, eine Woche darauf blühten bereits 70% und noch vor der letzten Fütterung alle gefütterten Pflanzen, von den ungefütterten dagegen keine einzige. In dem anschließenden halben Jahr entwickelten die 20 gefütterten Pflanzen im ganzen 127 Blüten; die größte Blütenzahl einer Einzelpflanze was 14. Die nichtgefütterten Pflanzen waren auch jetzt noch rein vegetativ.

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Flowering of in vitro cultures of Pinguicula lusitanica after feeding with pinus pollen

Summary Plants of Pinguicula lusitanica were grown in individual Erlenmeyer flasks on an inorganic agar medium containing no nitrogen or phosphorus. After 8 weeks of culture, twenty of the plants were fed Pinus pollen 4 times over a period of 5 weeks.As a result of the feeding, the number of leaves as well as the diameters of the rosettes were increased. The leaves became turned a deeper green and aged more slowly.The most spectacular effect of the pollen feeding was an initiation of flowering. The first buds were already visible after the second feeding. All of the treated plants flowered before the last feeding, whereas none of the untreated plants flowered. During the following 6 months, the treated plants developed 127 flowers, the largest number on a single specimen being 14. Even after this period of time the untreated plants remained vegetative.

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft     

Assessment of Prosthesis Alignment after Revision Total Knee Arthroplasty Using EOS 2D and 3D Imaging: A Reliability Study

Introduction

A new low-dose X-ray device, called EOS, has been introduced for determining lower-limb alignment in 2D and 3D. Reliability has not yet been assessed when using EOS on lower limbs containing a knee prosthesis. Therefore purpose of this study was to determine intraobserver and interobserver reliability of EOS 2D and 3D knee prosthesis alignment measurements after revision total knee arthroplasty (rTKA).

Methods

Forty anteroposterior and lateral images of 37 rTKA patients were included. Two observers independently performed measurements on these images twice. Varus/valgus angles were measured in 2D (VV2D) and 3D (VV3D). Intraclass correlation coefficients and the Bland and Altman method were used to determine reliability. T-tests were used to test potential differences.

Results

Intraobserver and interobserver reliability were excellent for VV2D and VV3D. No significant difference or bias between the first and second measurements or the two observers was found. A significant mean and absolute difference of respectively 1.00° and 1.61° existed between 2D and 3D measurements.

Conclusions

EOS provides reliable varus/valgus measurements in 2D and 3D for the alignment of the knee joint with a knee prosthesis. However, significant differences exist between varus/valgus measurements in 2D and 3D.