Discrete cleavage motifs of constitutive and immunoproteasomes revealed by quantitative analysis of cleavage products.

Proteasomes are the main proteases responsible for cytosolic protein degradation and the production of major histocompatibility complex class I ligands. Incorporation of the interferon gamma--inducible subunits low molecular weight protein (LMP)-2, LMP-7, and multicatalytic endopeptidase complex--like (MECL)-1 leads to the formation of immunoproteasomes which have been associated with more efficient class I antigen processing. Although differences in cleavage specificities of constitutive and immunoproteasomes have been observed frequently, cleavage motifs have not been described previously. We now report that cells expressing immunoproteasomes display a different peptide repertoire changing the overall cytotoxic T cell--specificity as indicated by the observation that LMP-7(-/-) mice react against cells of LMP-7 wild-type mice. Moreover, using the 436 amino acid protein enolase-1 as an unmodified model substrate in combination with a quantitative approach, we analyzed a large collection of peptides generated by either set of proteasomes. Inspection of the amino acids flanking proteasomal cleavage sites allowed the description of two different cleavage motifs. These motifs finally explain recent findings describing differential processing of epitopes by constitutive and immunoproteasomes and are important to the understanding of peripheral T cell tolerization/activation as well as for effective vaccine development.

The mitochondrial ClpB homolog Hsp78 cooperates with matrix Hsp70 in maintenance of mitochondrial function.

The mitochondrial heat shock protein Hsp78 is a member of the Hsp104/Clp family with unknown function. Saccharomyces cerevisiae deletion mutants of HSP78 show wild-type like growth. We report that deletion of the HSP78 gene in yeast strains with point mutations in the SSC1 gene (encoding matrix Hsp70) led to loss of mitochondrial DNA, indicating that at least one of the heat shock proteins Hsp78 and mt-Hsp70 is needed to maintain a rho+ state of the mitochondrial genome. Mitochondria isolated from these double mutants had a strongly reduced membrane potential, explaining defects in the rate of preprotein import. The lack of Hsp78 led to aggregation of the mutant mt-Hsp70 while other matrix chaperones stayed soluble. We conclude that Hsp78 is required to keep mutant forms of mt-Hsp70 soluble and suggest a cooperation of Hsp78 and mt-Hsp70 in maintenance of essential mitochondrial functions.

Anisotropy in cellular automata.

Time-dependent and stationary patterns produced by standard cellular automata used for various qualitative modeling purposes show various geometric features that reflect the anisotropy of the underlying grid and neighborhood rather than the properties of the physical model itself. We investigate several modifications of cellular automata such as random grids, stochastic local functions, asynchronous evaluations that are designed to avoid anisotropy. We apply these tools to a class of simple totalistic automata simulating contact processes. It appears that random grids are most useful.

Synchronous and asynchronous updating in cellular automata.

We analyze the properties of a synchronous and of various asynchronous methods to iterate cellular automata. Asynchronous methods in which the time variable is not explicitly defined, operate by specifying an updating order of the cells. The statistical properties of this order have significant consequences for the dynamics and the patterns generated by the cellular automata. Stronger correlations between consecutive steps in the updating order result in more, artificial structure in the patterns. Among these step-driven methods, using random choice with replacement to pick the next cell for updating, yields results that are least influenced by the updating method. We also analyse a time-driven method in which the state transitions of single cells are governed by a probability per unit time that determines an exponential distribution of the waiting time until the next transition. The statistical properties of this method are completely independent of the size of the grid. Consecutive updating steps therefore show no correlation at all. The stationary states of a cellular automaton do not depend on whether a synchronous or asynchronous updating method is used. Their basins of attraction might, however, be vastly different under synchronous and asynchronous iteration. Cyclic dynamics occur only with synchronous updating.

Cervical Detachment Using Monopolar SupraLoop™ Electrode versus Monopolar Needle in Laparoscopic Supracervical Hysterectomy (LSH): An Interventional, Comparative Cohort Study.

Objective: Currently available monopolar loop electrodes are difficult to handle in laparoscopic supracervical hysterectomy (LSH) and are entirely disposable devices, generating additional operating costs. The aim of this interventional study was the comparison of the efficiency and safety of cervical detachment with a newly developed monopolar loop electrode (SupraLoop™) with a conventional method of cervical detachment in LSH. Material and Methods: Our study sample included 1598 patients; 1070 patients that underwent LSH with cervical detachment using the monopolar SupraLoop™ (study group) and 528 patients that underwent LSH with cervical detachment using the monopolar needle (control group). We also assessed cervical detachment time and total device application and cutting time in a subgroup of 49 patients (23 patients from the study group and 26 patients from the control group). Results: Total operation time for LSH was significantly shorter among SupraLoop™ patients (93 ± 41 minutes) when compared to patients in whom cervical detachment was performed with the needle (105 ± 44 minutes) (p < 0.001). Cervical detachment time and total device application including cutting time was significantly shorter for the SupraLoop™ group (SupraLoop vs. needle; 0.12 ± 0.21 min vs. 5.1 ± 4.4 min [p < 0.001]; 2.3 ± 1.8 min vs. 5.4 ± 2.4 min [p < 0.001]). There were no major or minor complications directly related to the use of the SupraLoop™ device, whereas two intraoperative complications were directly related to the application of the monopolar needle. Conclusion: The newly developed monopolar loop electrode (SupraLoop™) is both an effective and safe instrument for cervical detachment in laparoscopic supracervical hysterectomy, and performed better than the needle, offering a significantly shorter operating time and less complications for the hysterectomy compared to the conventional method.

[Reproducibility of readings of ISO C 3D and CT lumbar pedicle screw scans]. / Reproduzierbarkeit der Befundung von lumbalen Pedikelschraubenpositionen mit ISO C 3D und CT.

PURPOSE: This study was conducted to evaluate the reproducibility of the reading of lumbar pedicle screw scans using a C-arm-based imaging system in comparison to computed tomography. The influence of the technique and the experience of the rater should be determined. MATERIALS AND METHODS: The lumbar spines of 23 patients were stabilized using 102 pedicle screws. The position of the screws was controlled intraoperatively using an Arcadis Orbic 3D scanner. All scans were evaluated independently by three raters. The position of the implants in reference to the pedicle walls was described. Additionally, another 100 lumbar pedicle screws in 16 patients were evaluated postoperatively with a multirow CT. Kappa according to Fleiss was calculated for the reproducibility of the rater statements. Each rater repeated the analysis of 24 screws to assess the intraobserver variance. RESULTS: The reports of the CT scans showed significantly less variation. The consent of all 3 raters was achieved in 79.4 vs. 65.1 % of cases. The Kappa values were 0.56 and 0.29, respectively. Poor results were obtained especially for the medial pedicle wall (consent 70.0 vs. 50.0 %). The influence of the experience of the rater was not able to be verified. CONCLUSION: The image quality of the ISO C 3D is worse than that of multirow CT scans for the evaluation of lumbar pedicle screws. This causes greater variance among the rater reports. We stopped using the ISO C 3D technique intraoperatively for the implantation of lumbar pedicle screws.

[Undergraduate education on chronic wound care at German medical schools--the role of surgical disciplines]. / Stand der medizinischen Lehre im Bereich chronischer Wunden/Wundbehandlung--der Beitrag der chirurgischen Fächer.

INTRODUCTION: In response to the problem of chronic wounds, we decided to investigate the status of chronic wound care in undergraduate medical programs in Germany. MATERIAL AND METHODS: The deans of medical schools in Germany and all relevant instructors of dermatology, surgery, internal medicine and general medicine, pathology and health care of the elderly at 32 universities in Germany were asked to fill out a questionnaire. RESULTS: The mean teaching time is 7 hours [0.75-16.5]. The lessons covered clinical entities such as venous ulcer (21 %), diabetic (18 %) and arterial ulcer (12 %), decubital ulcers (13 %), ulcers with vasculitis (11 %), burns (9 %) and neoplastic ulcers (4 %), or others (12 %). The respective material is taught in approximately two-thirds as a lecture and one-third in seminars and practical sessions. Only in 6 % of the cases a practical examination is performed. DISCUSSION: Undoubtedly 7 hours of teaching on chronic wound care are not sufficient. Stringent coordination of the medical education at each university and practical examinations may improve student education.

Ring vaccination.

Based on the description of an outbreak of foot-and-mouth disease (FMD), a particle model is developed describing the most important properties of this epidemic. Also control measures (mass and ring vaccination) are implemented. This model shows the expected behavior in simulations. Since it is impossible to treat this model analytically, we use ideas of branching processes on two levels to derive a caricature of the particle model. In simulations it is shown that this caricature exhibits similar behavior as the particle system. It is possible to analyze the caricature and, in this way, to obtain expressions for the most important quantities like the reproduction number or the expected final number of infected individuals etc. In this way mass vaccination and ring vaccination can be compared and control strategies can be optimized.

Biogenesis of the yeast frataxin homolog Yfh1p. Tim44-dependent transfer to mtHsp70 facilitates folding of newly imported proteins in mitochondria.

Tim44 is an essential component of the mitochondrial inner membrane protein import machinery. In this study we asked if Tim44 is of relevance in intramitochondrial protein folding. We investigated the role of Tim44 in the biogenesis of the authentic mitochondrial protein Yfh1p, the yeast homolog of mammalian frataxin, which was recently implicated in Friedreich ataxia. After inactivation of Tim44, binding of mitochondrial heat shock protein (mtHsp)70 to translocating Yfh1p and subsequent folding to the native state was nearly completely blocked. Residual amounts of imported Yfh1p showed an increased tendency to aggregate. To further characterize the functions of Tim44 in the matrix, we imported dihydrofolate reductase (DHFR) as a model protein. Depletion of Tim44 allowed import of DHFR, although folding of the newly imported DHFR was delayed. Moreover, the depletion of Tim44 caused a strongly reduced binding of mtHsp70 and Mge1 to the translocating polypeptide. Subsequent dissociation of mtHsp70 from imported DHFR was delayed, indicating that mtHsp70-substrate complexes formed independently of Tim44 differ from the complexes that form under the control of Tim44. We conclude that Tim44 not only plays a role in protein translocation but also in the pathways of mitochondrial protein folding.

Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins.

The essential gene TIM44 encodes a subunit of the inner mitochondrial membrane preprotein translocase that forms a complex with the matrix heat-shock protein Hsp70. The specific role of Tim44 in protein import has not yet been defined because of the lack of means to block its function. Here we report on a Saccharomyces cerevisiae mutant allele of TIM44 that allows selective and efficient inactivation of Tim44 in organello. Surprisingly, the mutant mitochondria are still able to import preproteins. The import rate is only reduced by approximately 30% compared with wild-type as long as the preproteins do not carry stably folded domains. Moreover, the number of import sites is not reduced. However, the mutant mitochondria are strongly impaired in pulling folded domains of preproteins close to the outer membrane and in promoting their unfolding. Our results demonstrate that Tim44 is not an essential structural component of the import channel, but is crucial for import of folded domains. We suggest that the concerted action of Tim44 and mtHsp70 drives unfolding of preproteins and accelerates translocation of loosely folded preproteins. While mtHsp70 is essential for import of both tightly and loosly folded preproteins, Tim44 plays a more specialized role in translocation of tightly folded domains.