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Histone 3 lysine27-to-methionine (H3-K27M) mutations most frequently occur in diffuse midline gliomas (DMGs) of the childhood pons but are also increasingly recognized in adults. Their potential heterogeneity at different ages and midline locations is vastly understudied. Here, through dissecting the single-cell transcriptomic, epigenomic and spatial architectures of a comprehensive cohort of patient H3-K27M DMGs, we delineate how age and anatomical location shape glioma cell-intrinsic and -extrinsic features in light of the shared driver mutation. We show that stem-like oligodendroglial precursor-like cells, present across all clinico-anatomical groups, display varying levels of maturation dependent on location. We reveal a previously underappreciated relationship between mesenchymal cancer cell states and age, linked to age-dependent differences in the immune microenvironment. Further, we resolve the spatial organization of H3-K27M DMG cell populations and identify a mitotic oligodendroglial-lineage niche. Collectively, our study provides a powerful framework for rational modeling and therapeutic interventions.

Today's machines tools routinely operate on a 24/7 basis with little, if any, downtime allowed for routine maintenance. They're also working faster with increased metal-removal capabilities and are more versatile [...]

When transmitting power involves metal-on-metal contact, it's not enough to douse your system with oil or grease. Here we examine the subtler details of lubrication selection, using the ballscrew as [...]

Grinding, rolling, and whirling are the most common ways to thread ball screws. Some manufacturers use a combination of the three methods in an effort to lower cost, raise production, [...]

Ball screws can transmit several kilowatts of power. This doesn't sound like a lot, and it is insignificant compared to the power consumption of a linear motor. Most of the […]

Molybdenum alkylidyne N-heterocyclic carbene (NHC) complexes of the type [Mo(C-p-C6H4Y)(OC(R)(CF3)2)2(L)(NHC)][B(ArF)4] (Y = OMe, NO2; R = CH3, CF3; L = none, pivalonitrile, tetrahydrofuran; NHC = 1,3-dimesitylimidazol-2-ylidene (IMes), 1,3-dimesityl-3,4-dihydroimidazol-2-ylidene (IMesH2), 1,3-dimesityl-3,4-dichloroimidazol-2-ylidene (IMesCl2), 1,3-diisopropylimidazol-2-ylidene (IiPr); B(ArF)4–= tetrakis(3,5-bis(trifluoromethyl)phen-1-yl)borate) were used in the ring expansion metathesis polymerization (REMP) of cyclic olefins. With cis-cyclooctene (cCOE) cyclic, low molecular weight oligomers were obtained at low monomer concentrations and the cyclic nature of the polymer was confirmed by MALDI-TOF measurements. High-molecular weight cyclic poly(cCOE) became available at high monomer concentrations. Also, post-REMP allowed for converting low-molecular-weight cyclic poly(cCOE) into high-molecular-weight cyclic poly(cCOE). Tailored catalysts together with suitable additives offered access to the stereoselective REMP of functional norbornenes providing functional cis-isotactic (cis-it), cis-syndiotactic (cis-st) and trans-itpoly(norbornene)s with up to 99% stereoselectivity. Mechanistic details supported by density functional theory (DFT) calculations are outlined.

Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIRΔJM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIRΔJM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation. Diabetes 49:889-895, 2000, Binding of insulin to the [Alpha]-subunit of the human insulin receptor (HIR)induces rapid tyrosine phosphorylation of the insulin receptor Β-subunit. Insulin-stimulated tyrosine phosphorylation occurs at 6 tyrosine residues, the tyrosines [...]

Stiffness or rigidity is a key factor in evaluating ball-screw drives for specific needs. Rigidity, in this context, describes a ball screw's elastic deformation under load and directly affects the [...]

Engineers working with ball screws usually use conventional life-expectancy equations for nuts with two-point contact. When it comes to four-point contact nuts, however, many manufacturers that sell them do not [...]

Ball screws are preloaded using different methods, resulting in either two or four-point contact of load balls. And all preload methods, whether by using oversized balls, shifting pitch, or using [...]

Ball nuts are generally classified as either 'singles' or 'doubles.' But what one vendor calls a single nut may be called a 'double' nut by another. So what is a [...]

Staying competitive in the global marketplace requires production equipment that operates around the clock with little, if any, time for routine maintenance. This places substantial demands on machine components, especially [...]

Ground ball screws surpass rolled ball screws in accuracy although they cost more. However, this does not mean that it is always best to choose ground ball screws. Accuracy is the most common parameter for comparing ball screws and there are at least three values required to specify lead accuracy. Lead deviation over the full travel of the nut on the screw, lead variation in one revolution of the screw thread, production methods and heat treatment play important roles in selecting one screw type over the other., Ground ball-screw accuracy always beats rolled ball screws, but lead deviation, production methods, and heat treatment also play significant roles in choosing one over the other. Is the following true [...]

Managing risks in large information infrastructures is often tied to inevitable simplification of the system, to make a risk analysis feasible. One common way of “compacting” matters for efficient decision making is to aggregate vulnerabilities and risks identified for distinct components into an overall risk measure related to an entire subsystem and the system as a whole. Traditionally, this aggregation is done pessimistically by taking the overall risk as the maximum of all individual risks, following the heuristic understanding that the “security chain” is only as strong as its weakest link. As that method is quite wasteful of information, this work proposes a new approach, which uses neural networks to resemble human expert’s decision making in the same regard. To validate the concept, we conducted an empirical study on human expert’s risk assessments, and trained several candidate networks on the empirical data to identify the best approximation to the opinions in our expert group.

Es ist Tradition, Auszüge aus den Reden der drei Kongresspräsidenten an dieser Stelle wiederzugeben. In diesem Jahr bot es sich natürlich an, sich mit dem Kongressmotto spielerisch auseinanderzusetzen: „Bewegung ist Leben!“. Verfolgen Sie die Gedankengänge von Prof. Ingo Marzi, Prof. Andrea Meurer und Prof. Alexander Beck.

Our original aim was to evaluate in vivo nuclear uptake of a conjugate containing the SV40 T Antigen nuclear localization sequence (NLS), 6 aminohexanoic acids alternating with 7 arginines, the fluorescence dye fluorescein isothiocyanate (FITC) and the gadolinium chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). During the confocal laser scanning microscopy examination of frozen sections of the kidneys of mice injected intravenously with the conjugate we found unexpected conjugate relocation. In the dry frozen sections no nuclear staining was found. After covering the sections with physiological saline and a coverslip, the conjugate was relocated to the cell nuclei. Causation of this effect by FITC and DOTA could be excluded with appropriate controls. In the assumption that other positively charged conjugates behave in the same manner we tested two further conjugates in vivo. An octaarginine coupled to a lysine carrying FITC on the -amino-group was found in the cell nuclei of the frozen kidney sections before and after covering the slices with saline. By contrast, a conjugate containing myristoylated HIV-Tat was found not to stain the cell nuclei but only the cytoplasm before and after covering with saline. This demonstrates that not all positively charged conjugates are relocated to the cell nucleus after covering the frozen sections with saline. This was confirmed by direct incubation of frozen sections from untreated kidneys with the same conjugates and additional positively charged FITC conjugates. By coincubating the FITC conjugates with a cytoplasm directed rhodamine conjugate on untreated kidney sections we found that such conjugates could be used as nuclear FITC counterstain. In summary, one should bear in mind that positively charged conjugates applied in vivo can relocate artificially after covering with saline.

RIN1 is a regulator of epithelial cell migration. We identify serine 292 as a novel phosphorylation site for PKD in RIN1. Phosphorylation at this site controls RIN1-mediated inhibition of cell migration by modulating the direct activation of Abl kinases.

Triiodobenzoic acid (TIBA) represents the core structure of most clinically used contrast agents for computed tomography and other X-ray procedures. To construct an intracellular radiopaque contrast agent, TIBA was coupled to various different positively and negatively charged fluorescein iothiocyanate (FITC)-labelled peptides. TIBA coupled to the SV40 T Antigen nuclear localization sequence (NLS) stained 80 of human glioma cells and caused cell death. This occurred with C- or N-terminal binding of TIBA and with the correct or mutant NLS. No cell death and only small numbers of stained cells (below 3 ) were observed after incubation with NLS conjugates lacking TIBA or after incubation with TIBA-conjugates containing a negatively charged polyglutamic acid stretch. TIBA-conjugates containing the Antennapedia-derived cell-penetrating peptide penetratin were only nuclearly taken up when TIBA and FITC were coupled to lysines outside the 16-amino acid peptide sequence. The study shows that intracellular TIBA may have potential as a chemotherapeutic agent rather than a contrast agent.

Ziel: Analyse der Sicherheit und Effektivität CT-gesteuerter Brachytherapie zur Ablation von Lebermalignomen. Patienten und Methodik: 21 Patienten mit 21 Lebermalignomen (19 Metastasen, zwei primäre Lebermalignome) wurden mit perkutaner, CT-gesteuerter interstitieller Brachytherapie mit 192Ir behandelt. Alle Patienten wiesen Umstände auf, die eine bildgeführte thermische Ablation mit Radiofrequenz oder laserinduzierter Thermotherapie (LITT) einschränkten: Tumordurchmesser = 5 cm bei sieben, enge Lagebeziehung zu Pfortader oder großen Lebervenen bei zehn, enge Lagebeziehung zur Hepatikusgabel bei vier Patienten. Die Dosimetrie erfolgte über unmittelbar nach CT-geführter Applikatorpositionierung akquirierte dreidimensionale CT-Datensätze. Ergebnisse: Die mittlere Tumorgröße betrug 4,6 cm (2,5–11 cm). Die minimale Dosis innerhalb des Tumorvolumens lag im Mittel bei 17 Gy (12–20 Gy). Der Volumenanteil gesunden Leberparenchyms mit einer Exposition > 5 Gy betrug 18% (5–39%) vom Lebergesamtvolumen abzüglich Tumorlast. Übelkeit und Erbrechen traten bei sechs Patienten auf (28%). Ein Patient entwickelte eine Obstruktion des Ductus choledochus durch ein strahleninduziertes Tumorödem in unmittelbarer Nachbarschaft. Leberenzymparameter und Bilirubinwerte zeigten regelhaft diskrete Anstiege ohne klinisches Korrelat. Die lokale Tumorkontrolle betrug 87% nach 6 Monaten und 70% nach 12 Monaten. Schlussfolgerung: CT-gesteuerte Brachytherapie ist sicher und effektiv. Die Technik ermöglicht gegenüber thermischen Ablationsverfahren eine breitere Indikationsstellung hinsichtlich Tumorgröße und Tumorlokalisation.

This paper evaluates precipitation forecasts by the operational limited area model ALADIN (grid spacing 10 km) in the region of the European Alps. The observational reference is a daily high-resolution rain-gauge analysis (grid spacing about 25 km) available for the Mesoscale Alpine Programme Special Observation Period (MAP SOP,Sept. 7 to Nov. 15, 1999). The daily analysis is disaggregated in time using data from hourly reporting rain stations. Accordingly, the evaluation is performed for hourly, daily, and SOP mean precipitation fields and at grid scales of 25, 50, and 100 km, since skill of both the ALADIN forecasts as well as the analysis increases with scale. Applying standard continuous statistics for comparisons of means, variances, and patterns the results are: ALADIN and analysis converge with increasing scale of comparison (e.g., at 25 km and hourly resolution ALADIN explains 14% of the spatial field variance and 79% at 100 km at the SOP mean, the global means are not separable), but surprisingly the ALADIN performance of hourly and daily grid box time series are comparable, and forecasts of time series are generally better than of spatial fields (e.g., daily ALADIN forecasts overestimates spatial field variance by almost 50 %, but grid box time variance by only about 10%). The smaller spatial skill of ALADIN is attributed to a spatial bias pattern.

The primary structures of two biantennary N-glycans of the glycoprotein Rapana venosa (marine snail) haemocyanin were determined. Two different structural subunits have been found in R. venosa haemocyanin: RvH1 and RvH2. The carbohydrate content of the N-terminal functional unit RvH1-a of RvH1 was studied and compared with the N-terminal functional unit RvH2-a of RvH2. Oligosaccharide fragments were released from the glycoprotein by Smith degradation of a haemocyanin pronase digest and separated on a Superdex 300 column. The glycopeptide fragments, giving a positive reaction for the orcinol/H2SO4 method, were separated by HPLC. In order to determine the linked sugar chains to the hinge glycopeptides isolated from the functional unit RvH1-a, several techniques were applied, including capillary electrophoresis, matrix-assisted laser desorption ionization-MS and electrospray ionization-MS in combination with glycosidase digestion. On the basis of these results and amino acid sequence analysis, we concluded that the functional unit RvH1-a contains 7% oligosaccharides N-glycosidically attached to Asn262 and Asn401, and the following structures were suggested:

Background and Objective:Although in recent years methods in emergency medicine have been remarkably improved, thoracic trauma remains associated with high mortality. To select adequate treatment in multitrauma patients, early diagnosis of lung contusion is of great importance. Upon hospital admission, however, even modern imaging techniques frequently fail to correctly estimate both severity and extent of chest trauma. Therefore, the aim of our study was to define laboratory markers indicating chest trauma almost immediately after accident. Patients and Methods:107 trauma patients were enrolled in this prospective study. Blood samples were taken immediately at the site of accident, on hospital admission, after 30 and 60 min, and 2-hourly for an interval of 12 h, then daily. In addition to the biochemical analysis of 20 different substances, the following data were drawn and correlated to the laboratory results: Injury Severity Score (ISS), Polytrauma Score (PTS), Ulm score HTAPE (trauma pattern-specific: head, thorax, abdomen, pelvis, extremities; 0–3 degrees each), Multiple Organ Failure (MOF) score accordings to Goris et al, overall lethality, primary and secondary lethality. All trauma scores were subject to descriptive analysis. Our main analysis focuses on the thorax trauma score including variable selection by logistic regression as well as ROC analysis. Results:In nonsurvivors, overall trauma severity and head injury were remarkably higher (15.9%) than in survivors. Whereas there was a strong correlation between head injury and primary lethality (≤ 3 days), secondary lethality (> 3 days post trauma) was charged by thoracic trauma as well as by limb trauma. Of all mediators investigated, it was interleukin-6 (IL-6) that showed highest correlations (p < 0.0001) to the extent of chest trauma, especially during the first hour after hospital admission. Further correlations, yet clearly less pronounced, were found between IL-6 and PTS, between ISS, abdominal pelvic and limb trauma, but not to head injury. Our findings suggest that there is a correlation between endotoxin and 6-keto-prostaglandin F1αand the severity of thoracic injury, too, but it is of lower statistical impact compared to IL-6. The practical value of both mediators, however, is limited because of the sophisticated and time-consuming laboratory analysis they demand. Conclusion:The study results suggest IL-6 to be an early marker for the extent of chest trauma.

The phosphorylation of serine-/threonine-residues of insulin receptor substrate-1 (IRS-1) by protein kinase C-ζ (PKC-ζ), which leads to an increased tyrosine dephosphorylation of IRS-1, has been recently identified as a novel hyperinsulinemia-induced negative feed-back regulation of the insulin signalling pathway. To identify the underlying molecular mechanism, we investigated the serine-/threonine-residues in IRS-1 phosphorylated by PKC-ζ. Because the activity of the tyrosine phosphatase SHP-2, which dephosphorylates IRS-1, is regulated by the binding to the C-terminal part of IRS-1 (IRS-1^c) this region was studied. In the in vitro assay the atypical PKC ζ phosphorylates IRS-1^c comparable to PKC-θ and -β1, whereas PKC-β2 showed lower and PKC-i and PKC-α showed negligible phosphorylation. We observed one major ³²P-motif labelled by PKC-ζ, containing four phosphorylated serine/threonine residues (Ser 1215, 1216, 1220 and Thr 1221) near the C-terminal end of IRS-1. These sites are closely adjacent to tyrosine 1222, which represents one of the two binding sites for the protein tyrosine phosphatase SHP-2. In conclusion, the PKC-ζ-dependent in vitro phosphorylation of IRS-1 at a site closely situated to a crucial binding site of the tyrosine phosphatase SHP-2 suggests the modulation of the dephosphorylation activity of SHP-2 by PKC-ζ. Furthermore, our findings can contribute to the explanation of the complex positive and negative regulatory action of PKC-ζ on insulin signalling.

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS).1, 2 Phosphorylation-specific marker ions (m/z 79, PO3⁻, and m/z 97, H2PO4⁻) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO3⁻) and m/z 97 (H2PO4⁻) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C2F3O⁻ fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic β-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by µLC/ESI-sCID-MS and µLC/ESI-MS analysis. Copyright © 2001 John Wiley & Sons, Ltd.

The phosphorylation of serine-/threonine-residues of insulin receptor substrate-1 (IRS-1) by protein kinase C-ζ (PKC-ζ), which leads to an increased tyrosine dephosphorylation of IRS-1, has been recently identified as a novel hyperinsulinemia-induced negative feed-back regulation of the insulin signalling pathway. To identify the underlying molecular mechanism, we investigated the serine-/threonine-residues in IRS-1 phosphorylated by PKC-ζ. Because the activity of the tyrosine phosphatase SHP-2, which dephosphorylates IRS-1, is regulated by the binding to the C-terminal part of IRS-1 (IRS-1^c) this region was studied. In the in vitro assay the atypical PKC ζ phosphorylates IRS-1^c comparable to PKC-θ and -β1, whereas PKC-β2 showed lower and PKC-i and PKC-α showed negligible phosphorylation. We observed one major ³²P-motif labelled by PKC-ζ, containing four phosphorylated serine/threonine residues (Ser 1215, 1216, 1220 and Thr 1221) near the C-terminal end of IRS-1. These sites are closely adjacent to tyrosine 1222, which represents one of the two binding sites for the protein tyrosine phosphatase SHP-2. In conclusion, the PKC-ζ-dependent in vitro phosphorylation of IRS-1 at a site closely situated to a crucial binding site of the tyrosine phosphatase SHP-2 suggests the modulation of the dephosphorylation activity of SHP-2 by PKC-ζ. Furthermore, our findings can contribute to the explanation of the complex positive and negative regulatory action of PKC-ζ on insulin signalling.

Patients and Methods: During a 12-year period (1/88-12/99), 4,526 arthroscopic (ASC) of the knee joint were performed at our clinic. 279 of the patients involved were aged between 0 an 17 years (at the time of the procedure). 162 out of these 279 ASCs were performed posttraumatically after fresh injuries; 32 patients were children (age 6-12 years), 130 adolescents (age 13-17 years). Results: In 131 patients (81%), we established findings requiring treatment, which led to immediate arthroscopic intervention or open surgery. Findings requiring no direct therapeutic action were established during the procedure in only 31 patients (19%), with hemostasis (eight patients = 5%) and/or flushing-out of the hemarthrosis with insertion of a drain being regarded as an extended diagnostic ASC. On the basis of the findings established during the procecure, a clarification by magnetic resonance imaging (MRI) would have been promising and sufficient in only one patient (0.6%) with an isolated synovitis. In the other 30 out of these 31 patients with extended diagnostic ASC (18%), sufficient diagnostic clarification was only achieved arthroscopically. Conclusions: In view of these results, we consider that only in rare, exceptional cases (e. g., relative or absolute contraindications to arthroscopy or anesthesia), there is a posttraumatic indication for a preoperative knee MRI in children and adolescents.

The chemical behavior of two bis(3,5-dialkylpyrazol-1-yl)acetic acids and their coordination properties towards ZnCl2 and FeCl2 was studied. Reaction of bis(3,5-dimethylpyrazol-1-yl)acetic acid (bdmpza) (1) with ZnCl2 and FeCl2 gave the 2:1 complexes [(bdmpza)2Zn] (4) and [(bdmpza)2Fe] (5). Crystal structures of both complexes were obtained. In contrast, the sterically more hindered ligand bis(3,5-di-tert-butylpyrazol-1-yl)acetic acid (bdtbpza) (3) coordinates only once to zinc(II) resulting in the complex [(bdtbpza)ZnCl] (6). This provides a structural model complex for the active sites of zinc enzymes such as thermolysin or carboxypeptidase. The good agreement with these active sites was demonstrated by a crystal structure of 6. To the best of our knowledge, this is the first report of zinc complexes with a monoanionic N,N,O-tridentate ligand using a carboxylate O-donor. Substitution of benzylthiolate for the chloride ligand 6 yielded [(bdtbpza)Zn(SCH2Ph)] (8) indicating that the bulky tert-butyl groups do not prevent access to the zinc atom. Reaction of bdtbpza (3) with FeCl2 led to [(bdtbpza)FeCl] (7), which might serve as a structural model complex for the active sites of mononuclear non-heme iron oxidases and oxygenases such as IPNS, DAOCS or CAS.

AbstractThe prosclerotic transforming growth factor β1 (TGF-β1) is a key factor in the induction and maintenance of fibrosis in different organs. To assess relative changes in TGF-β1 mRNA levels, the comparative kinetic reverse transcription-polymerase chain reaction strategy was used. In this method, cellular mRNA levels of the target and a house-keeping gene are reverse transcribed, amplified by the polymerase chain reaction (PCR) and the kinetics of PCR amplification are compared. Since the current determination of the PCR products, using electrophoretic separation in polyacrylamide gel, staining and scanning of the gel, is timeconsuming (≥ 5 hours) and inaccurate, we have developed a method using capillary gel electrophoresis (CGE) in combination with laser-induced fluorescence (LIF) detection for quantification of PCR-products. Using the CGE-LIF method, a minute aliquot of the PCR reaction mixture is separated and quantified within 10 min. Comparison of the values with those obtained by polyacrylamide gel electrophoresis demonstrates the improved sensitivity (> 1000 fold) and accuracy of the proposed method. The CGE-LIF procedure offers a convenient way of automated, comparative analysis of low levels of mRNA via reverse transcription PCR in low cell numbers or small amounts of tissue samples.

Supplemental Digital Content is available in the text.

Arbeitszeitgesetz und Kommerzialisierung erschweren die Weiterbildung der künftigen Fachärzte. Der DGOU-Ausschuss Versorgung, Qualität und Sicherheit hat am 9. Mai 2018 in Frankfurt/Main einen Workshop zu Versorgungsstrukturen in O und U durchgeführt.

Proteasomal proteolysis is based on the activity of six different catalytic proteasomal subunits (β1, β2, β5, β1i, β2i, β5i) as well as ubiquitin-mediated recruitment of cytosolic proteins to the proteasomal complex. The latter is regulated by a balance between ubiquitinating enzymes and a variety of ubiquitin-specific proteases (USP). Although proteasomal proteolysis has become a major therapeutic target, the individual activity profiles of proteasomal subunits and USP in primary human malignant cells are unknown. Recently, an activity-based, cell-permeable affinity probe has been developed, which allows to visualize the individual proteasomal subunit activity in living cells in a semiquantitative fashion (Berkers et al., Nature Methods, May 2005). Similarly, a ubiquitin-based synthetic probe (HAUbVS) allows to visualize a broad range of USP based on their activity in cellular lysates. We have used both types of probes to assess i) the differential activity of the proteasomal catalytic subunits in intact cells and ii) the activity profiles of a panel of USP in primary cells of human hematologic malignancies. Individual subunits of both the constitutive and the immuoproteasome were tagged by the probe in live cells in situ, resolved by 1D or 2D SDS-PAGE, and identified by mass spectrometry. Comparison of different cases of ALL and CLL revealed a remarkable variability in the relative activities of the β1/1i–, β2/2i–, and β5/5i–type of subunits, respectively, which contrasted with their stochiometrically constant relation of the respective polypeptides in the architecture of the proteasome. Of note, live primary myeloma cells lacked active β1/1i–type subunits and contained only extremely low amounts of β5/5i–type active subunits, while active β2/2i subunits were present in amounts comparable with primary leukaemia cells or monocytes. The variability in active proteasomal subunits contrasted with a relatively homogeneous distribution of the activity profiles of seven different USP that were targeted by the USP-specific probe. In particular, and in contrast to control cell lines, we failed to confirm the substantial upregulation of UCH-L1 in primary cells, which had been suggested to be a characteristic of the proteasomal machinery in malignant cells, based on studies using human cell lines. In addition, we here for the first time directly visualize the effect of bortezomib on individual proteasome subunits in primary human cells: although Bortezomib is reported to selectively target the β5 subunit, we here show that both the β1 and β1i subunits are also inhibited by the substance, the latter with even higher affinity (IC50 approx. 2.5 nM). In one case of ALL that lacked detectable active β5/5i subunits in intact cells, Bortezomib induced cytotoxic death at the concentration where β1/β1i activity was eliminated. In summary, our study for the first time visualizes the differential activity of proteasomal subunits in living primary human cells, demonstrates significant variability in their activity profiles in leukaemia and myeloma cells and suggests that differential proteasomal activity might contribute to the variable sensitivity of cancer cells towards proteasome inhibitors such as Bortezomib.

Because cell lines can be adapted to proteasome inhibition in vitro (Pfeifer G. et al., Science 283: 978–981, 1999.), secondary resistance of malignant cells towards Bortezomib (Velcade®) in vivo is a likely scenario. We have succeeded to adapt the human AML cell line HL-60 to Bortezomib in vitro, so that the adapted subline (HL-60a) shows normal viability and growth rate at 40 nM Velcade®, while cell death is induced above 10nM in the parental line. We hypothesized that alternative proteolytic pathways might allow the continued proliferation and survival of Bortezomib-adapted cells, and assessed the activity-profiles of proteasomal subunits, ubiquitin-specific proteases and lysosomal cysteine proteases in a functional proteomics approach, using recently developed synthetic affinity probes. These tools for the first time allow semiquantitative visualization of the individual members of these protease families, based on their activity, in contrast to western blot which lacks activity information or to the turnover of fluorogenic substrates, which is not truly protease-specific. After 72h of culture in Bortezomib-free medium (wash out phase), HL-60a cells contained significantly reduced levels of active proteasomal β1,β2, and β5 subunits, compared to the parental line, as confirmed by a reduced turnover of the β5-selective fluorogenic substrate Suc-LLVY-AMC. A panel of 7 different ubiquitin-specific proteases (USP) was visualized in both types of cells, using the affinity probe HAUbVS. Of these, a 97 kD USP that we have identified as USP14 by mass spectrometry-based sequencing, was significantly upregulated in HL-60a cells. By contrast, the activity of lysosomal cysteine proteases remained unchanged in HL-60a cells. Because the cytosolic protease tripeptidyl peptidase II (TPPII) can partially substitute for proteasome activity in lactacystine-treated cell lines, we assessed TPPII-activity using a fluorogenic substrate. We observed a significant upregulation of TPPII-activity in HL-60a cells, compared to non-adapted controls. Inhibition of TPPII, however, was not sufficient to restore Bortezomib-sensitivity in HL-60a cells. Interestingly, the combination of Bortezomib with the HIV protease inhibitor Ritonavir induced synergistic cytotoxicity both in HL-60 and HL-60a cells. Thus, Bortezomib-resistance is accompabied by upregulation of protease activities in alternative pathways. Combining different protease inhibitors like Bortezomib and Ritonavir might be a promising option to overcome primary or secondary Bortezomib resistance.

Dendritic cells (DC) initiatiate immunity and maintain tolerance. They internalize exogenous antigen and convert it into immunogenic peptides by lysosomal proteolytic degradation, ultimately followed by presentation to CD4 T cells. Monocyte-derived DC (MO-DC) generated in vitrowith GM-CSF and IL-4 serve as prototype DC to analyse the cellular biology and biochemistry of DC. However, different types of primary DC, whose functional role in vivoand relationship to MO-DC generated in vitrois unclear, reside in human tissue as well as peripheral blood. The composition of lysosomal proteases in these primary human DC1 and DC2-cells and the way they handle a clinically relevant antigen are unknown, and there is no comparison of the lysosomal processing of antigen by these primary DC to that in primary human B cells or MO-DC generated ex vivo. We have isolated human peripheral blood (PB) DC1 and DC2 cells as well as primary B lymphocytes by magnetic separation and isolated lysosomal compartments from these cells, as well as from MO-DC. Expression and activity of endocytic proteases were assessed by western blot and active site-restricted affinity labelling using a synthetic probe that selectively binds to the active centre of cysteine proteases and allows a simultaneous semiquantitaive assessment and identification of multiple active protease species. In this analysis, PB-DC1 and DC2-cells lacked significant active Cathepsins (Cat) S, L and B as well as asparagine-specific endoprotease AEP, the major enzymes involved in antigen processing in the MHC II-compartment. Surprisingly, lysosomal extracts from PB-DC1 were by far more effective than MO-DC in processing the muliple sclerosis-associated autoantigen myelin basic protein (MBP) in vitro. When analyzed on a molecular scale using mass spectrometry, MBP processing was dominated by CatS, CatD and AEP in MO-DC, as expected, similar to B-lymphoblastoid cells (BLC). PB-DC, however, did not generate proteolytic processing intermediates indicative of CatS or AEP activity but showed the same pattern as primary B-lymphocyte-derived lysosomes, i.e. processing was performed by two cleavage sites that can be reproduced by purified CatG in vitro, suggesting a CatG like dominant lysosomal protease. While active CatG was present in primary human B cells, PB-DC1 cells lacked CatG protein by western blot, suggesting the presence of an as yet unknown dominant endoprotease with CatG-like activity in PB-DC1. By cleaving MBP after pos F90 and F114, this protease directly eliminates the integrity of the major immunodominant MBP epitope MBP85-99. This might lead to poor presentation of this epitope to regulatory T cells resulting in inefficient silencing of MBP-autoreactive T cells during the development of autoimmunity. Our results emphasize the need to apply state-of-the-art biochemical tools to primary human types of APC for the understanding of antigen processing and the rational design of tolerogenic or immunotherapy approaches towards human malignant and autoimmune disorders.