Bericht über die 135. Jahresversammlung in Münster/Westfalen

Ohne Zusammenfassung     

Effects of chemical modification of lysine residues in trypsin

Chemical modifications are a simple method to identify and modify functional determinants of enzymes. In the case of serine proteases, it is possible to induce characteristics which are advantageous for peptide synthesis. In this work, we investigated the influence of guanylation and succinylation of lysine residues on the S′-subsite specificity, the catalytic behavior and stability of trypsin. We have found, that succinylation leads to an about 10-fold better acceptance of basic residues in P1′, whereas guanylation shows no remarkable effects. Furthermore, guanylation enhances, succinylation reduces the general enzyme–substrate interactions in P2′. The structural fundamentals of these specificity changes are discussed. The catalytic behavior of trypsin was not influenced by guanylation and succinylation but an enhancement of the stability against autolytic processes by introducing additional negative charges into the protein was observed.     

A new class of long-acting hormonal steroid preparation: synthesis of dimeric androgens coupled at C3-C3 and C-17-C3 and of an androgen-progestogen combination

3β-Hydroxy-4-androsten-17-one was prepared from 4-androsten-3, 17-dione according to the method of Klimstra and Colton (1) and dimerized by means of esterification with succinic acid. The reduction with lithium-tri-t-butoxyaluminium hydride gave a testosterone derivative coupled between C₃-C₃ which showed after a single Injection of 10 mg a protracted but relatively weak androgenic effect in castrated male rats. The direct esterification of testosterone hemisuccinate with 4-androsten-3β,17β-diol gave the testosterone derivative coupled between C₁₇-C₃ which showed a more even and more protracted time response curve than testosterone enanthate. The testosterone-ethynodiol succinate also coupled between C₁₇-C₃, showed an androgenic depot-effect similar to that of the dimeric C₁₇-C₃ testosterone derivative.     

An Atomic Model of the Tropomyosin Cable on F-actin

Tropomyosin regulates a wide variety of actin filament functions and is best known for the role that it plays together with troponin in controlling muscle activity. For effective performance on actin filaments, adjacent 42-nm-long tropomyosin molecules are joined together by a 9- to 10-residue head-to-tail overlapping domain to form a continuous cable that wraps around the F-actin helix. Yet, despite the apparent simplicity of tropomyosin’s coiled-coil structure and its well-known periodic association with successive actin subunits along F-actin, the structure of the tropomyosin cable on actin is uncertain. This is because the conformation of the overlap region that joins neighboring molecules is poorly understood, thus leaving a significant gap in our understanding of thin-filament structure and regulation. However, recent molecular-dynamics simulations of overlap segments defined their overall shape and provided unique and sufficient cues to model the whole actin-tropomyosin filament assembly in atomic detail. In this study, we show that these MD structures merge seamlessly onto the ends of tropomyosin coiled-coils. Adjacent tropomyosin molecules can then be joined together to provide a comprehensive model of the tropomyosin cable running continuously on F-actin. The resulting complete model presented here describes for the first time (to our knowledge) an atomic-level structure of αα-striated muscle tropomyosin bound to an actin filament that includes the critical overlap domain. Thus, the model provides a structural correlate to evaluate thin-filament mechanics, self-assembly mechanisms, and the effect of disease-causing mutations.     

The effect of adaptation temperature on the metabolic level of the eelAnguilla vulgaris L.

Summary 1. The present paper reviews some investigations on the problem of temperature adaptation of the eel. The experiments were made in order to find out why the metabolic rate of muscle in vitro does not reflect the capacity adaptation of the intact eel.2. Oxygen tension in the muscle tissue and in the venous blood has been measured by inserting micro oxygen electrodes. Oxygen tension in the muscle of the tail is very low; tension in the large caudal vein is more than ten times higher.3. Oxygen tension in the muscle is not altered by changing the adaptation temperature. The cold-acclimated eel shows a lower oxygen tension in the venous blood than the warm-adapted fish.4. Oxygen tension in the caudal vein depends largely on the breathing rate; this can be seen when the experimental temperature is changed and differently adapted individuals are tested. Therefore we suggest that the metabolic rate is certainly influenced by adaptive changes in the nervous system.

---

Der Einfluß der Adaptationstemperatur auf die Stoffwechselhöhe des AalesAnguilla vulgaris L.

Kurzfassung Beim Aal weisen der Grundstoffwechsel, gemessen am Sauerstoffverbrauch des Ganztieres, und die Atmung des Muskelgewebes in vitro eine partielle Kompensation auf. Die Höhe des Standardsauerstoffverbrauchs ist aber unabhängig von der Stoffwechselaktivität eines größeren Teiles der Skelettmuskulatur. Es wird über Messungen des Sauerstoffpartialdrucks in der Muskulatur und im venösen Blut unterschiedlich adaptierter Aale berichtet. Die niedrige Sauerstoffspannung im Gewebe und die starke Abhängigkeit des Sauerstoffpartialdrucks im venösen Blut von der Atmungsintensität sprechen dafür, daß ein Mechanismus, der den Standardsauerstoffverbrauch des adaptierenden Ganztieres steuert, in einer Regulation des Kreislaufs und der Atmung zu sehen ist.

---

---

This paper was presented at the Fourth International Biometeorological Congress, New Brunswick (N.J.), U.S.A., August 26 to September 2, 1966.     

Leptin impairs myogenesis in C2C12 cells through JAK/STAT and MEK signaling pathways

Reduced lean body mass in genetically obese (ob/ob) or anorectic/cachectic subjects prompted us to verify the hypothesis whether leptin, white adipose tissue cytokine, might be a negative organizer of myogenesis. Recombinant leptin (100 ng/mL) stimulated mitogenesis together with the raise in T^202/Y²⁰⁴P-ERK1/2 protein expression. Concomitantly, it impaired cell viability and muscle fiber formation from C2C12 mouse myoblasts. Detailed acute and chronic studies with the use of metabolic inhibitors revealed that both JAK/STAT3 and MEK/MAPK but not PI3-K/AKT/GSK-3β signaling pathways were activated by leptin, and that STAT3 (Y⁷⁰⁵P-STAT3) and MEK (T^202/Y²⁰⁴P-ERK1/2) mediate these effects. In contrary, insulin evoked PI3-K-dependent phosphorylation of AKT (S⁴⁷³) and GSK-3β (S⁹) and insulin surpassed leptin-dependent inhibition of myogenic differentiation in PI3-K-dependent manner. GSK-3β seems to play dual role in muscle development. Insulin-dependent effect on GSK-3β (S⁹P-GSK-3β) led to accelerated myotube construction. In contrary, leptin through MEK-dependent manner caused GSK-3β phosphorylation (Y²¹⁶P-GSK-3β) with resultant drop in myoblast fusion. Summing up, partially opposite effects of insulin and leptin on skeletal muscle growth emphasize the importance of interplay between these cytokines. They determine how muscle mass is gained or lost.     

Good Manufacturing Practices (GMP) manufacturing of advanced therapy medicinal products: a novel tailored model for optimizing performance and estimating costs

Background aimsAdvanced therapy medicinal products (ATMP) have gained considerable attention in academia due to their therapeutic potential. Good Manufacturing Practice (GMP) principles ensure the quality and sterility of manufacturing these products. We developed a model for estimating the manufacturing costs of cell therapy products and optimizing the performance of academic GMP-facilities.MethodsThe “Clean-Room Technology Assessment Technique” (CTAT) was tested prospectively in the GMP facility of BCRT, Berlin, Germany, then retrospectively in the GMP facility of the University of California-Davis, California, USA. CTAT is a two-level model: level one identifies operational (core) processes and measures their fixed costs; level two identifies production (supporting) processes and measures their variable costs. The model comprises several tools to measure and optimize performance of these processes. Manufacturing costs were itemized using adjusted micro-costing system.ResultsCTAT identified GMP activities with strong correlation to the manufacturing process of cell-based products. Building best practice standards allowed for performance improvement and elimination of human errors. The model also demonstrated the unidirectional dependencies that may exist among the core GMP activities. When compared to traditional business models, the CTAT assessment resulted in a more accurate allocation of annual expenses. The estimated expenses were used to set a fee structure for both GMP facilities. A mathematical equation was also developed to provide the final product cost.ConclusionsCTAT can be a useful tool in estimating accurate costs for the ATMPs manufactured in an optimized GMP process. These estimates are useful when analyzing the cost-effectiveness of these novel interventions.     

Reverse Action of Ribonuclease T1 Variants In ICE

Abstract

Synthesis of guanylyl(3′→5′)cytidine catalysed by RNase T1 variants (Tyr42Trp, Tyr24Trp and GluSSAla) was studied in frozen aqueous systems at-10°C and in solution at 0°C. Freezing the reaction mixture resulted in significantly enhanced dinucleoside monophosphate yields independently of the effect of mutation on substrate binding and catalytic mechanism. We assume that the protonation state of the catalytic residues is influenced by freezing, possibly due to conformational changes of the enzyme proteins.     

A traversodont cynodont from the Middle Triassic (Ladinian) of Baden-Württemberg (Germany)

Nanogomphodon wildi n. gen., n. sp. is based on a tiny lower postcanine tooth from the lower Lettenkeuper (Lower Keuper or Erfurt Formation; Ladinian) of Michelbach an der Bilz (Baden-Württemberg). It represents the first record of a traversodont cynodont from the Middle Triassic of Europe and exhibits a distinctive combination of dental features. Along with recent discoveries of other traversodont taxa from the Upper Triassic of eastern North America,Nanogomphodon indicates the existence of a distinct lineage of these cynodonts in the Northern Hemisphere.