STOFFWECHSELUNTERSUCHUNGEN DES CEREBRALEN ANFALLSGESCHEHENS

Abstract— The activity of glutamate decarboxylase in the brain of rats during and prior to experimentally produced cerebral seizures was compared with that of control rats. An inhibition of enzyme activity during the tonic convulsions after intracisternal injection of l -glutamate or pyridoxal-5-phosphate, after audiogenic stimulation, after intraperitoneal injection of pentamethylenetetrazole and during the electroshock could be observed. During the preconvulsive stage the enzyme was strongly inhibited after an intracisternal injection of l -glutamate, l -aspartate, and after audiogenic stimulation. Only after the intracisternal injection of pyridoxal-5-phosphate the enzyme activity as compared with that of control rats was unchanged. The different effects of l -glutamate and pyridoxal-5-phosphate in vivo and in vitro on the glutamate decarboxylase are pointed out in particular. The inhibition of this enzyme in vivo is believed to be one of the possible causes of cerebral seizures.     

13C NMR studies of the fluxes in the central metabolism of Corynebacterium glutamicum during growth and overproduction of amino acids in batch cultures

The carbon flux distribution in the central metabolism of Corynebacterium glutamicum was studied in batch cultures using [1-¹³C]- and [6-¹³C]glucose as substrate during exponential growth as well as during overproduction of l-lysine and l-glutamate. Using the ¹³C NMR data in conjunction with stoichiometric metabolite balances, molar fluxes were quantified and normalised to the glucose uptake rate, which was set to 100. The normalised molar flux via the hexose monophosphate pathway was 40 during exponential growth, whereas it was only 17 during l-glutamate production. During l-lysine production, the normalised hexose monophosphate pathway flux was elevated to 47. Thus, the carbon flux via this pathway correlated with the NADPH demand for bacterial growth and l-lysine overproduction. The normalised molar flux in the tricarboxylic acid cycle at the level of 2-oxoglutarate dehydrogenase was 100 during exponential growth and 103 during l-lysine secretion. During l-glutamate formation, the normalised flux through the tricarboxylic acid cycle was reduced to 60. In contrast to earlier NMR studies with C. glutamicum, no significant activity of the glyoxylate pathway could be detected. All experiments indicated a strong in vivo flux from oxaloacetate back to phosphoenolpyruvate and/or pyruvate, which might be due to phosphoenolpyruvate carboxykinase activity in C. glutamicum.     

Acute psychological stress increases plasma levels of cortisol, prolactin and TSH.

The effects of acute stress during a parachute jump on hormonal responses were studied in 12 experienced and 11 inexperienced military parachutists. Each subject performed two jumps. Prior to and immediately after each jump blood samples were drawn and analysed for plasma levels of cortisol, prolactin, thyrotropin (TSH), somatotropin (STH), and luteinizing hormone (LH). While there was a significant increase in cortisol, prolactin and TSH levels after both jumps, no alterations could be observed in STH and LH levels. Stress-induced hormonal responses were not affected by jump experience. There was also no association between the endocrine variables and anxiety scores.     

Microbioreactor-assisted cultivation workflows for time-efficient phenotyping of protein producing Aspergillus niger in batch and fed-batch mode

In recent years, many fungal genomes have become publicly available. In combination with novel gene editing tools, this allows for accelerated strain construction, making filamentous fungi even more interesting for the production of valuable products. However, besides their extraordinary production and secretion capacities, fungi most often exhibit challenging morphologies, which need to be screened for the best operational window. Thereby, combining genetic diversity with various environmental parameters results in a large parameter space, creating a strong demand for time-efficient phenotyping technologies. Microbioreactor systems, which have been well established for bacterial organisms, enable an increased cultivation throughput via parallelization and miniaturization, as well as enhanced process insight via non-invasive online monitoring. Nevertheless, only few reports about microtiter plate cultivation for filamentous fungi in general and even less with online monitoring exist in literature. Moreover, screening under batch conditions in microscale, when a fed-batch process is performed in large-scale might even lead to the wrong identification of optimized parameters. Therefore, in this study a novel workflow for Aspergillus niger was developed, allowing for up to 48 parallel microbioreactor cultivations in batch as well as fed-batch mode. This workflow was validated against lab-scale bioreactor cultivations to proof scalability. With the optimized cultivation protocol, three different micro-scale fed-batch strategies were tested to identify the best protein production conditions for intracellular model product GFP. Subsequently, the best feeding strategy was again validated in a lab-scale bioreactor.     

Rapid and comprehensive evaluation of microalgal fatty acids via untargeted gas chromatography and time‐of‐flight mass spectrometry

Due to their high triacylglyceride content, microalgae are intensively investigated for bio‐economy and food applications. However, lipid analysis is a laborious task incorporating extraction, transesterification and typically gas chromatographic measurement. Co‐elution induces a significant risk of fatty acid misidentification and thus, additional purification steps like thin layer chromatography are needed. Contrary to database matching approaches, solely targeted analysis is facilitated as compound identification is driven by matching retention times or indices with standard substances. In this context, a rapid workflow for the analysis of algal fatty acids is presented. In‐situ transesterification was used to simplify sample preparation and conditions were optimized towards fast processing. If results are needed at the very day of sampling, direct processing without a preceding drying step is feasible to obtain a rough estimate about the occurrence of the major compounds. Coupling gas chromatography and time‐of‐flight mass spectrometry enables untargeted analysis. Unknown compounds may be identified by structural reconstruction of their respective fragmentation patterns and by database matching to routinely avoid mismatches by co‐elution of disturbing agents. The developed workflow was successfully applied to derive the exact stereochemistry of all fatty acids from Chlorella vulgaris and a systematic shift depending on physiological state of the cells was confirmed.     

Visualizing regulatory interactions in metabolic networks

Background  

Direct visualization of data sets in the context of biochemical network drawings is one of the most appealing approaches in the field of data evaluation within systems biology. One important type of information that is very helpful in interpreting and understanding metabolic networks has been overlooked so far. Here we focus on the representation of this type of information given by the strength of regulatory interactions between metabolite pools and reaction steps.     

24-h Langendorff-perfused neonatal rat heart used to study the impact of adenoviral gene transfer

The human genome project has increased the demand for simple experimental systems that allow the impact of gene manipulations to be studied under controlled ex vivo conditions. We hypothesized that, in contrast to adult hearts, neonatal hearts allow long-term perfusion and efficient gene transfer ex vivo. A Langendorff perfusion system was modified to allow perfusion for >24 h with particular emphasis on uncompromised contractile activity, sterility, online measurement of force of contraction, inotropic response to beta-adrenergic stimulation, and efficient gene transfer. The hearts were perfused with serum-free medium (DMEM + medium 199, 4 + 1) supplemented with hydrocortisone, triiodothyronine, ascorbic acid, insulin, pyruvate, l-carnitine, creatine, taurine, l-glutamine, mannitol, and antibiotics recirculating (500 ml/2 hearts) at 1 ml/min. Hearts from 2 day-old rats beat constantly at 135-155 beats/min and developed active force of 1-2 mN. During 24 h of perfusion, twitch tension increased to approximately 165% of initial values (P < 0.05), whereas the inotropic response to isoprenaline remained constant. A decrease in total protein content of 10% and histological examination indicated moderate edema, but actin and calsequestrin concentration remained unchanged and perfusion pressure remained constant at 7-11 mmHg. Perfusion with a LacZ-encoding adenovirus at 3 x 108 active virus particles yielded homogeneous transfection of approximately 80% throughout the heart and did not affect heart rate, force of contraction, or response to isoprenaline compared with uninfected controls (n = 7 each). Taken together, the 24-h Langendorff-perfused neonatal rat heart is a relatively simple, inexpensive, and robust new heart model that appears feasible as a test bed for functional genomics.     

Metabolic isotopomer labeling systems. Part II: structural flux identifiability analysis

Metabolic flux analysis using carbon labeling experiments (CLEs) is an important tool in metabolic engineering where the intracellular fluxes have to be computed from the measured extracellular fluxes and the partially measured distribution of 13C labeling within the intracellular metabolite pools. The relation between unknown fluxes and measurements is described by an isotopomer labeling system (ILS) (see Part I [Math. Biosci. 169 (2001) 173]). Part II deals with the structural flux identifiability of measured ILSs in the steady state. The central question is whether the measured data contains sufficient information to determine the unknown intracellular fluxes. This question has to be decided a priori, i.e. before the CLE is carried out. In structural identifiability analysis the measurements are assumed to be noise-free. A general theory of structural flux identifiability for measured ILSs is presented and several algorithms are developed to solve the identifiability problem. In the particular case of maximal measurement information, a symbolical algorithm is presented that decides the identifiability question by means of linear methods. Several upper bounds of the number of identifiable fluxes are derived, and the influence of the chosen inputs is evaluated. By introducing integer arithmetic this algorithm can even be applied to large networks. For the general case of arbitrary measurement information, identifiability is decided by a local criterion. A new algorithm based on integer arithmetic enables an a priori local identifiability analysis to be performed for networks of arbitrary size. All algorithms have been implemented and flux identifiability is investigated for the network of the central metabolic pathways of a microorganism. Moreover, several small examples are worked out to illustrate the influence of input metabolite labeling and the paradox of information loss due to network simplification.