In situ FTIR ATR spectroscopic study of the interaction of immobilized human tumor necrosis factor-alpha with a monoclonal antibody in aqueous environment

By in situ FTIR ATR measurements, the antibody (AB) recognition of human tumor necrosis factor-alpha (TNFalpha) immobilized on the Ge surface of a multiple internal reflection element (MIRE) was investigated. The experiments were performed in aqueous environment in a flow-through cell. After immobilization of TNFalpha on the Ge-MIRE by direct adsorption from aqueous solution, the immobilisate reached stability after about 1 h under flow-through conditions. The remaining sites of the Ge surface were saturated by bovine serum albumin (BSA) in order to prevent unspecific binding of anti-TNFalpha AB which was then added. The obtained FTIR ATR spectra were shown to result exclusively from AB specifically interacting with TNFalpha, since the absence of immunoglobulin binding to BSA adsorbed to the Ge MIRE was verified by a reference experiment. Finally, the stability of all adsorbed protein immobilisates was monitored under flow-through conditions for 10.5 h. The TNFalpha-AB complex showed a decrease of 7.4%, whereas the BSA adsorbate remained stable. IR measurements were performed with polarized light in order to study orientational effects of the immobilized proteins. The dichroic ratios and surface concentrations of all used proteins are available after quantitative analysis of the amide II bands.     

Life time—circadian clocks, mitochondria and metabolism

A functional circadian clock has long been considered a selective advantage. Accumulating evidence shows that the clock coordinates a variety of physiological processes in order to schedule them to the optimal time of day and thus to synchronize metabolism to changes in external conditions. In mitochondria, both metabolic and cellular defense mechanisms are carefully regulated. Abnormal clock function, might influence mitochondrial function, resulting in decreased fitness of an organism.     

Nimesulide-induced hepatic mitochondrial injury in heterozygous Sod2(+/-) mice

Nimesulide, a preferential COX-2 inhibitor, has been associated with rare idiosyncratic hepatotoxicity. The underlying mechanisms of liver injury are unknown, but experimental evidence has identified oxidative stress as a potential hazard and mitochondria as a target. The aim of this study was to explore whether genetic mitochondrial abnormalities, resulting in impaired mitochondrial function and mildly increased oxidative stress, might sensitize mice to the hepatic adverse effects of nimesulide. We used heterozygous superoxide dismutase 2 (Sod2(+/-)) mice as a model, as these mice develop clinically silent mitochondrial stress but otherwise appear normal. Nimesulide was administered for 4 weeks (10 mg/kg, ip, bid), at a dose equivalent to human therapeutic dosage. We found that the drug potentiated hepatic mitochondrial oxidative injury (decreased aconitase activity, increased protein carbonyls) in Sod2(+/-), but not wild-type, mice. Furthermore, the nimesulide-treated mutant mice exhibited increased hepatic cytosolic levels of cytochrome c and caspase-3 activity, as well as increased numbers of apoptotic hepatocytes. Finally, nimesulide in vitro caused a concentration-dependent net increase in superoxide anion in mitochondria from Sod2(+/-), but not Sod2(+/+) mice. In conclusion, repeated administration of nimesulide can superimpose an oxidant stress, potentiate mitochondrial damage, and activate proapoptotic factors in mice with genetically compromised mitochondrial function.     

A novel flow based hollow-fiber blood-brain barrier in vitro model with immortalised cell line PBMEC/C1-2

A flow based hollow-fiber in vitro model of the blood-brain barrier (BBB) was established. The immortalised porcine brain microvascular endothelial cell line PBMEC/C1-2 was cultured in a pulsatile hollow-fiber cartridge system (Cellmax Quad). The usability of PBMEC/C1-2 in the flow based hollow-fiber model was increased from three days in the originally used Transwell model up to four months due to the application of shear stress and co-culturing with glioma cell line C6. It was shown that the tightness of PBMEC/C1-2 layers was enhanced significantly in astrocyte conditioned medium (ACM) and in co-culture. The morphology of PBMEC/C1-2 and C6 was visualised by environmental scanning electron microscopy (ESEM). Permeation studies were accomplished with a set of benzodiazepines. The raw data were processed with three different calculation models and the results were compared with permeability coefficients obtained with an established Transwell model. In summary a flow based hollow-fiber BBB in vitro model was developed, which can be used to perform experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and dynamics) and pathophysiological (e.g., effects of diseases on BBB permeability and vice versa) objectives.     

Thermodynamics of microbial growth and metabolism: an analysis of the current situation

This paper attempts to review in how far thermodynamic analysis can be used to understand and predict the performance of microorganisms with respect to growth and bio-product synthesis. In the first part, a simple thermodynamic model of microbial growth is developed which explains the relationship between the driving force for growth in terms of Gibbs energy dissipation and biomass yield. From the currently available literature, it appears that the Gibbs energy dissipation per C-mol of biomass grown, which represents the driving force for chemotrophic growth, may have been adapted by evolutionary processes to strike a reasonable compromise between metabolic rate and growth efficiency. Based on empirical correlations of the C-molar Gibbs energy dissipation, the wide variety of biomass yields observed in nature can be explained and roughly predicted. This type of analysis may be highly useful in environmental applications, where such wide variations occur. It is however not able to predict biomass yields in very complex systems such as mammalian cells nor is it able to predict or to assess bio-product or recombinant protein yields. For this purpose, a much more sophisticated treatment that accounts for individual metabolic pathways separately is required. Based on glycolysis as a test example, it is shown in the last part that simple thermodynamic analysis leads to erroneous conclusions even in well-known, simple cases. Potential sources for errors have been analyzed and can be used to identify the most important needs for future research.     

Impaired daily glucocorticoid rhythm in Per1 Brd mice

Biological clocks have evolved in all kinds of organisms in order to anticipate and adjust to the daily light–dark cycle. Within the last decade, the molecular machinery underlying the circadian system was unraveled. In the present study, the impact of the loss of the Per1 or Per2 genes, key components of the core clock oscillator, on body mass, food and water intake, glucose metabolism, and hypothalamic-pituitary-adrenal axis, was investigated in the Per1 ^Brd and Per2 ^Brd mouse models. The results reveal that the lack of Per1 but not Per2 has severe consequences for the regulation of these parameters. Specifically, in Per1 ^Brd animals, we found an impaired daily glucocorticoid rhythm, with markedly elevated levels during the day compared to control animals. In addition, Per1 ^Brd mice showed significant differences in body mass as well as food and water intake. Although the Per1 ^Brd are lighter than wildtype mice, food and water intake per gram body mass is elevated. In addition, the Per1 ^Brd mice exhibit an increased glucose metabolism after i.p. injection with glucose. In conclusion, our study presents first evidence for a link between an altered metabolism in Per1 and Per2 deficient mice, which in the case of the Per1 ^Brd animals might be due to an impaired corticosterone rhythm.     

Just the beginning: novel functions for angiotensin-converting enzymes

Cardiovascular disease is predicted to be the commonest cause of death worldwide by the year 2020. Diabetes, smoking and hypertension are the main risk factors. The renin-angiotensin system plays a key role in regulating blood pressure and fluid and electrolyte homeostasis in mammals. The discovery of specific drugs that block either the key enzyme of the renin-angiotensin system, angiotensin-converting enzyme (ACE), or the receptor for its main effector angiotensin II, was a major step forward in the treatment of hypertension and heart failure. In recent years, however, the renin-angiotensin system has been shown to be a far more complex system than initially thought. It has become clear that additional peptide mediators are involved. Furthermore, a new ACE, angiotensin-converting enzyme 2 (ACE2), has been discovered which appears to negatively regulate the renin-angiotensin system. In the heart, ACE2 deficiency results in severe impairment of cardiac contractility and upregulation of hypoxia-induced genes. We shall discuss the interplay of the various effector peptides generated by angiotensin-converting enzymes ACE and ACE2, highlighting the role of ACE2 as a negative regulator of the renin-angiotensin system.     

8-Aryl xanthines potent inhibitors of phosphodiesterase 5

In clinical studies, several inhibitors of phosphodiesterase 5 (PDE5) have demonstrated utility in the treatment of erectile dysfunction. We describe herein a series of 8-aryl xanthine derivatives which function as potent PDE5 inhibitors with, in many cases, high levels of selectivity versus other PDE isoforms.