The photocycle of the photophobic receptor sensory rhodopsin II from N. pharaonis was analyzed by varying measuring wavelengths, temperature, and pH, and by exchanging H2O with D2O. The data can be satisfactorily modeled by eight exponents over the whole range of modified parameters. The kinetic data support a model similar to that of bacteriorhodopsin (BR) if a scheme of irreversible first-order reactions is assumed. Eight kinetically distinct protein states can then be identified. These states are formed from five spectrally distinct species. The chromophore states Si correspond in their spectral properties to those of the BR photocycle, namely pSRII510 (K), pSRII495 (L), pSRII400 (M), pSRII485 (N), and pSRII535 (O). In comparison to BR, pSRII400 is formed approximately 10 times faster than the M state; however, the back-reaction is almost 100 times slower. Comparison of the temperature dependence of the rate constants with those from the BR photocycle suggests that the differences are caused by changes of DeltaS. The rate constants of the pSRII photocycle are almost insensitive to the pH variation from 9.0 to 5.5, and show only a small H2O/D2O effect. This analysis supports the idea that the conformational dynamics of pSRII controls the kinetics of the photocycle of pSRII.

In the present work the light-activated proton transfer reactions of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) and those of the channel-mutants D75N-pSRII and F86D-pSRII are investigated using flash photolysis and black lipid membrane (BLM) techniques. Whereas the photocycle of the F86D-pSRII mutant is quite similar to that of the wild-type protein, the photocycle of D75N-pSRII consists of only two intermediates. The addition of external proton donors such as azide, or in the case of F86D-pSRII, imidazole, accelerates the reprotonation of the Schiff base, but not the turnover. The electrical measurements prove that pSRII and F86D-pSRII can function as outwardly directed proton pumps, whereas the mutation in the extracellular channel (D75N-pSRII) leads to an inwardly directed transient current. The almost negligible size of the photostationary current is explained by the long-lasting photocycle of about a second. Although the M decay, but not the photocycle turnover, of pSRII and F86D-pSRII is accelerated by the addition of azide, the photostationary current is considerably increased. It is discussed that in a two-photon process a late intermediate (N- and/or O-like species) is photoconverted back to the original resting state; thereby the long photocycle is cut short, giving rise to the large increase of the photostationary current. The results presented in this work indicate that the function to generate ion gradients across membranes is a general property of archaeal rhodopsins.

Sensory rhodopsin II (pSRII), the photophobic receptor from Natronobacterium pharaonis, has been studied by time-resolved resonance Raman (RR) spectroscopy using the rotating cell technique. Upon excitation with low laser power, the RR spectra largely reflect the parent state pSRII 500 whereas an increase of the laser power leads to a substantial accumulation of long-lived intermediates contributing to the RR spectra. All RR spectra could consistently be analysed in terms of four component spectra which were assigned to the parent state pSRII 500 and the long-lived intermediates M 400 , N 485 and O 535 based on the correlation between the CN NC stretching frequency and the absorption maximum. The parent state and the intermediates N 485 and O 535 exhibit a protonated Schiff base. The CN NN stretching frequencies and the H/D isotopic shifts indicate strong hydrogen bonding interactions of the Schiff base in pSRII 500 and O 535 whereas these interactions are most likely very weak in N 485 .

Vitamin C and vitamin E are known as important cellular antioxidants and are involved in several other non-antioxidant processes. Generally vitamin C and vitamin E are not synthesized by humans and therefore have to be applied by nutrition. The absence or deficiency of the vitamins can lead to several dysfunctions and even diseases (e.g. scurvy). The main interest in this study is that vitamin C and E are known to influence bone formation, e.g. vitamin C plays the key role in the synthesis of collagen, the major component of the extracellular bone matrix.In the present study we evaluate the effect of ascorbic acid (vitamin C) and α-tocopherol (vitamin E) on the proliferation and differentiation of primary bovine osteoblasts in vitro. Starting from standard growth medium we minimized the foetal calf serum to reduce their stimulatory effect on proliferation.An improved growth and an increased synthesis of the extracellular matrix proteins collagen type I, osteonectin and osteocalcin was observed while increasing the ascorbic acid concentration up to 200 μg/ml. Furthermore the effects of α-tocopherol on cell growth and cell differentiation were examined, whereby neither improved growth nor increased synthesis of the extracellular matrix proteins collagen type I, osteonectin and osteocalcin were detected.Further investigations are necessary to target at better supportive effect of vitamins on bone regeneration, and healing.

It remains unexplored in what way osteogenic stimulation with dexamethasone, ascorbic acid and β-glycerol phosphate (DAG) influences the process of mineralization, the composition and structure of the assembled mineral. Therefore, we analyzed and characterized biomineralization in DAG-stimulated and unstimulated 3D human unrestricted somatic stem cell (USSC) cultures. Microspheres were analyzed by histological staining, scanning electron microscopy (SEM), semi-quantitative energy-dispersive X-ray spectroscopy (EDX), quantitative wavelength-dispersive X-ray spectroscopy (WDX), transmission electron microscopy (TEM), selected area electron diffraction (SAED) and Raman spectroscopy. Mineral material was detected by SEM and histological staining in both groups, and showed structural differences. DAG influenced the differentiation of USSCs and the formation, structure and composition of the assembled mineral. SEM showed that cells of the +DAG spheres exhibited morphological signs of osteoblast-like cells. EDX and WDX confirmed a Ca-P mineral in both groups. Overall, the mineral material found showed structural similarities to the mineral substance of bony material.