Photosynthetically oxygenated salicylate biodegradation in a continuous stirred tank photobioreactor

A consortium consisting of a Chlorella sorokiniana strain and a Ralstonia basilensis strain was able to carry out sodium salicylate biodegradation in a continuous stirred tank reactor (CSTR) using exclusively photosynthetic oxygenation. Salicylate biodegradation depended on algal activity, which itself was a function of microalgal concentration, light intensity, and temperature. Biomass recirculation improved the photobioreactor performance by up to 44% but the results showed the existence of an optimal biomass concentration above which dark respiration started to occur and the process efficiency started to decline. The salicylate removal efficiency increased by a factor of 3 when illumination was increased from 50-300 microE/m2.s. In addition, the removal rate of sodium salicylate was shown to be temperature-dependent, increasing from 14 to 27 mg/l.h when the temperature was raised from 26.5 to 31.5 degrees C. Under optimized conditions (300 microE/m2.s, 30 degrees C, 1 g sodium salicylate/l in the feed and biomass recirculation) sodium salicylate was removed at a maximum constant rate of 87 mg/l.h, corresponding to an estimated oxygenation capacity of 77 mg O2/l.h (based on a BOD value of 0.88 g O2/g sodium salicylate for the tested bacterium), which is in the range of the oxygen transfer capacity of large-scale mechanical surface aerators. Thus, although higher degradation rates were attained in the control reactor, the photobioreactor is a cost-efficient process which reduces the cost of aeration and prevents volatilization problems associated with the degradation of toxic volatile organic compounds under aerobic conditions.     

Generation of mature fat pads in vitro and in vivo utilizing 3-D long-term culture of 3T3-L1 preadipocytes

Tissue-inherent factors such as cell-cell and cell-extracellular matrix interactions are regarded to exert a potentially large impact on adipogenesis as well as on secretory functions of adipose tissue. However, an appropriate 3-D adipogenesis model useful for addressing such interactions is still lacking. In this study, using tissue-engineering techniques, we demonstrate for the first time the development of coherent fat pads consisting of unilocular signet-ring cells in vitro. The constructs were generated by differentiating 3T3-L1 preadipocytes on 3-D polymeric scaffolds for either 9, 21, or 35 days in vitro. Only long-term culture yielded uniform tissues histologically comparable to native fat. Light and scanning electron microscopy provided direct evidence of 3-D tissue coherence and cell-cell contact in a tissue context, which was in strong contrast to conventional 2-D monolayer culture. Further differences between the two culture systems included enhanced secretion of leptin in 3-D tissue culture and differences in laminin expression (mRNA and protein level). Increase of triglyceride content over culture time and mRNA expression of other adipocyte genes, such as PPARgamma and Glut-4, were found to be similar. Implantation of long-term differentiated tissue constructs in nude mice resulted in further development and maintenance of fat pads. The presented model system is suggested to contribute to a better understanding of adipose tissue development and function facilitating studies on tissue-inherent interactions in vitro and in vivo.     

Crystal structure of human monoamine oxidase B, a drug target enzyme monotopically inserted into the mitochondrial outer membrane

Monoamine oxidase B (MAO B) is an outer mitochondrial membrane protein that oxidizes arylalkylamine neurotransmitters and has been a valuable drug target for many neurological disorders. The 1.7 angstrom resolution structure of human MAO B shows the enzyme is dimeric with a C-terminal transmembrane helix protruding from each monomer and anchoring the protein to the membrane. This helix departs perpendicularly from the base of the structure in a different way with respect to other monotopic membrane proteins. Several apolar loops exposed on the protein surface are located in proximity of the C-terminal helix, providing additional membrane-binding interactions. One of these loops (residues 99-112) also functions in opening and closing the MAO B active site cavity, which suggests that the membrane may have a role in controlling substrate binding.     

Volume-regulated Cl- channels in human pleural mesothelioma cells

A novel xyloglucan-specific endo-β-1,4-glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a pI of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3-1,4-β-glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (−2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full-length cDNA encoding XEG was cloned and sequenced. It consists of a 2436-bp open reading frame encoding a 776-amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli, and enzymatically active recombinant XEG was obtained.     

Drosophila melanogaster P Transposable Elements: Mechanisms of Transposition and Regulation

The molecular mechanisms that control P element transposition and determine its tissue specificity remain incompletely understood, although much information has been compiled about this element in the last decade. This review summarizes the currently available information about P element transposition, P-M hybrid dysgenesis and P cytotype features, P element-encoded repressors, and regulation of transposition.     

Absence of the RGS9.Gbeta5 GTPase-activating complex in photoreceptors of the R9AP knockout mouse

Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein beta subunit (Gbeta5). RGS9.Gbeta5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or Gbeta5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9.Gbeta5 complex, and therefore all three proteins, RGS9, Gbeta5 , and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP.     

Influence of GrpE on DnaK-substrate interactions

The DnaK chaperone of Escherichia coli assists protein folding by an ATP-dependent interaction with short peptide stretches within substrate polypeptides. This interaction is regulated by the DnaJ and GrpE co-chaperones, which stimulate ATP hydrolysis and nucleotide exchange by DnaK, respectively. Furthermore, GrpE has been claimed to trigger substrate release independent of its role as a nucleotide exchange factor. However, we show here that GrpE can accelerate substrate release from DnaK exclusively in the presence of ATP. In addition, GrpE prevented the association of peptide substrates with DnaK through an activity of its N-terminal 33 amino acids. A ternary complex of GrpE, DnaK, and a peptide substrate could be observed only when the peptide binding to DnaK precedes GrpE binding. Furthermore, we demonstrate that GrpE slows down the release of a protein substrate, sigma(32), from DnaK in the absence of ATP. These findings suggest that the ATP-triggered dissociation of GrpE and substrates from DnaK occurs in a concerted fashion.     

The life span determinant p66Shc localizes to mitochondria where it associates with mitochondrial heat shock protein 70 and regulates trans-membrane potential

P66Shc regulates life span in mammals and is a critical component of the apoptotic response to oxidative stress. It functions as a downstream target of the tumor suppressor p53 and is indispensable for the ability of oxidative stress-activated p53 to induce apoptosis. The molecular mechanisms underlying the apoptogenic effect of p66Shc are unknown. Here we report the following three findings. (i) The apoptosome can be properly activated in vitro in the absence of p66Shc only if purified cytochrome c is supplied. (ii) Cytochrome c release after oxidative signals is impaired in the absence of p66Shc. (iii) p66Shc induces the collapse of the mitochondrial trans-membrane potential after oxidative stress. Furthermore, we showed that a fraction of cytosolic p66Shc localizes within mitochondria where it forms a complex with mitochondrial Hsp70. Treatment of cells with ultraviolet radiation induced the dissociation of this complex and the release of monomeric p66Shc. We propose that p66Shc regulates the mitochondrial pathway of apoptosis by inducing mitochondrial damage after dissociation from an inhibitory protein complex. Genetic and biochemical evidence suggests that mitochondria regulate life span through their effects on the energetic metabolism (mitochondrial theory of aging). Our data suggest that mitochondrial regulation of apoptosis might also contribute to life span determination.