A comparative evaluation of oxygen mass transfer and broth viscosity using Cephalosporin-C production as a case strategy

The production of Cephalosporin-C (CPC) a secondary metabolite, using a mold Acremonium chrysogenum was studied in a lab scale Internal loop air lift reactor. Cephalosporin-C production process is a highly aerobic fermentation process. Volumetric gas–liquid mass transfer coefficient (k_La) and viscosity (η) were evaluated, during the growth and production phases of the microbial physiology. An attempt has been made to correlate the broth viscosity, η and volumetric oxygen transfer coefficient, k_La during the Cephalosporin-C production in an air lift reactor. The impact of biomass concentration and mycelial morphology on broth viscosity has been also evaluated. The broth exhibits a typical non-Newtonian fermentation broth. Rheology parameters like consistency index and fluidity index are also studied.     

Histochemical staining and quantification of plant mitochondrial respiratory chain complexes using blue-native polyacrylamide gel electrophoresis

Our knowledge of the respiratory chain and associated defects depends on the study of the multisubunit protein complexes in the inner mitochondrial membrane. Functional analysis of the plant mitochondrial respiratory chain has been successfully achieved by a combination of blue-native polyacrylamide gel electrophoresis (BN-PAGE) for separation of the protein complexes, and in-gel histochemical staining of the enzyme activities. We have optimized this powerful technique by determining linear ranges of amount of protein and enzyme activity for each respiratory complex. Time courses of the in-gel enzyme activities were also performed to determine optimal reaction times. Using the in-gel activity staining method we have previously shown decreased activity of complex V (F(1)F(0)-ATPase) in male-sterile sunflowers (Sabar et al., 2003). Here we have identified unique supercomplexes comprising complex IV (cytochrome c oxidase) in sunflower mitochondria. This method therefore represents a reliable tool for the diagnosis of respiratory dysfunction. In addition, the wider application of BN-PAGE in combination with enzyme activity staining is discussed.     

The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein-protein docking simulations have been performed for the binary cytochrome b5-cytochrome c and ternary (cytochrome b5)-(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.     

Orthogonal site-specific protein modification by engineering reversible thiol protection mechanisms

Covalent modification is an important strategy for introducing new functions into proteins. As engineered proteins become more sophisticated, it is often desirable to introduce multiple, modifications involving several different functionalities in a site-specific manner. Such orthogonal labeling schemes require independent labeling of differentially reactive nucleophilic amino acid side chains. We have developed two protein-mediated protection schemes that permit independent labeling of multiple thiols. These schemes exploit metal coordination or disulfide bond formation to reversibly protect cysteines in a Cys(2)His(2) zinc finger domain. We constructed a variety of N- and C-terminal fusions of these domains with maltose-binding protein, which were labeled with two or three different fluorophores. Multiple modifications were made by reacting an unprotected cysteine in MBP first, deprotecting the zinc finger, and then reacting the zinc finger cysteines. The fusion proteins were orthogonally labeled with two different fluorophores, which exhibited intramolecular fluorescene resonance energy transfer (FRET). These conjugates showed up to a threefold ratiometric change in emission intensities in response to maltose binding. We also demonstrated that the metal- and redox-mediated protection methods can be combined to produce triple independent modifications, and prepared a protein labeled with three different fluorophores that exhibited a FRET relay. Finally, labeled glucose-binding protein was covalently patterned on glass slides using thiol-mediated immobilization chemistries. Together, these experiments demonstrated that reversible thiol protection schemes provide a rapid, straightforward method for producing multiple, site-specific modifications.     

Protein dynamics control proton transfer from bulk solvent to protein interior: a case study with a green fluorescent protein

The kinetics of proton transfer in Green Fluorescent Protein (GFP) have been studied as a model system for characterizing the correlation between dynamics and function of proteins in general. The kinetics in EGFP (a variant of GFP) were monitored by using a laser-induced pH jump method. The pH was jumped from 8 to 5 by nanosecond flash photolysis of the "caged proton," o-nitrobenzaldehyde, and subsequent proton transfer was monitored by following the decrease in fluorescence intensity. The modulation of proton transfer kinetics by external perturbants such as viscosity, pH, and subdenaturing concentrations of GdnHCl as well as of salts was studied. The rate of proton transfer was inversely proportional to solvent viscosity, suggesting that the rate-limiting step is the transfer of protons through the protein matrix. The rate is accelerated at lower pH values, and measurements of the fluorescence properties of tryptophan 57 suggest that the enhancement in rate is associated with an enhancement in protein dynamics. The rate of proton transfer is nearly independent of temperature, unlike the rate of the reverse process. When the stability of the protein was either decreased or increased by the addition of co-solutes, including the salts KCl, KNO(3), and K(2)SO(4), a significant decrease in the rate of proton transfer was observed in all cases. The lack of correlation between the rate of proton transfer and the stability of the protein suggests that the structure is tuned to ensure maximum efficiency of the dynamics that control the proton transfer function of the protein.     

Eider females form non-kin brood-rearing coalitions

Kin selection is a powerful tool for understanding cooperation among individuals, yet its role as the sole explanation of cooperative societies has recently been challenged on empirical grounds. These studies suggest that direct benefits of cooperation are often overlooked, and that partner choice may be a widespread mechanism of cooperation. Female eider ducks (Somateria mollissima) may rear broods alone, or they may pool their broods and share brood-rearing. Females are philopatric, and it has been suggested that colonies may largely consist of related females, which could promote interactions among relatives. Alternatively, shared brood care could be random with respect to relatedness, either because brood amalgamations are accidental and nonadaptive, or through group augmentation, assuming that the fitness of all group members increases with group size. We tested these alternatives by measuring the relatedness of co-tending eider females in enduring coalitions with microsatellite markers. Females formed enduring brood-rearing coalitions with each other at random with respect to relatedness. However, based on previous data, partner choice is nonrandom and dependent on female body condition. We discuss potential mechanisms underlying eider communal brood-rearing decisions, which may be driven by the specific ecological conditions under which sociality has evolved in this species.     

Extrusion and removal of lipid from the cytoplasm of porcine oocytes at the germinal vesicle stage: centrifugation under hypertonic conditions influences vitrification

In the present study, we examined a novel lipid removal method, centrifugation in solutions made hypertonic by adding 0.27 M sugar. This allowed the lipid to be extruded and removed without the loss of active mitochondria or extra cytoplasm. The type of sugar influenced the proportion of oocytes that could be stratified by centrifugation. Glucose induced the highest extrusion rate of lipid droplets. After vitrification the rates of survival, germinal vesicle breakdown and metaphase II were 30, 26, and 7%, respectively, for lipid-removed GV oocytes; this was significantly higher (P<0.05) than for corresponding vitrified lipid-intact oocytes (2, 0, and 0%, respectively). These results indicated that this method is useful to remove whole lipid droplets without losing mitochondria and improves cryotolerance of porcine GV oocytes.     

Oocyte nucleus controls progression through meiotic maturation

We analyzed progression through the meiotic maturation in oocytes manipulated to replace the prophase oocyte nucleus with the nucleus from a cumulus cell, a pachytene spermatocyte or the pronucleus from a fertilized egg. Removal of the oocyte nucleus led to a significant reduction in histone H1 kinase activity. Replacement of the oocyte nucleus by a pronucleus followed by culture resulted in premature pseudomeiotic division and occasional abnormal cytokinesis; however, histone H1 kinase activity was rescued, microtubules formed a bipolar spindle, and chromosomes were condensed. In addition to the anomalies observed after pronuclear transfer, those after transfer of the nucleus from a cumulus cell or spermatocyte included a dramatically impaired ability to form the bipolar spindle or to condense chromosomes, and histone H1 kinase activity was not rescued. Expression of a cyclin B-YFP in enucleated oocytes receiving the cumulus cell nucleus rescued histone H1 kinase activity, but spindle formation and chromosome condensation remained impaired, indicating a pleiotropic effect of oocyte nucleus removal. However, when the cumulus cell nucleus was first transformed into pronuclei (transfer into a metaphase II oocyte followed by activation), such pronuclei supported maturation after transfer into the oocyte in a manner similar to that of normal pronuclei. These results show that the oocyte nucleus contains specific components required for the control of progression through the meiotic maturation and that some of these components are also present in pronuclei.     

Oogenesis in Hydra: nurse cells transfer cytoplasm directly to the growing oocyte

Oogenesis in Hydra occurs in so-called egg patches containing several thousand germ cells. Only one oocyte is formed per egg patch; the remaining germ cells differentiate as nurse cells. Whether and how nurse cells contribute cytoplasm to the developing oocyte has been unclear. We have used tissue maceration to characterize the differentiation of oocytes and nurse cells in developing egg patches. We show that nurse cells decrease in size at the same time that developing oocytes increase dramatically in volume. Nurse cells are also tightly attached to oocytes at this stage and confocal images of egg patches stained with the fluorescent membrane dye FM 4-64 clearly show large gaps (10 microm) in the cell membranes separating nurse cells from the developing oocyte. We conclude that nurse cells directly transfer cytoplasm to the developing oocyte. Following this transfer of cytoplasm, nurse cells undergo apoptosis and are phagocytosed by the oocyte. These results demonstrate that basic mechanisms of alimentary oogenesis typical of Caenorhabditis and Drosophila are already present in the early metazoan Hydra.