Enhancement of oxygen mass transfer in stirred bioreactors using oxygen-vectors. 1. Simulated fermentation broths

Oxygen mass transfer represents the most important parameter involved in the design and operation of mixing-sparging equipment for bioreactors. It can be described and analyzed by means of the mass transfer coefficient, k_La. The k_La values are affected by many factors such as geometrical and operational characteristics of the vessels, media composition, type, concentration and microorganism morphology, and biocatalysts properties. The efficiency of oxygen transfer could be enhanced by adding oxygen-vectors in broths, such as hydrocarbons or fluorocarbons, without increasing the energy consumption for mixing or aeration. The experimental results obtained for simulated broths indicated a considerable increase of k_La in the presence of n-dodecane, and the existence of a certain value of n-dodecane concentration that corresponds to a maximum mass transfer rate of oxygen. The magnitude of the positive effect of n-dodecane depends both on the broths characteristics and operational conditions of the bioreactor.Notation d stirrer diameter, mm - d oxygen electrode diameter, mm - D bioreactor diameter, mm - h distance from the inferior stirrer to the bioreactor bottom, mm - H bioreactor height, mm - k_La oxygen mass transfer coefficient, s^-1 - l impeller blade length, mm - I oxygen electrode immersed length, mm - P power consumption for mixing of non-aerated broths, W - P_a power consumption for mixing of aerated broths, W - (P_a/V) specific power input, W/m³ - s baffle width, mm - v_S superficial air velocity, m/s - V volume of medium, m³ - w impeller blade height, mm - volumetric fraction of oxygen-vector - _a apparent viscosity, Pa*s - density, kg/m³     

Immunologically induced,complement-dependent up-regulation of the prion protein in the mouse spleen: follicular dendritic cells versus capsule and trabeculae

The expression of the prion protein (PrP) in the follicular dendritic cell network of germinal centers in the spleen is critical for the splenic propagation of the causative agent of prion diseases. However, a physiological role of the prion protein in the periphery remains elusive. To investigate the role and function of PrP expression in the lymphoid system we treated naive mice i.v. with preformed immune complexes or vesicular stomatitis virus. Immunohistochemistry and Western blot analysis of the spleen revealed that 8 days after immunization, immune complexes and vesicular stomatitis virus had both induced a strong increase of PrP expression in the follicular dendritic cell network. Remarkably, this up-regulation did not occur in mice that lack an early factor of the complement cascade, C1q, a component which has been shown previously to facilitate early prion pathogenesis. In addition to the variable PrP level in the germinal centers, we detected steady and abundant PrP expression in the splenic capsule and trabeculae, which are structural elements that have not been associated before with PrP localization. The abundant trabeculo-capsular PrP expression was also evident in spleens of Rag-1-deficient mice, which have been shown before to be incapable of prion expansion. We conclude that trabeculocapsular PrP is not sufficient for splenic prion propagation. Furthermore, our observations may provide important clues for a physiological function of the prion protein and allow a new view on the role of complement and PrP in peripheral prion pathogenesis.     

Biosynthesis,glycosylation, and enzymatic processing in vivo of human tripeptidyl-peptidase I

Human tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal serine protease that removes tripeptides from the free N termini of small polypeptides and also shows a minor endoprotease activity. Due to various naturally occurring mutations, an inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2). In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells. Human TPP I was initially identified as a single precursor polypeptide of approximately 68 kDa, which, within a few hours, was converted to the mature enzyme of approximately 48 kDa. Compounds affecting the pH of intracellular acidic compartments, those interfering with the intracellular vesicular transport as well as inhibition of the fusion between late endosomes and lysosomes by temperature block or 3-methyladenine, hampered the conversion of TPP I proenzyme into the mature form, suggesting that this process takes place in lysosomal compartments. Digestion of immunoprecipitated TPP I proenzyme with both N-glycosidase F and endoglycosidase H as well as treatment of the cells with tunicamycin reduced the molecular mass of TPP I proenzyme by approximately 10 kDa, which indicates that all five potential N-glycosylation sites in TPP I are utilized. Mature TPP I was found to be partially resistant to endo H treatment; thus, some of its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser(475) on human TPP I maturation in cultured cells demonstrated that although TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo.     

Identification of a functionally critical protein kinase C phosphorylation residue of cardiac troponin T

Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-alpha or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-alpha or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt.     

Application of singular value decomposition to the analysis of time-resolved macromolecular x-ray data

Singular value decomposition (SVD) is a technique commonly used in the analysis of spectroscopic data that both acts as a noise filter and reduces the dimensionality of subsequent least-squares fits. To establish the applicability of SVD to crystallographic data, we applied SVD to calculated difference Fourier maps simulating those to be obtained in a time-resolved crystallographic study of photoactive yellow protein. The atomic structures of one dark state and three intermediates were used in qualitatively different kinetic mechanisms to generate time-dependent difference maps at specific time points. Random noise of varying levels in the difference structure factor amplitudes, different extents of reaction initiation, and different numbers of time points were all employed to simulate a range of realistic experimental conditions. Our results show that SVD allows for an unbiased differentiation between signal and noise; a small subset of singular values and vectors represents the signal well, reducing the random noise in the data. Due to this, phase information of the difference structure factors can be obtained. After identifying and fitting a kinetic mechanism, the time-independent structures of the intermediates could be recovered. This demonstrates that SVD will be a powerful tool in the analysis of experimental time-resolved crystallographic data.     

Targeting presenilin-type aspartic protease signal peptide peptidase with gamma-secretase inhibitors

Presenilin is implicated in the pathogenesis of Alzheimer's disease. It is thought to constitute the catalytic subunit of the gamma-secretase complex that catalyzes intramembrane cleavage of beta-amyloid precursor protein, the last step in the generation of amyloidogenic Abeta peptides. The latter are major constituents of amyloid plaques in the brain of Alzheimer's disease patients. Inhibitors of gamma-secretase are considered potential therapeutics for the treatment of this disease because they prevent production of Abeta peptides. Recently, we discovered a family of presenilin-type aspartic proteases. The founding member, signal peptide peptidase, catalyzes intramembrane cleavage of distinct signal peptides in the endoplasmic reticulum membrane of animals. In humans, the protease plays a crucial role in the immune system. Moreover, it is exploited by the hepatitis C virus for the processing of the structural components of the virion and hence is an attractive target for anti-infective intervention. Signal peptide peptidase and presenilin share identical active site motifs and both catalyze intramembrane proteolysis. These common features let us speculate that gamma-secretase inhibitors directed against presenilin may also inhibit signal peptide peptidase. Here we demonstrate that some of the most potent known gamma-secretase inhibitors efficiently inhibit signal peptide peptidase. However, we found compounds that showed higher specificity for one or the other protease. Our findings highlight the possibility of developing selective inhibitors aimed at reducing Abeta generation without affecting other intramembrane-cleaving aspartic proteases.     

The promiscuity of heterospecific lox sites increases dramatically in the presence of palindromic DNA

Heterospecific lox sites are mutated lox sites that in the presence of Cre recombinase recombine with themselves but not or much less with wildtype loxP. We here show that in Escherichia coli both lox511 and lox2272 sites become highly promiscuous with respect to loxP when in the presence of Cre one of the recombination partners is present in a larger stretch of an inverted repeat of non-lox DNA. In such a palindromic DNA configuration, also the occurrence of other DNA repeat-mediated recombination events is somewhat increased in the presence of Cre. The results indicate that in recombinase mediated cassette exchange or other double lox applications based on the exclusivity of heterospecific lox sites, or in research combining Cre-lox approaches with hairpin RNA for gene silencing, the presence of duplicated DNA around lox sites has to be taken into account. It is proposed that the presence of palindromic non-lox DNA interferes with the homology search of the Cre enzyme prior to the actual recombination event.     

Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41

The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36(Mut(e,g)) and N36(Mut(a,d)), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36(Mut(e,g)) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36(Mut(a,d)) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36(Mut(a,d)) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628-661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36(Mut(e,g)) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC(50) approximately 308 nm versus approximately 16 microm). These results indicate that N36(Mut(e,g)) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the pre-hairpin intermediate to form heterotrimers. Thus N36(Mut(e,g)) represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36(Mut(e,g)) with the pre-hairpin intermediate is presented.     

Requirements for signal peptide peptidase-catalyzed intramembrane proteolysis

The presenilin-type aspartic protease signal peptide peptidase (SPP) can cleave signal peptides within their transmembrane region. SPP is essential for generation of signal peptide-derived HLA-E epitopes in humans and is exploited by Hepatitis C virus for processing of the viral polyprotein. Here we analyzed requirements of substrates for intramembrane cleavage by SPP. Comparing signal peptides that are substrates with those that are not revealed that helix-breaking residues within the transmembrane region are required for cleavage, and flanking regions can affect processing. Furthermore, signal peptides have to be liberated from the precursor protein by cleavage with signal peptidase in order to become substrates for SPP. We propose that signal peptides require flexibility in the lipid bilayer to exhibit an accessible peptide bond for intramembrane proteolysis.