Functional characterization of generalized Langevin equations

We present an exact functional formalism to deal with linear Langevin equations with arbitrary memory kernels and driven by any noise structure characterized through its characteristic functional. No others hypothesis are assumed over the noise, neither the fluctuation dissipation theorem. We found that the characteristic functional of the linear process can be expressed in terms of noise's functional and the Green function of the deterministic (memory-like) dissipative dynamics. This object allow us to get a procedure to calculate all the Kolmogorov hierarchy of the non-Markov process. As examples we have characterized through the 1-time probability a noise-induced interplay between the dissipative dynamics and the structure of different noises. Conditions that lead to non-Gaussian statistics and distributions with long tails are analyzed. The introduction of arbitrary fluctuations in fractional Langevin equations have also been pointed out

Functional characterization of hexokinases in the moss Physcomitrella patens

Carbohydrates are important nutrients and structural components in all living organisms. In plants they affect the developmental and metabolic processes throughout the plant life cycle. However, the mechanisms by which plants recognise and respond to carbohydrates are mainly unknown. Hexokinase, an enzyme that mediates the first catalytic step in hexose metabolism, has recently been suggested to be involved in sugar sensing and signalling in plants. The moss Physcomitrella patens has recently emerged as a powerful model system in plant functional genomics following the discovery that gene targeting works in it with frequencies comparable to those in yeast. The aim of this thesis was to learn more about the function of plant hexokinases, both as key metabolic enzymes and in their putative role as sensors in sugar signalling, using Physcomitrella patens as a model system. Five hexokinases from Physcomitrella patens were cloned and studied with respect to their subcellular localizations. PpHxk1 and PpHxk5 are located in the stroma of chloroplasts and are dependent on N-terminal transit peptides for correct localization. PpHxk2 and PpHxk3 both contain hydrophobic membrane anchors that localize the proteins to the outer envelope of chloroplasts. PpHxk4 contain neither a transit peptide nor an anchor, and is found in the cytosol. A targeted knockout revealed that PpHxk1 is the major hexokinase in Physcomitrella, accounting for 80% of the glucose phosphorylating activity. Consistent with this, the knockout mutant exhibits an artificial starvation phenotype with altered sensitivities to plant hormones and a disturbed development

Functional characterization of orbicularis oculi and extraocular muscles

The orbicularis oculi are the sphincter muscles of the eyelids and are involved in modulating facial expression. They differ from both limb and extraocular muscles (EOMs) in their histology and biochemistry. Weakness of the orbicularis oculi muscles is a feature of neuromuscular disorders affecting the neuromuscular junction, and weakness of facial muscles and ptosis have also been described in patients with mutations in the ryanodine receptor gene. Here, we investigate human orbicularis oculi muscles and find that they are functionally more similar to quadriceps than to EOMs in terms of excitation-contraction coupling components. In particular, they do not express the cardiac isoform of the dihydropyridine receptor, which we find to be highly expressed in EOMs where it is likely responsible for the large depolarization-induced calcium influx. We further show that human orbicularis oculi and EOMs express high levels of utrophin and low levels of dystrophin, whereas quadriceps express dystrophin and low levels of utrophin. The results of this study highlight the notion that myotubes obtained by explanting satellite cells from different muscles are not functionally identical and retain the physiological characteristics of their muscle of origin. Furthermore, our results indicate that sparing of facial and EOMs in patients with Duchenne muscular dystrophy is the result of the higher levels of utrophin expression

Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones

Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for,10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e. g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup [1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 A crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5

Functional characterization of infiltrating T lymphocytes in human hepatic allografts

We have employed recently developed techniques in T-cell culturing to study the nature and function of infiltrating hepatic allograft T cells. Using the rationale that intragraft T cells are activated during cell mediated damage to the allograft, we were able to show that these cells would propagate and remain functionally active in the presence of the T-cell growth factor, IL-2. In several instances, phenotyiic analysis of cells grown in this manner was very similar to that found within the graft. Both proliferative and cytotoxic responses could be detected from the cultured cell lines. The majority of the proliferative responses were donor-directed and immunogenetic analysis could define donor-directed HLA reactivity, to either class I or class II antigens, or both. Monoclonal anti-HLA antibodies inhibition profiles verified the apparent HLA reactivity. In a smaller percentage of cases, only IL-2 responsiveness could be detected, and no HLA reactivity could be determined. Cytotoxicity could be detected against both class I and class II antigens, however, those cells which demonstrated a greater magnitude of donor-directed cytotoxicity appeared to be directed against class I antigens. A significant correlation between donor-directed proliferation of biopsy cultured lymphocytes and cellular rejection was found. This model appears to be useful in delineating functions of the intragraft T-cell population during rejection. © 1986

Functional characterization of synthetic leukotriene B and its stereochemical isomers.

Leukotriene B (LTB), a potent lipid chemotactic factor for neutrophils, is 5S,12R-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid (Fig 1), based upon direct comparison of natural LTB with synthetic 5S,12R-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-di-HETE) stereoisomers in three biological assays. Of the six synthetic stereoisomers evaluated, only the 5S,12R,6,14-cis,8,10-trans compound had chemotactic potency for human neutrophils in vitro that was comparable to that of natural LTB, with a concentration of 3 X 10(9-9) M eliciting a one-half maximum response. In contrast, the racemic mixture of 5R,12R- and 5S,12S-6,10-trans,8,14-cis, the racemic mixture of 5S,12R- and 5R,12S-6,10-trans,8,14-cis, the 5S,12R-6,8-trans,10,14-cis, the 5S,12R-6,8,10-trans,14-cis, and the 5S,12S-6,8,10-trans,14-cis stereoisomers required concentrations of 3 X 10(-7) to 1 X 10(-6) M to elicit comparable responses. Only natural LTB and its synthetic counterpart elicited a local neutrophil infiltration when injected into the skin of the rhesus monkey at 10 ng and 100 ng per site. Natural and synthetic LTB at a concentration of 3 X 10(-8) M each provoked an EC25 contractile response of guinea pig pulmonary parenchymal strips in vitro, whereas the other four tested stereoisomers of 5,12-di-HETE were inactive at this concentration. Structure-function analyses suggest that the neutrophil chemotactic activity depends critically upon the C-1 to C-12 domain, including the stereochemistry of the 6-,8-,and 10-olefinic bonds and the presence of both hydroxyl groups

Functional Characterization of the Eukaryotic Cysteine Desulfurase Nfs1p from Saccharomyces cerevisiae

Previous studies have indicated that the essential protein Nfs1 performs a crucial role in cellular iron-sulfur (Fe/S) protein maturation. The protein is located predominantly in mitochondria, yet low amounts are present in cytosol and nucleus. Here we examined several aspects concerning the molecular function of yeast Nfs1p as a model protein. First, we demonstrated that purified Nfs1p facilitates the in vitro assembly of Fe/S proteins by using cysteine as its specific substrate. Thus, eukaryotic Nfs1 is a functional orthologue of the bacterial cysteine desulfurase IscS. Second, we showed that only the mitochondrial version but not the extramitochondrial version of Nfs1p is functional in generating cytosolic and nuclear Fe/S proteins. Mutation of the nuclear targeting signal of Nfs1p did not affect the maturation of cytosolic and nuclear Fe/S proteins, despite a severe growth defect under this condition. Nfs1p could not assemble an Fe/S cluster on the Isu scaffold proteins when they were located in the yeast cytosol. The lack of function of these central Fe/S cluster assembly components suggests that the maturation of extramitochondrial Fe/S protein does not involve functional copies of the mitochondrial Fe/S cluster assembly machinery in the yeast cytosol. Third, the extramitochondrial version of Nfs1p was shown to play a direct role in the thiomodification of tRNAs. Finally, we identified a highly conserved N-terminal {beta}-sheet of Nfs1p as a functionally essential part of the protein. The implication of these findings for the structural stability of Nfs1p and for its targeting mechanism to mitochondria and cytosol/nucleus will be discussed