Molecular Insights into Frataxin-Mediated Iron Supply for Heme Biosynthesis in Bacillus subtilis

Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in Bacillus subtilis and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme b biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen–deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the in vitro-generated heme b co-factor upon Fra-mediated iron transfer was confirmed by using the B. subtilis nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme b co-factor, hence representing a key player in the iron-dependent physiology of B. subtilis.     

Elucidation of the Interaction Mechanism with Liposomes of gH625-Peptide Functionalized Dendrimers

We have demonstrated that amide-based dendrimers functionalized with the membrane-interacting peptide gH625 derived from the herpes simplex virus type 1 (HSV-1) envelope glycoprotein H enter cells mainly through a non-active translocation mechanism. Herein, we investigate the interaction between the peptide-functionalized dendrimer and liposomes composed of PC/Chol using fluorescence spectroscopy, isothermal titration calorimetry, and surface plasmon resonance to get insights into the mechanism of internalization. The affinity for the membrane bilayer is very high and the interaction between the peptide-dendrimer and liposomes took place without evidence of pore formation. These results suggest that the presented peptidodendrimeric scaffold may be a promising material for efficient drug delivery.     

An optimal control model for maximum-height human jumping

To understand how intermuscular control, inertial interactions among body segments, and musculotendon dynamics coordinate human movement, we have chosen to study maximum-height jumping. Because this activity presents a relatively unambiguous performance criterion, it fits well into the framework of optimal control theory. The human body is modeled as a four-segment, planar, articulated linkage, with adjacent links joined together by frictionless revolutes. Driving the skeletal system are eight musculotendon actuators, each muscle modeled as a three-element, lumped-parameter entity, in series with tendon. Tendon is assumed to be elastic, and its properties are defined by a stress-strain curve. The mechanical behavior of muscle is described by a Hill-type contractile element, including both series and parallel elasticity. Driving the musculotendon model is a first-order representation of excitation-contraction (activation) dynamics. The optimal control problem is to maximize the height reached by the center of mass of the body subject to body-segmental, musculotendon, and activation dynamics, a zero vertical ground reaction force at lift-off, and constraints which limit the magnitude of the incoming neural control signals to lie between zero (no excitation) and one (full excitation). A computational solution to this problem was found on the basis of a Mayne-Polak dynamic optimization algorithm. Qualitative comparisons between the predictions of the model and previously reported experimental findings indicate that the model reproduces the major features of a maximum-height squat jump (i.e. limb-segmental angular displacements, vertical and horizontal ground reaction forces, sequence of muscular activity, overall jump height, and final lift-off time).     

Role of spermatozoal platelet-activating factor in fertilization

Platelet-activating factor (PAF), a potent lipid mediator of inflammation, has been shown to play a role in both the implantation and viability of mammalian embryos. We examined whether human and mouse spermatozoa release PAF during in vitro incubation and assessed the effect of exogenous PAF and the PAF receptor antagonist WEB 2086, a thieno-triazolodiazepine, on mouse in vitro fertilization (IVF) rate. PAF biological activity was detected in 11 samples of leukocyte-free, purified human spermatozoa (28 pg PAF/10(6) cells/24 hr) and 5 samples of epididymal mouse spermatozoa (7.8 pg PAF/10(6) cells/3 hr). Exogenous PAF (10(-8) and 10(-6) M) increased (p less than 0.01) the fertilization rate 2- and 3-fold, respectively of mouse oocytes by mouse epididymal spermatozoa. 10(-4) M PAF, however, reduced sperm motility and decreased (p less than 0.05) the fertilization rate. 10(-6) M WEB 2086, decreased IVF to approximately 50% of the control fertilization rate (42% vs. 89%). WEB 2086 treatment also promoted the attachment of supernumerary spermatozoa to both fertilized and unfertilized oocytes. The fertilization rate in the presence of WEB 2086 returned to control levels when zona-pellucida-free oocytes were employed, indicating that WEB 2086 did not interfere with the spermatozoal acrosome reaction. These data suggest that PAF, of spermatozoal origin, may be important in mammalian fertilization.     

DNase I sensitivity in facultative and constitutive heterochromatin

In situ nick translation allows the detection of DNase I sensitive and insensitive regions in fixed mammalian mitotic chromosomes. We have determined the difference in DNase I sensitivity between the active and inactive X chromosomes inMicrotus agrestis (rodent) cells, along both their euchromatic and constitutive heterochromatic regions. In addition, we analysed the DNase I sensitivity of the constitutive heterochromatic regions in mouse chromosomes. InMicrotus agrestis female cells the active X chromosome is sensitive to DNase I along its euchromatic region while the inactive X chromosome is insensitive except for an early replicating region at its distal end. The late replicating constitutive heterochromatic regions, however, in both the active and inactive X chromosome are sensitive to DNase I. In mouse cells on the other hand, the constitutive heterochromatin is insensitive to DNase I both in mitotic chromosomes and interphase nuclei.     

DNA hypomethylation causes an increase in DNase-I sensitivity and an advance in the time of replication of the entire inactive X chromosome

Summary We have examined the effect of 5-azacytidine (5-aza-C) induced hypomethylation of DNA on the time of replication and DNase I sensitivity of the X chromosomes of female Gerbillus gerbillus (rodent) lung fibroblast cells. Using in situ nick translation to visualise the potential state of activity of large regions of metaphase chromosomes we show that 5-aza-C causes a dramatic increase in the DNase-I sensitivity of the entire inactive X chromosome of female G. gerbillus cells and this increase in nuclease sensitivity correlates with a large shift in the time of replication of the inactive X chromosome from late S phase to early S phase. These effects of 5-aza-C on the inactive X chromosome are associated with a 15% decrease in DNA methylation. Our results indicate that DNA methylation concomitantly affects both the time of replication and the chromatin conformation of the inactive X chromosome.     

Comparison of classical Lowry, modified Lowry and a dye-binding assay for the estimation of protein in allergen extracts and influence of different parameters on the modified Lowry assay

Protein values of dialysed allergen extracts determined by Lowry, modified Lowry (trichloroacetic acid precipitation of the proteins) and dye-binding assay were compared. The influence of different parameters on the modified Lowry was examined. The reproducibility of the modified Lowry was checked with three independent measurements. For the examination of recovery a constant amount of 6-grass pollen allergen proteins was added to the samples of the standardized human serum albumin prepared for the calibration curve. The samples were measured by modified Lowry. The mean of the ratio between the protein values of the dialysed allergen extracts obtained by modified Lowry and those obtained by classical Lowry was 3.59 (coefficient of variation Cv = 45%). The mean of the ratio between the protein values of the allergen extracts obtained by modified Lowry and dye-binding assay was 1:0.71 (Cv = 31%). Phenol interfered with the modified Lowry. Phenolic allergen extracts showed higher "protein values" than non-phenolic allergen extracts. This influence could be reduced by a second precipitation of the dissolved precipitate. The precipitation of non-phenolic dialysed aqueous allergen extracts was complete after the first trichloroacetic acid precipitation. By incubating samples with the Folin-Ciocalteu's reagent at 55 degrees C in a waterbath, the time necessary for developing the colour could be reduced from 45 min to 5 min. Protein measurements by modified Lowry of a 6-grass pollen allergen extract in three different laboratories showed good reproducibility. For these extract 785 micrograms protein/ml (Cv = 4%) could be measured.(ABSTRACT TRUNCATED AT 250 WORDS)