Breaking up and making up: The secret life of the vacuolar H+‐ATPase

The vacuolar ATPase (V‐ATPase; V₁V_o‐ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin‐coated vesicles. V‐ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V‐ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V‐ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high‐resolution structure determination by cryo electron microscopy and X‐ray crystallography together with sophisticated single‐molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V‐ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.     

Foldon-guided self-assembly of ultra-stable protein fibers

A common objective in protein engineering is the enhancement of the thermodynamic properties of recombinant proteins for possible applications in nanobiotechnology. The performance of proteins can be improved by the rational design of chimeras that contain structural elements with the desired properties, thus resulting in a more effective exploitation of protein folds designed by nature. In this paper, we report the design and characterization of an ultra-stable self-refolding protein fiber, which rapidly reassembles in solution after denaturation induced by harsh chemical treatment or high temperature. This engineered protein fiber was constructed on the molecular framework of bacteriophage P22 tail needle gp26, by fusing its helical core to the foldon domain of phage T4 fibritin. Using protein engineering, we rationally permuted the foldon upstream and downstream from the gp26 helical core and characterized gp26-foldon chimeras by biophysical analysis. Our data demonstrate that one specific protein chimera containing the foldon immediately downstream from the gp26 helical core, gp26(1-140)-F, displays the highest thermodynamic and structural stability and refolds spontaneously in solution following denaturation. The gp26-foldon chimeric fiber remains stable in 6.0 M guanidine hydrochloride, or at 80 degrees C, rapidly refolds after denaturation, and has both N and C termini accessible for chemical/biological modification, thereby representing an ideal platform for the design of self-assembling nanoblocks.     

Scanning irradiation device for mice in vivo with pulsed and continuous proton beams

A technical set-up for irradiation of subcutaneous tumours in mice with nanosecond-pulsed proton beams or continuous proton beams is described and was successfully used in a first experiment to explore future potential of laser-driven particle beams, which are pulsed due to the acceleration process, for radiation therapy. The chosen concept uses a microbeam approach. By focusing the beam to approximately 100 × 100 μm(2), the necessary fluence of 10(9) protons per cm(2) to deliver a dose of 20 Gy with one-nanosecond shot in the Bragg peak of 23 MeV protons is achieved. Electrical and mechanical beam scanning combines rapid dose delivery with large scan ranges. Aluminium sheets one millimetre in front of the target are used as beam energy degrader, necessary for adjusting the depth-dose profile. The required procedures for treatment planning and dose verification are presented. In a first experiment, 24 tumours in mice were successfully irradiated with 23 MeV protons and a single dose of 20 Gy in pulsed or continuous mode with dose differences between both modes of 10%. So far, no significant difference in tumour growth delay was observed.     

Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination

Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ～14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.     

Genome-Wide Expression Analysis in Fibroblast Cell Lines from Probands with Pallister Killian Syndrome

Pallister Killian syndrome (OMIM: # 601803) is a rare multisystem disorder typically caused by tissue limited mosaic tetrasomy of chromosome 12p (isochromosome 12p). The clinical manifestations of Pallister Killian syndrome are variable with the most common findings including craniofacial dysmorphia, hypotonia, cognitive impairment, hearing loss, skin pigmentary differences and epilepsy. Isochromosome 12p is identified primarily in skin fibroblast cultures and in chorionic villus and amniotic fluid cell samples and may be identified in blood lymphocytes during the neonatal and early childhood period. We performed genomic expression profiling correlated with interphase fluorescent in situ hybridization and single nucleotide polymorphism array quantification of degree of mosaicism in fibroblasts from 17 Caucasian probands with Pallister Killian syndrome and 9 healthy age, gender and ethnicity matched controls. We identified a characteristic profile of 354 (180 up- and 174 down-regulated) differentially expressed genes in Pallister Killian syndrome probands and supportive evidence for a Pallister Killian syndrome critical region on 12p13.31. The differentially expressed genes were enriched for developmentally important genes such as homeobox genes. Among the differentially expressed genes, we identified several genes whose misexpression may be associated with the clinical phenotype of Pallister Killian syndrome such as downregulation of ZFPM2, GATA6 and SOX9, and overexpression of IGFBP2.     

Low-Dimensional Dynamics in Sensory Biology 1: Thermally Sensitive Electroreceptors of the Catfish

We report the results of a search for evidence of periodic unstableorbits in the electroreceptors of the catfish. The function of thesereceptor organs is to sense weak external electric fields. Inaddition, they respond to the ambient temperature and to the ioniccomposition of the water. These quantities are encoded by receptorsthat make use of an internal oscillator operating at the level of themembrane potential. If such oscillators have three or more degreesof freedom, and at least one of which also exhibits a nonlinearity,they are potentially capable of chaotic dynamics. By detecting theexistence of stable and unstable periodic orbits, we demonstratebifurcations between noisy stable and chaotic behavior using theambient temperature as a parameter. We suggest that the techniquedeveloped herein be regarded as an additional tool for the analysisof data in sensory biology and thus can be potentially useful instudies of functional responses to external stimuli. We speculatethat the appearance of unstable orbits may be indicative of a stateof heightened sensory awareness by the animal.     

Selectivity of the surface binding site (SBS) on barley starch synthase I

Starch synthase I (SSI) from various sources has been shown to preferentially elongate branch chains of degree of polymerisation (DP) from 6–7 to produce chains of DP 8–12. In the recently determined crystal structure of barley starch synthase I (HvSSI) a so-called surface binding site (SBS) was seen, which was found by mutational analysis to be essential for the activity of HvSSI on glycogen. We now show in binding studies using surface plasmon resonance that HvSSI has no detectable affinity for malto-triose and -tetraose, but clearly binds maltopentaose, -hexaose, -heptaose (M7) and β-cyclodextrin (β-CD) albeit with a measurable K _D for only β-CD and M7. Moreover, an HvSSI SBS mutant F538A lost the ability to bind β-CD and maltooligosaccharides. This behaviour suggests that a chain in the α-glucan molecule (amylopectin) that is undergoing extension attaches itself at the SBS and that the active site itself, likely working on a different end chain, has low affinity for both substrate and product.     

Structure of theEscherichia coli ATP synthase and role of the γ and ε subunits in coupling catalytic site and proton channeling functions

The structure of theEscherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the and subunits of the F₁ part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 Å at its widest point. The and subunits are interdigitated in side view for around 60 Å of the 90 Å length of the molecule. The F₁ narrows and has three-fold symmetry at the end furthest from the F₀ part. The F₁ is linked to F₀ by a stalk approximately 45 Å long and 25–30 Å in diameter. The F₀ part is mostly buried in the lipid bilayer. The subunit provides a domain that extends into the central cavity of the F₁ part. The and subunits are in a different conformation when ATP+Mg²⁺ are present in catalytic sites than when ATP+EDTA are present. This is consistent with these two small subunits switching conformations as a function of whether or not phosphate is bound to the enzyme at the position of the phosphate of ATP. We suggest that this switching is the key to the coupling of catalytic site events with proton translocation in the F₀ part of the complex.     

<Emphasis Type="Italic">Oswaldocruzia lanfrediae</Emphasis> n. sp. (Strongylida: Molineidae), a parasite of <Emphasis Type="Italic">Leptodactylus paraensis</Emphasis> Heyer (Anura: Leptodactylidae) in Brazil

Leptodactylus paraensis Heyer, is a Neotropical anuran species that inhabits Rainforest habitats in the eastern Amazon, but because it has only been recently separated from the Leptodactylus pentadactylus (Laurenti) species group, little is known about its helminth fauna. This study describes a new species of Oswaldocruzia Travassos, 1917 and records the first occurrence of this genus parasitising L. paraensis and the second species for the Caxiuanã National Forest in the eastern Amazon, Brazil. Oswaldocruzia lanfrediae n. sp. is characterised by having an anterior extremity with a smooth cephalic vesicle divided into two portions, a claviform oesophagus, well-developed cuticular longitudinal ridges and lateral alae. Females have a well-developed ovojector, with didelphic and amphidelphic uteri. Males show complex robust spicules divided into a slightly curved shoe, a bifurcated fork and a blade terminating in 2–3 processes. The new species differs from its congeners especially regarding the lateral alae and the morphology of the spicules, in addition to morphometric characters such as body size, oesophagus length, deirid position, nerve-ring position and relative position of the vulva in females.