Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and partial characterization of the b-type cytochrome from human polymorphonuclear leukocytes

Polymorphonuclear leukocytes contain an oxidase system that can be activated to produce superoxide radicals and hydrogen peroxide. A nonmitochondrial b cytochrome, functioning in the generation of these oxygen species, has been purified to apparent homogeneity from human polymorphonuclear phagocytes. After solubilization of the cytochrome with Triton X-100, the cell extract was subsequently chromatographed on Blue Sepharose and Sephacryl S-300. The final preparation was maximally purified 170-fold with a specific content of 5.33 +/- 2.03 nmol mg-1 of protein (mean +/- S.D.; n = 7) and a yield of 21 +/- 13% (n = 5). The apparent molecular mass of the nondenatured cytochrome was estimated by gel filtration to be 235 kDa. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single polypeptide was found with a molecular mass of 127 kDa. From the pyridine hemochrome spectrum 1 protoheme IX/polypeptide was calculated. The light absorbance bands of the dithionite-reduced cytochrome were found to be at 558.5 (alpha), 529 (beta), and 426 nm (Soret), and that of the oxidized cytochrome at 413.5 nm. The difference absorbance coefficients are delta epsilon (426.5 - 440 nm) = 160.6 +/- 11 mM-1 cm-1 and delta epsilon (558.5 - 542 nm) = 29.3 +/- 2 mM-1 cm-1 (mean +/- S.D.; n = 5). Carbon monoxide binds to the cytochrome in a time-dependent fashion (maximum binding after 50-60 min). The midpoint potential of the solubilized nonpurified cytochrome is identical to the cytochrome in situ (Em7.0 = -218 +/- 7 mV (mean +/- S.D.; n = 5)). However, purified cytochrome b shows a significantly decreased midpoint potential, estimated at -407 +/- 18 mV (n = 4). The protein does not contain noncovalently bound FAD or FMN, and no spectral evidence was obtained for the presence of covalently bound flavin. Preliminary amino acid analysis of the cytochrome shows a high content of hydrophilic residues.     

Phytoplankton dynamics and sedimentation processes during spring and summer in Balsfjord,Northern Norway

Summary Chlorophyll a, phytoplankton species composition and carbon (PPC) estimated from cell-counts, were monitored together with hydrographic parameters and nutrients in the upper 50 m of Balsfjord (ca. 70° N), northern Norway between 08 February and 29 June 1982. Sediment traps were placed at 10, 50, 100, and 170 m (10 m above bottom) for intervals of 5–20 days during the study period. Trap contents were analyzed for phytoplankton as above; dry weight, particulate organic material (POM), particulate organic nitrogen and carbon (PON and POC), ash, and particulate phosphorus were also measured. The phytoplankton community exhibited three main phases: During the first (02–15 April, chiefly surface biomass) and the second (20 April–10 May, deep biomass-maximum and spring bloom peak) periods, Phaeocystis pouchetii dominated biomass (ca. 50% of PPC) followed by vegetative cells of Chaetoceros socialis. In the third period (10 May onwards, characterized by surface estuarinecir-culation), dino- and microflagellates dominated the low post-bloom biomass. Protozooplankton comprising tintinnids, other ciliates and heterotrophic dinoflagellates increased in abundance. Vegetative cells of phytoplankton were scarce in trap collections at 50 m or below; resting cells of Chaetoceros comprised nearly all the intact sedimenting phytoplankton. Krill faeces accounted for >90% by volume of the total faecal material trapped, despite a >21 biomass dominance of copepods in the fjord. The greatest sedimentation rates of krill faeces were at > 100 m, reflecting the downward migration of krill during the day. In all, 2–3 g Cm^–2 of krill faeces were collected, representing ca. twice that from intact phytoplankton cells. POC in the traps at 50 m was ca. 11 gm^–2, accounting for ca. 17% of the estimated primary production during the study period. As the secondary production is high, a large proportion of the production of P. pouchetii must be grazed by herbivores. Copepod faeces are probably remineralized in the euphotic zone, while those of krill provide the major coupling between the pelagial and the benthos. The implications of such a sedimentation model for partitioning energy flow between the pelagial and the benthos is discussed.     

Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 6.5 A resolution.

ATP synthase, the assembly which makes ATP in mitochondria, chloroplasts and bacteria, uses transmembrane proton gradients generated by respiration or photosynthesis to drive the phosphorylation of ADP. Its membrane domain is joined by a slender stalk to a peripheral catalytic domain, F1-ATPase. This domain is made of five subunits with stoichiometries of 3 alpha: 3 beta: 1 gamma: 1 delta: 1 epsilon, and in bovine mitochondria has a molecular mass of 371,000. We have determined the 3-dimensional structure of bovine mitochondrial F1-ATPase to 6.5 A resolution by X-ray crystallography. It is an approximately spherical globule 110 A in diameter, on a 40 A stem which contains two alpha-helices in a coiled-coil. This stem is presumed to be part of the stalk that connects F1 with the membrane domain in the intact ATP synthase. A pit next to the stem penetrates approximately 35 A into the F1 particle. The stem and the pit are two examples of the many asymmetric features of the structure. The central element in the asymmetry is the longer of the two alpha-helices in the stem, which extends for 90 A through the centre of the assembly and emerges on top into a dimple 15 A deep. Features with threefold and sixfold symmetry, presumed to be parts of homologous alpha and beta subunits, are arranged around the central rod and pit, but the overall structure is asymmetric. The central helix provides a possible mechanism for transmission of conformational changes induced by the proton gradient from the stalk to the catalytic sites of the enzyme.     

A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo

The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of translation by mapping ribosomes with high resolution on a genome-wide scale. We present a variation on this approach that offers a rapid and cost-effective alternative for the genome-wide profiling of chloroplast ribosomes. Ribosome footprints from leaf tissue are hybridized to oligonucleotide tiling microarrays of the plastid ORFeome and report the abundance and translational status of every chloroplast mRNA. Each assay replaces several time-consuming traditional methods while also providing information that was previously inaccessible. To illustrate the utility of the approach, we show that it detects known defects in chloroplast gene expression in several nuclear mutants of maize (Zea mays) and that it reveals previously unsuspected defects. Furthermore, it provided firm answers to several lingering questions in chloroplast gene expression: (1) the overlapping atpB/atpE open reading frames, whose translation had been proposed to be coupled, are translated independently in vivo; (2) splicing is not a prerequisite for translation initiation on an intron-containing chloroplast RNA; and (3) a feedback control mechanism that links the synthesis of ATP synthase subunits in Chlamydomonas reinhardtii does not exist in maize. An analogous approach is likely to be useful for studies of mitochondrial gene expression.     

Ultra high risk status and transition to psychosis in 22q11.2 deletion syndrome

The 22q11.2 deletion syndrome (22q11DS) is characterized by high rates of psychotic symptoms and schizophrenia, making this condition a promising human model for studying risk factors for psychosis. We explored the predictive value of ultra high risk (UHR) criteria in a sample of patients with 22q11DS. We also examined the additional contribution of socio‐demographic, clinical and cognitive variables to predict transition to psychosis within a mean interval of 32.5 ± 17.6 months after initial assessment. Eighty‐nine participants with 22q11DS (age range: 8‐30 years; mean 16.1 ± 4.7) were assessed using the Structured Interview for Psychosis‐Risk Syndromes. Information on Axis I diagnoses, internalizing and externalizing symptoms, level of functioning and IQ was also collected. At baseline, 22 (24.7%) participants met UHR criteria. Compared to those without a UHR condition, they had a significantly lower functioning, more frequent anxiety disorders, and more severe psychopathology. Transition rate to psychosis was 27.3% in UHR and 4.5% in non‐UHR participants. Cox regression analyses revealed that UHR status significantly predicted conversion to psychosis. Baseline level of functioning was the only other additional predictor. This is the first study investigating the predictive value of UHR criteria in 22q11DS. It indicates that the clinical path leading to psychosis is broadly comparable to that observed in other clinical high‐risk samples. Nevertheless, the relatively high transition rate in non‐UHR individuals suggests that other risk markers should be explored in this population. The role of low functioning as a predictor of transition to psychosis should also be investigated more in depth.     

Forest biomass,soil and biodiversity relationships originate from biogeographic affinity and direct ecological effects

Ecosystem biomass, soil conditions and the diversity of different taxa are often interrelated. These relationships could originate from biogeographic affinity (varying species pools) or from direct ecological effects within local communities. Disentangling regional and local causes is challenging as the former might mask the latter in natural ecosystems with varying habitat conditions. However, when the species pool contribution is considered in statistics, local ecological effects might be detected. In this study we disentangle the indirect effects of the species pool and direct ecological effects on the complex relationships among wood volume, soil conditions and diversities of different plant and fungal groups in 100 old‐growth forest sites (10 × 10 m) at the border of boreal and nemoral zones in northern Europe. We recorded all species for different vegetation groups: woody and herbaceous vascular plants, terricolous and epiphytic bryophytes and lichens. Fungal communities were detected by DNA‐based analyses from soil samples. Above‐ground wood volume was used as a proxy of biomass. We measured soil pH and nutrient content and obtained modelled climate parameters for each site. Species pool effect was considered by dividing sites into boreal and nemoral groups based on community composition. In order to disentangle direct and indirect effects, we applied variation partitioning, and raw and partial correlations. We found many significant positive relationships among studied variables. Many of these relationships were associated to boreal and nemoral species pools, thus indicating that biogeographic affinity of interacting plants and fungi largely defines forest diversity and functioning. At the same time, several relationships were significant also after considering biogeography: woody plant and ectomycorrhizal fungi diversities with wood volume, many plant and fungal groups with each other, or with soil conditions. These direct ecological interactions could be considered in forestry practices to achieve both economic gain and maintenance of biodiversity.     

The molecular mechanism of induction of unfolded protein response by Chlamydia

The unfolded protein response (UPR) contributes to chlamydial pathogenesis, as a source of lipids and ATP during replication, and for establishing the initial anti-apoptotic state of host cell that ensures successful inclusion development. The molecular mechanism(s) of UPR induction by Chlamydia is unknown. Chlamydia use type III secretion system (T3SS) effector proteins (e.g, the Translocated Actin-Recruiting Phosphoprotein (Tarp) to stimulate host cell's cytoskeletal reorganization that facilitates invasion and inclusion development. We investigated the hypothesis that T3SS effector-mediated assembly of myosin-II complex produces activated non-muscle myosin heavy chain II (NMMHC-II), which then binds the UPR master regulator (BiP) and/or transducers to induce UPR. Our results revealed the interaction of the chlamydial effector proteins (CT228 and Tarp) with components of the myosin II complex and UPR regulator and transducer during infection. These interactions caused the activation and binding of NMMHC-II to BiP and IRE1α leading to UPR induction. In addition, specific inhibitors of myosin light chain kinase, Tarp oligomerization and myosin ATPase significantly reduced UPR activation and Chlamydia replication. Thus, Chlamydia induce UPR through T3SS effector-mediated activation of NMMHC-II components of the myosin complex to facilitate infectivity. The finding provides greater insights into chlamydial pathogenesis with the potential to identify therapeutic targets and formulations.     

A real-time PCR assay for DNA-methylation using methylation-specific blockers

DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.