Molecular cloning and structural analysis of the phosphate translocator from pea chloroplasts and its comparison to the spinach phosphate translocator

Using an 5-AvaII fragment of the spinach (Spinacia oleracea L.) phosphate translocator cDNA as a probe for a hybridization screening of a pea (Pisum sativum L.) cDNA library we have cloned and sequenced a cDNA clone coding for the phosphate translocator precursor protein from pea chloroplasts. The full-length cDNA clone comprises 42 base pairs (bp) at the 5-non-coding region, a 1206-bp coding region corresponding to a polypeptide of 402 amino-acid residues (relative molecular mass 43 671) and 244 bp at the non-coding 3-region. Determination of the N-terminal sequence of the phosphate translocator from both pea and spinach chloroplasts revealed that the transit peptides consist of 72 and 80 amino-acid residues, respectively. These transit peptides are different from those of other chloroplastic transit peptides in that they both contain an amphiphilic -helix which is located either in close proximity to the processing site in pea or at the N-terminus in spinach. The mature proteins from pea and spinach both contain about 87% identical amino-acid residues and about seven putative membrane-spanning -helices. Some of these -helices have an amphiphilic character and might serve to form a hydrophilic translocation channel through the membrane. The in-vitro synthesized pea precursor protein is directed to the chloroplast and inserted into the chloroplast envelope membrane.Abbreviations bp base pairs - kDa kilodaltons - M_r relative moleculas mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We wish to thank Dr D. Pappin and R. Jakes (AFRC Sequencing Laboratory, Department of Biochemistry, University of Leeds, UK) for performing the N-terminal sequence determinations and are greatful to Dr J. S. Gantt (Botany Department, University of Georgia, Athens, USA) for a pea leaf cDNA library and to Professor J. C. Gray (University of Cambridge, Department of Botany, Cambridge, UK) for helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Science and Engineering Research Council and the Royal Society. D.L.W. was the recipient of the Royal Society Rosenheim research fellowship and K.F. was supported by a fellowship from the Studienstiftung des deutschen Volkes.     

Histones inhibit human chorionic gonadotrophin-stimulated but not atrial peptide-stimulated testosterone production and cyclic nucleotide formation by isolated mouse Leydig cells

Recently it has been reported that histone type H2A can inhibit gonadotrophin-stimulated cAMP formation and steroidogenesis by ovarian cells. In the present study we have investigated if similar antigonadotrophic effects of commercially available histones can also be demonstrated on testicular steroidogenic cells. Using percoll-purified mouse Leydig cells, we have demonstrated that several types of histones could almost completely inhibit hCG-stimulated testosterone production and cAMP formation. The inhibition was dose-dependent and could be reversed by the addition of excess of hCG. The most potent histone types were H2AS and H8S, both of which could inhibit hCG-stimulated cAMP formation half-maximally at concentrations of 4-5 micrograms/ml. Forskolin-stimulated cAMP formation was not affected by histones. When the cells were stimulated with either db-cAMP or rAP-II, histone H2AS and H8S failed to inhibit the testosterone production. In fact there was a marked increase in the amount of testosterone produced, the reason for which is not yet understood. The amount of cGMP accumulated in response to rAP-II was not affected by the presence of H2AS or H8S. In unstimulated cells, neither the cyclic nucleotide level nor the amount of steroid produced was affected by the histones. Based on the [125I]hCG binding data it is possible to conclude that histone H2AS inhibits the binding of hCG to its receptors on Leydig cells and thereby causes the inhibition of hCG-stimulated cAMP formation and steroidogenesis.     

Functional interaction of constant and variable domains of human immunodeficiency virus type 1 gp120.

A previously reported amino acid substitution within the second conserved domain of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope results in the production of noninfectious particles. Molecular characterization of spontaneous revertant viruses, which arose during long-term cocultures of this env mutant, revealed that an amino acid change within another region of gp120 could functionally compensate for the mutation and restore infectivity. In the current study, we have introduced a conservative amino acid substitution at this second-site revertant codon and observed a marked reduction in HIV-1 infectivity. During the passage of this defective virus in cocultures, yet another revertant appeared which contained an amino acid change within a variable region of gp120 which restored infectivity to near wild-type levels. These results, in combination with other point mutations that have been introduced into the HIV-1 envelope, suggest that at least three discrete regions of gp120 may interact during the establishment of a productive viral infection. This critical step occurs subsequent to the adsorption of virions to the cell surface and either prior to or concomitant with the fusion of viral and cellular membranes.     

Stress protein of cyanobacteria CP36: interaction with photoactive complexes and formation of supramolecular structures

Cyanobacterium Anacystis nidulans R2, Synechocystis sp. PCC 6803 (wild-type strain and mutants Delta2 and Delta3 lacking PSII and PSI, respectively), and Synechocystis sp. BO 9201 synthesize the pigment--protein complex CP36 (CPIV-4, CP43') under iron deficiency in the medium. Accumulation of CP36 is accompanied by structural reorganizations in the photosynthetic membranes. Integrating mean times of excitation relaxation (quenching) are 2.2 nsec (CP36), 1 nsec (PSI), and 420 psec (PSII in Fm state). The energy migration between CP36 and the photosystems can be described by a model of a one-layer ring of CP36 around core-complexes. The excitation from CP36 to PSI is transferred within <10 psec. The energy transfer from CP36 to PSII occurs during 170 psec. Cells with low content of CP36 probably contain only a latent fraction of unbound to phycobilisomes PSII which is the analog of PSIIbeta of higher plants. In PSI there are four binding sites for CP36 monomers per RC. PSII can bind up to 32 molecules of CP36 per RC. Cells with a large amount of CP36 contain monomer form of PSII core-complex which can bind eight tetramers of CP36 (8 binding sites). In conditions of iron deficiency only one monomer of a dimer PSII core-complex is destroyed and released chlorophyll is accumulated in CP36. Accumulation of CP36 in A. nidulans cells can be accompanied by membrane stacking which is similar to the stacking in chlorophyll b-containing organisms. The stacking can occur in the region of localization of PSII latent fraction bound to CP36. The membrane stacking shields PSII stromal surfaces from the aqueous phase for activation of electron transfer on the acceptor side of PSII.     

COLACIUM LIBELLAE SP. NOV. (EUGLENOPHYCEAE), A PHOTOSYNTHETIC INHABITANT OF THE LARVAL DAMSELFLY RECTUM1

Colacium libellae sp. nov., in the non-flagellated palmella stage and lacking stalks, lines the cuticle of the rectum of damselfly larvae, forming a conspicuous green plug. In culture this species forms highly-branched colonies of stalked cells. The cells tire often elongated to twice the size of most motile cells (40–50 × 6–10 μm), with parallel sides and rounded apices. These features are distinctive when comparisons are made among clones of related species in soil-water-pea medium. C. libellae established itself in the rectum of previously Colacium-free larvae of the damsel fly Anomalagrion hastatum whereas a clone of C. vesiculosum and C. mucronatum did not.     

Response to Rimiterol and Salbutamol Aerosols Administered by Intermittent Positive-pressure Ventilation

The bronchodilator and cardiac effects produced by aerosols of 0·5% salbutamol and 0·5% and 1% rimiterol administered for three minutes in 40% oxygen by intermittent positive-pressure ventilation (I.P.P.V.) were compared in 15 asthmatic patients. Salbutamol and both the concentrations of rimiterol were equipotent in peak bronchodilator effect, but salbutamol had a significantly longer duration of bronchodilator action. There was significantly less increase in heart rate after rimiterol than after salbutamol. Aerosols of 0·5% rimiterol, 0·5% salbutamol, and saline were administered by I.P.P.V. to 10 normal volunteers. There was no difference between the mean heart rates after 0·5% rimiterol and saline but a highly significant increase in mean heart rate was observed after 0·5% salbutamol. It was concluded that 0·5% rimiterol was an effective short-acting bronchodilator drug with little or no cardiac beta₁-adrenergic activity when administered for three minutes by I.P.P.V. in 40% oxygen.     

Discrimination of pathogenic clinical isolates and laboratory strains of Bacillus cereus by NMR-based metabolomic profiling

Six different Bacillus cereus strains were selected from two different ecotypes: (1) three commonly used laboratory strains that are considered avirulent, and (2) three clinical isolates from meningitis patients. Screening of genomic DNA for the presence of genes encoding known toxins gave no candidate genes that were unambiguously able to distinguish between the two groups. However, the application of multivariate pattern-recognition methods to metabolite profiles derived from the different strains using 1H nuclear magnetic resonance spectroscopy (metabolomics) was able to classify the different profiles. The two different ecotypes were clearly separated on the basis of their metabolite profiles, showing that it is possible to use metabolomic methods to classify pathogens on the basis of their expressed physiology, even when it is not possible to infer a direct mechanistic link to specific virulence factors. This metabolomic approach could also have a wide range of possible applications in both general microbiology and microbial ecology for distinguishing and identifying different functional/physiological ecotypes of bacterial strains or species.     

Food web structure in the high Arctic Canada Basin: evidence from δ13C and δ15N analysis

The food-web structure of the Arctic deep Canada Basin was investigated in summer 2002 using carbon and nitrogen stable isotope tracers. Overall food-web length of the range of organisms sampled occupied four trophic levels, based on 3.8 trophic level enrichment (¹⁵N range: 5.3–17.7). It was, thus, 0.5–1 trophic levels longer than food webs in both Arctic shelf and temperate deep-sea systems. The food sources, pelagic particulate organic matter (POM) (¹³C=–25.8, ¹⁵N=5.3) and ice POM (¹³C=–26.9, ¹⁵N=4.1), were not significantly different. Organisms of all habitats, ice-associated, pelagic and benthic, covered a large range of ¹⁵N values. In general, ice-associated crustaceans (¹⁵N range 4.6–12.4, mean 6.9) and pelagic species (¹⁵N range 5.9–16.5, mean 11.5) were depleted relative to benthic invertebrates (¹⁵N range 4.6–17.7, mean 13.2). The predominantly herbivorous and predatory sympagic and pelagic species constitute a shorter food chain that is based on fresh material produced in the water column. Many benthic invertebrates were deposit feeders, relying on largely refractory material. However, sufficient fresh phytodetritus appeared to arrive at the seafloor to support some benthic suspension and surface deposit feeders on a low trophic level (e.g., crinoids, cumaceans). The enriched signatures of benthic deposit feeders and predators may be a consequence of low primary production in the high Arctic and the subsequent high degree of reworking of organic material.     

Supercritical fluid CO2 extraction accelerates isolation of humic acid from livePilayella littoralis (Phaeophyta)

Supercritical fluid CO₂ extraction of the live, dried algaPilayella littoralis efficiently removes non-humic substances in hours instead of the days required for sequential extraction with ether, acetone, ethanol and dioxane. Completion of extraction (including aqueous removal of alginic acids) with either preliminary in a standard procedure gives humic acid samples with closely comparable properties.Authors for correspondence