Diatom fucoxanthin chlorophyll a/c-binding protein (FCP) and land plant light-harvesting proteins use a similar pathway for thylakoid membrane Insertion.

The light-harvesting proteins in plastids of different lineages including algae and land plants represent a superfamily of chlorophyll-binding proteins that seem to be phylogenetically related, although some of the light-harvesting complex (LHC) proteins bind different carotenoids. LHCs can be divided into chlorophyll a/b-binding proteins found in green algae, euglenoids, and higher plants and into chlorophyll a/c-binding proteins of various algal taxa. LHC proteins from diatoms are named fucoxanthin-chlorophyll a/c-binding proteins (FCP). In contrast to chlorophyll a/b-binding proteins, there is no information so far about the way FCPs integrate into thylakoid membranes. The diatom FCP preproteins have a bipartite presequence that is necessary to enable transport into the four membrane-bound diatom plastids, but similar to chlorophyll a/b-binding proteins there is apparently no presequence present for targeting to the thylakoid membrane. By establishing an in vitro import assay for diatom thylakoids, we demonstrated that thylakoid integration of diatom FCP depends on the presence of stromal factors and GTP. This indicates that a pathway involving signal recognition particles (SRP) is involved in membrane integration just as shown for LHCs in higher plants. We also demonstrate integration of diatom FCP into thylakoids of higher plants and vice versa SRP-dependent targeting of LHCs from pea and Arabidopsis into diatom thylakoids. The similar SRP-dependent modes of thylakoid integration of land plant LHCs and FCPs support recent analyses indicating a common origin of chlorophyll a/b- and a/c-binding proteins.

Bedside vital signs capture for the non-ICU setting--an open source, PC-based solution.

The efficient and reliable capture of vital signs and other bedside data in the non-ICU setting has been a challenging problem for the medical informatics community. The problem is compounded by the complexities associated with storage of this data into an electronic medical record system (EMRS). There are a lack of off-the-shelf solutions that satisfy the basic system requirements of bedside data capture, user authentication, data validation prior to storage, error handling, and convenience. With the current state of technology available, we feel the solution to this problem requires the presence of a PC with custom interface software at the bedside. This allows for the successful interface between available vital signs capture devices, existing EMRS s, and the user. This report summarizes the alternatives we found and our proposed solution to this important problem.

Inverse regulation of F1-ATPase activity by a mutation at the regulatory region on the gamma subunit of chloroplast ATP synthase.

Chloroplast ATP synthase is a thiol-modulated enzyme whose DeltamuH(+)-linked activation is strongly influenced by reduction and the formation of a disulphide bridge between Cys(199) and Cys(205) on the gamma subunit. In solubilized chloroplast coupling factor 1 (CF(1)), reduction of the disulphide bond elicits the latent ATP-hydrolysing activity. To assess the regulatory importance of the amino acid residues around these cysteine residues, we focused on the three negatively charged residues Glu(210)-Asp-Glu(212) close to the two cysteine residues and also on the following region from Leu(213) to Ile(230), and investigated the modulation of ATPase activity by chloroplast thioredoxins. The mutant gamma subunits were reconstituted with the alpha and beta subunits from F(1) of the thermophilic bacterium Bacillus PS3; the active ATPase complexes obtained were purified by gel-filtration chromatography. The complex formed with a mutant gamma subunit in which Glu(210) to Glu(212) had been deleted was inactivated rather than activated by reduction of the disulphide bridge by reduced thioredoxin, indicating inverse regulation. This complex was insensitive to the inhibitory CF(1)-epsilon subunit when the mutant gamma subunit was oxidized. In contrast, the deletion of Glu(212) to Ile(230) converted the complex from a modulated state into a highly active state.

Protein transport into "complex" diatom plastids utilizes two different targeting signals.

The plastids found in diatoms and other chromophytic algae are completely enclosed by four membranes in contrast to chloroplasts of higher plants, which are surrounded by only two membranes. The bipartite targeting sequence of diatom nuclear-encoded plastid proteins contains an endoplasmic reticulum signal sequence and, based on sequence comparison, a transit peptide-like domain similar to that which targets proteins into the plastids of higher plants. By performing heterologous import experiments using the precursor of the gamma subunit of the chloroplast ATPase from the diatom Odontella sinensis we were able to show that protein import into diatom plastids is at least a two-step event. We demonstrate that the first step involves co-translational transport through endoplasmic reticulum membranes and that there is an additional targeting step which is similar to the import of precursor proteins into chloroplasts of higher plants and green algae indicating that the transit peptide-like domain of the diatom precursor is functionally equivalent to the respective targeting signal of higher plants. Our results suggest that the transit peptide depending targeting mechanism in plastids has apparently remained relatively unchanged over the course of evolution, with only the peptidase cleavage site significantly modified.

The formation or the reduction of a disulfide bridge on the gamma subunit of chloroplast ATP synthase affects the inhibitory effect of the epsilon subunit.

We have studied the change of the catalytic activity of chimeric complexes that were formed by chloroplast coupling factor 1 (CF1) -gamma, alpha and beta subunits of thermophilic bacterial F1 after formation or reduction of the disulfide bridge of different gamma subunits modified by oligonucleotide-directed mutagenesis techniques. For this purpose, three mutant gamma subunits were produced: gamma Delta194-230, here 37 amino acids from Pro-194 to Ile-230 are deleted, gammaC199A, Cys-199 is changed to Ala, and gamma Delta200-204, amino acids from Asp-200 to Lys-204 are deleted. All of the chimeric subunit complexes produced from each of these mutant CF1-gamma subunits and alpha and beta subunits from thermophilic bacterial F1 lost the sensitivity against thiol reagents when compared with the complex containing wild-type CF1-gamma. The pH optimum (pH 8.5-9.0) and the concentration of methanol to stimulate ATPase activities were not affected by these mutations. These indicate that the introduction of the mutations did not change the main features of ATPase activity of the chimeric complex. However, the interaction between gamma subunit and epsilon subunit was strongly influenced by the type of gamma subunit itself. Although the ATPase activity of the chimeric complex that contained gamma Delta200-204 or gammaC199A was inhibited by the addition of recombinant epsilon subunit from CF1 similarly to complexes containing the reduced wild-type gamma subunit, the recombinant epsilon subunit did not inhibit the ATPase of the complex, which contained the oxidized form of gamma subunit. Therefore the affinity of the epsilon subunit to the gamma subunit may be dependent on the state of the gamma subunit or the epsilon subunit may bind to the oxidized form of gamma subunit in a mode that does not inhibit the activity. The ATPase activity of the complex that contains gamma Delta194-230 was not efficiently inhibited by epsilon subunit. These results show that the formation or reduction of the disulfide bond on the gamma subunit may induce a conformational change in the region that directly affects the interaction of this subunit with the adjacent epsilon subunit.

[The occurrence of Clostridium difficile in fecal samples of dogs and cats]. / Untersuchungen zum Vorkommen von Clostridium difficile in Kotproben von Hunden und Katzen.

Fecal samples of 150 dogs and 175 cats originating from different veterinary practices were investigated for assessing the occurrence of Clostridium (Cl.) difficile by using a selective medium for cultural isolation. From dogs without enteric symptoms 7 (9.3%) of 75 samples were positive for Cl. difficile, with 2 strains being cytotoxic for bovine embryonic lung fibroblast cells, which could be neutralized by Cl. difficile-antitoxin. In samples of 75 dogs with enteric symptoms Cl. difficile could be isolated in 2 cases (2.7%). In cats 9 (9%) of 100 fecal samples deriving from animals without enteric symptoms contained Cl. difficile, while in 75 cats with enteric symptoms, the isolation rate was 6.7% (5 strains). Of either group only 1 Cl. difficile-strain showed cytotoxicity for tissue culture. The results of this study allow to conclude, that in contrast to the significance for man Cl. difficile is neither for dogs nor for cats an important enteric agent. However these pets can harbour and shed strains of Cl. difficile, even cytotoxigenic ones, in faeces. In view of these findings the possibility of occasional human infections by household dogs or cats needs attention and further investigation.