Alternative splice variants of rhomboid proteins: Comparative analysis of database entries for select model organisms and validation of functional potential.

Background: Rhomboid serine proteases are present across many species and are often encoded in each species by more than one predicted gene. Based on protein sequence comparisons, rhomboids can be differentiated into groups - secretases, presenilin-like associated rhomboid-like (PARL) proteases, iRhoms, and "inactive" rhomboid proteins. Although these rhomboid groups are distinct, the different types can operate simultaneously. Studies in Arabidopsis showed that the number of rhomboid proteins working simultaneously can be further diversified by alternative splicing. This phenomenon was confirmed for the Arabidopsis plastid rhomboid proteins At1g25290 and At1g74130. Although alternative splicing was determined to be a significant mechanism for diversifying these two Arabidopsis plastid rhomboids, there has yet to be an assessment as to whether this mechanism extends to other rhomboids and to other species.  Methods: We thus conducted a comparative analysis of select databases to determine if the alternative splicing mechanism observed for the two Arabidopsis plastid rhomboids was utilized in other species to expand the repertoire of rhomboid proteins. To help verify the in silico observations, select splice variants from different groups were tested for activity using transgenic- and additive-based assays. These assays aimed to uncover evidence that the selected splice variants display capacities to influence processes like antimicrobial sensitivity. Results: A comparison of database entries of six widely used eukaryotic experimental models  (human, mouse, Arabidopsis, Drosophila, nematode, and yeast) revealed robust usage of alternative splicing to diversify rhomboid protein structure across the various motifs or regions, especially in human, mouse and Arabidopsis. Subsequent validation studies uncover evidence that the splice variants selected for testing displayed functionality in the different activity assays. Conclusions: The combined results support the hypothesis that alternative splicing is likely used to diversify and expand rhomboid protein functionality, and this potentially occurred across the various motifs or regions of the protein.

Potential for mcl-PHA production from nonanoic and azelaic acids.

Greater than 65% of canola and high-oleic soy oil fatty acids is oleic acid, which is readily converted to nonanoic (NA) and azelaic (AzA) acids by ozonolysis. NA is an excellent substrate for medium-chain-length polyhydroxyalkanoate (mcl-PHA) production but AzA has few uses. Pseudomonas citronellolis DSM 50332 and Pseudomonas fluorescens ATCC 17400, both able to produce mcl-PHA from fatty acids and to grow on AzA as the sole source of carbon and energy, were assessed for the accumulation of mcl-PHA from AzA and NA. In N-limited shake flasks using NA, P. citronellolis produced 32% of its dry biomass as mcl-PHA containing 78% 3-hydroxynonanoate with 22% 3-hydroxyheptanoate. Pseudomonas fluorescens produced only 2% PHA. N-limited P. citronellolis on AzA produced 20% dry weight PHA containing 75% 3-hydroxydecanoate and 25% 3-hydroxyoctanoate, indicative of de novo synthesis. Although selective pressure, including ß-oxidation inhibition, under well-controlled (chemostat) conditions was applied to P. citronellolis, no side-chain carboxyl groups were detected. It was concluded that one or more of FabG and PhaJ or the PHA synthase cannot catalyze reactions involving ω-carboxy substrates. However, a process based on oleic acid could be established if Pseudomonas putida was engineered to grow on AzA.

Carbon-limited fed-batch production of medium-chain-length polyhydroxyalkanoates by a phaZ-knockout strain of Pseudomonas putida KT2440.

A medium-chain-length poly-3-hydroxyalkanote (MCL-PHA) depolymerase knockout mutant of Pseudomonas putida KT2440 was produced by double homologous recombination. A carbon-limited shake-flask study confirmed that depolymerase activity was eliminated. Lysis of both mutant and wild-type strains occurred under these conditions. In carbon-limited, fed-batch culture, the yield of unsaturated monomers from unsaturated substrate averaged only 0.62 mol mol(-1) for the phaZ minus strain compared to 0.72 mol mol(-1) for the wild type. The mutant strain also produced more CO2 and less residual biomass from the same amount of carbon substrate. However, most results indicated that elimination of PHA depolymerase activity had little impact on the overall yield of biomass and PHA.

Factors influencing consent to having videotaped mental health sessions.

OBJECTIVE: The authors critically reviewed the literature regarding factors influencing consent to having videotaped mental health sessions. METHODS: The authors searched the literature in PubMed, PsycINFO, Google Scholar, and Web of Science from the mid-1950s through February 2009. RESULTS: The authors identified 27 studies, of which 19 (73%) examined general practice. Only 4 (15%) were in mental health. Most patients agree to be videotaped when asked. Those who did not consent tended to be female and younger, with previous psychiatric history or psychological distress. The data are mixed about whether psychiatric patients felt inhibited in videotaped sessions. CONCLUSION: The mental health literature in this area is limited and dated. Implications for practice are drawn inferentially from the general-practice literature. Recommendations for increasing the consent rate include building a relationship with patients before asking them for videotaping and, when asking, explaining the educational value and specific purpose of the recording.

Biodegradation of 1,4-dioxane by a Flavobacterium.

A dioxane-degrading consortium was enriched from soil obtained from a contaminated groundwater plume. The enriched consortium did not use dioxane as the sole source of carbon and energy but co-metabolized dioxane in the presence of tetrahydrofuran (THF). THF and dioxane concentrations up to 1000 ppm were degraded by the enriched consortium in about 2 weeks with a longer lag phase observable at 1000 ppm. Three colonies from the enriched consortium were then obtained on agar plates containing basal salts and glucose as the carbon source. Only one of the three colonies was capable of dioxane degradation. Further enrichment of this colony in liquid media led to a pure culture that grew on glucose and co-metabolically degraded dioxane after THF degradation. The rate and extent of dioxane degradation of this isolate increased with increasing THF concentration. This isolate was subsequently identified as a Flavobacterium by 16S rDNA sequencing. Using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis of microbial populations, Flavobacterium was determined to be the dominant species in the enriched consortium and was distinct from the two other colonies that did not degrade dioxane. This is the first report of a dioxane-degrading Flavobacterium which is phylogenetically distinct from any previously identified dioxane degrader.

Uncovering a link between a plastid translocon component and rhomboid proteases using yeast mitochondria-based assays.

Rhomboid proteases are present in bacteria, insects, yeasts, parasites, mammals and plants. These proteases are part of the regulated intramembrane proteolysis mechanism for controlling processes such as development, stress response, lipid metabolism and mitochondrial membrane remodeling. Specific rhomboid protease substrates linked to these processes have been identified from insects to mammals, but not for plants. Identification of a link is a key step for elucidating the role of each rhomboid protease. Here, using a yeast mitochondria-based approach, we report evidence of a potential link between a plastid translocon component and organellar rhomboid proteases. This identification expands the types of processes involving regulated intramembrane proteolysis potentially to include at least one aspect of plastid protein transport.

Evidence that the plastid translocon Tic40 components possess modulating capabilities.

The transport of proteins into the plastid is a process that faces changing cellular needs such as the situation found in different plant organs or developing tissues. The plastid translocon must therefore be responsive to the changing cell environment to deliver efficiently different arrays of structurally diverse proteins. Although the Tic40-related envelope proteins appear to be translocon components designed to address the varying needs of protein translocation, details of their involvement remain elusive. This study was thus designed to combine plant-based experiments and yeast mitochondrion-based approaches for unveiling clues related to how the Tic40 components may behave during the protein translocation process. The main findings related to how Tic40 proteins may work are: 1) natural fluctuations are apparent in developing tissues, in different organs of the same plant, and in different species; 2) transgenic Arabidopsis seedlings can tolerate functionally a wide range of variations in Tic40 levels, from partial suppression to excessive production; 3) the Tic40 proteins themselves exhibit configurational changes in their association with yeast mitochondria in response to different carbon sources; 4) the presence of Tic40 proteins in yeast mitochondria influences regulatory aspects of the mitochondrial translocon; and 5) the Tic40 proteins associate with mitochondrial translocon components involved in regulatory-like events. The combined data provide evidence that Tic40 proteins possess modulating capabilities.

Constitutive expression of pea Lhcb 1-2 in tobacco affects plant development, morphology and photosynthetic capacity.

Lhcb1-2 from pea was constitutively expressed in transgenic tobacco plants and assessed for functional impact. The successful assembly of the encoded proteins into LHCII trimers was confirmed by electrospray tandem mass spectrometry. Constitutive production of LHCb1-2 led to increased number of thylakoid membranes per chloroplast, increased grana stacking, higher chloroplast numbers per palisade cell and increased photosynthetic capacity at low irradiance, both on a chlorophyll and leaf area basis. The transgenic plants also displayed increased cell volume, larger leaves, higher leaf number per plant at flowering, increased biomass and increased seed weight, when grown under low irradiance levels. Under high irradiance, both transgenic and wild type plants displayed similar photosynthetic rates when tested at 25 degrees C; however, the non-photochemical quenching (NPQ) and qE values increased in the transgenic plants. The exposure of transgenic plants to a photoinhibitory treatment (4 degrees C for 4 h, under continuous illumination) resulted in more detrimental impairment of photosynthesis, since recovery was slower than the non-transgenic plants. These data indicate that constitutive expression of additional Lhcb1-2 transgenes led to a series of changes at all levels of the plant (cellular, leaf and whole organism), and a delay in flowering and senescence. The additional production of the pea protein appears to be accommodated by increasing cellular structures such as the number of thylakoids per chloroplast, organelle volume, organelles per cell, and leaf expansion. The presence of the trimeric pea protein in the tobacco LHCII, however, caused a possible change in the organization of the associated super-complex, that in turn limited photosynthesis at low temperature.

The Tic40 translocon components exhibit preferential interactions with different forms of the Oee1 plastid protein precursor.

The transport of proteins across the plastid envelope involves a host of proteinaceous components that attend to varying structural needs of the process. This study focuses on interactions between two select forms (designated Tic40 and Toc36) of the Tic40-related components and different structural versions of the Oee1 precursor to dissect the components' mode of operation. Interaction profiling revealed several features pertaining to how Tic40-related components might work during the transport process. The main operational features revealed are: (1) Tic40 interacts preferentially with Oee1 precursors containing only the plastid-targeting domain, (2) Toc36 interacts preferentially with Oee1 precursors containing both plastid- and thylakoid lumen-targeting domains, (3)carboxyl truncations to either the entire Oee1 precursor or Toc36 affect interactions negatively, and (4) the general reduction of Tic40-related protein levels in transgenic plants appears to exert a greater negative impact on endogenous Oee1 levels than the other proteins assessed, a trend that corroborates the findings of the protein interaction experiments.

The plastid translocon component TOC36 exhibits an affinity for the bacterial protein translocation process.

The 44-kDa envelope polypeptides are active components of the plastid translocon, but their role in plastid protein import remains elusive. One form from Brassica napus (bnToc36B) was previously observed to exert a significant overall effect on bacterial protein translocation, but the nature of the influence requires further characterization. The experimental strategies employed in this study thus focus specifically on the nature of the bnToc36B-bacterial Sec translocon relationship to gain an understanding of Toc36's function. BnToc36B's presence in bacteria created a number of effects related to the protein transport process that together point to functional interactions with the bacterial Sec translocon. These effects are (1) reduced sensitivity to azide impairment as measured by a higher recovery rate from azide treatment, (2) reduced sensitivity to suboptimal temperatures manifesting as sustained levels of protein synthesis and translocation, (3) sustained levels of growth and beta-lactamase transport in high ampicillin concentrations, and (4) evidence for a physical affinity for the bacterial translocon. A reduction in overall SecA levels and a more stable SecA profile, when subjected to azide treatment, was observed in bnToc36B-containing bacteria. The implications of the bacterial data are discussed.

Chronic treatment with azide in situ leads to an irreversible loss of cytochrome c oxidase activity via holoenzyme dissociation.

Chronic treatment of cultured cells with very low levels of azide (I(50)<10 microm) leads to slow (t(12) = 6 h), irreversible loss of cytochrome c oxidase (COX) activity. Azide-mediated COX losses were not accompanied by inhibition of other mitochondrial enzymes and were not dependent upon electron flux through oxidative phosphorylation. Although azide treatment also reduced activity (but not content) of both CuZn superoxide dismutase and catalase, a spectrum of pro-oxidants (and anti-oxidants) failed to mimic (or prevent) azide effects, arguing that losses in COX activity were not due to resultant compromises in free radical scavenging. Loss of COX activity was not attributable to reduced rates of mitochondrial protein synthesis or declines in either COX subunit mRNA or protein levels (COX I, II, IV). Co-incubation experiments using copper (CuCl(2), Cu-His) and copper chelators (neocuproine, bathocuproine) indicated that azide effects were not mediated by interactions with either Cu(A) or Cu(B). In contrast, difference spectroscopy and high performance liquid chromatography analyses demonstrated azide-induced losses in cytochrome aa(3) content although not to the same extent as catalytic activity. Differential azide effects on COX content relative to COX activity were confirmed using a refined inhibition time course in combination with blue native electrophoresis, and established that holoenzyme dissociation occurs subsequent to losses in catalytic activity. Collectively, these data suggest that COX deficiency can arise through enhanced holoenzyme dissociation, possibly through interactions with the structure or coordination of its heme moieties.