Functional dichotomy of ribosomal proteins during the synthesis of mammalian 40S ribosomal subunits

Our knowledge of the functions of metazoan ribosomal proteins in ribosome synthesis remains fragmentary. Using siRNAs, we show that knockdown of 31 of the 32 ribosomal proteins of the human 40S subunit (ribosomal protein of the small subunit [RPS]) strongly affects pre–ribosomal RNA (rRNA) processing, which often correlates with nucleolar chromatin disorganization. 16 RPSs are strictly required for initiating processing of the sequences flanking the 18S rRNA in the pre-rRNA except at the metazoan-specific early cleavage site. The remaining 16 proteins are necessary for progression of the nuclear and cytoplasmic maturation steps and for nuclear export. Distribution of these two subsets of RPSs in the 40S subunit structure argues for a tight dependence of pre-rRNA processing initiation on the folding of both the body and the head of the forming subunit. Interestingly, the functional dichotomy of RPS proteins reported in this study is correlated with the mutation frequency of RPS genes in Diamond-Blackfan anemia.     

Impaired methylation as a novel mechanism for proteasome suppression in liver cells

The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell and hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25 kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.     

The development of a matrix-assisted laser desorption/ionization mass spectrometry-based method for the protein fingerprinting and identification of Aeromonas species using whole cells

This report describes the development of a method to detect the waterborne pathogen Aeromonas using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The genus Aeromonas is one of several medically significant genera that have gained prominence due to their evolving taxonomy and controversial role in human diseases. In this study, MALDI-MS was applied to the characterization of seventeen species of Aeromonas. These seventeen species were represented by thirty-two strains, which included type, reference and clinical isolates. Intact cells from each strain were used to generate a reproducible library of protein mass spectral fingerprints or m/z signatures. Under the test conditions used, peak lists of the mass ions observed in each species revealed that three mass ions were conserved among all the seventeen species tested. These common mass ions having an average m/z of 6301, 12,160 or 12,254, and 13,450, can be potentially used as genus-specific biomarkers to identify Aeromonas in unknown samples. A dendrogram generated using the m/z signatures of all the strains tested indicated that the mass spectral data contained sufficient information to distinguish between genera, species, and strains. There are several advantages of using MALDI-MS based protein mass spectral fingerprinting of whole cells for the identification of microorganisms as well as for their differentiation at the sub-species level: (1) the capability to detect proteins, (2) high throughput, and (3) relatively simple sample preparation techniques. The accuracy and speed with which data can be obtained makes MALDI-MS a powerful tool especially suited for environmental monitoring and detection of biological hazards.     

Expression in Escherichia coli and characterization of β-xylosidases GH39 and GH-43 from Bacillus halodurans C-125

To develop xylosidases as tools for the hydrolysis of wheat bran arabinoxylans, two β-xylosidases from Bacillus halodurans C-125 have been cloned and expressed in Escherichia coli. The recombinant (His)₆-tagged enzymes, designated as XylBH39 and XylBH43, were efficiently purified using Ni²⁺-affinity chromatography. Determination of native molecular masses indicated that XylBH43 is dimeric in solution, whereas a similar analysis of XylBH39 did not allow differentiation between the dimeric and trimeric states. Both enzymes had similar pH and temperature optima (pH 7.5 and 55 °C for XylBH39 and pH 8 and 60 °C for XylBH43) and were relatively stable over the pH range of 3.5–8.5. In contrast, XylBH39 was more thermostable. At 60 °C, XylBH39 and XylBH43 displayed approximate half-life values of 2.40 and 0.05 h, respectively. The comparison of the ratio k _cat/K _M revealed that XylBH43 hydrolyzed p-nitrophenyl-β-d-xyloside more efficiently (4.6-fold) than XylBH39. Similarly, while XylBH43 was 18-fold less active on p-nitrophenyl-α-l-arabinofuranoside, XylBH39 was essentially inactive on this substrate. Using either p-nitrophenyl-β-d-xyloside or xylotriose, XylBH39 performed transglycosylation, while xylobiose proved to be a poor substrate for both hydrolysis and transglycosylation. The use of XylBH39 and XylBH43 for the posttreatment of endoxylanase-generated wheat bran hydrolysates revealed that XylBH43 efficiently produced xylose monomers (385 μg/ml after 330 min incubation). Its activity was improved by the simultaneous deployment of an α-l-arabinofuranosidase. Together, these enzymes were able to release 521 μg/ml of xylose after 330 min. This constitutes an approximate yield improvement of 35%.     

On the dimensionality of ecological stability

Ecological stability is touted as a complex and multifaceted concept, including components such as variability, resistance, resilience, persistence and robustness. Even though a complete appreciation of the effects of perturbations on ecosystems requires the simultaneous measurement of these multiple components of stability, most ecological research has focused on one or a few of those components analysed in isolation. Here, we present a new view of ecological stability that recognises explicitly the non‐independence of components of stability. This provides an approach for simplifying the concept of stability. We illustrate the concept and approach using results from a field experiment, and show that the effective dimensionality of ecological stability is considerably lower than if the various components of stability were unrelated. However, strong perturbations can modify, and even decouple, relationships among individual components of stability. Thus, perturbations not only increase the dimensionality of stability but they can also alter the relationships among components of stability in different ways. Studies that focus on single forms of stability in isolation therefore risk underestimating significantly the potential of perturbations to destabilise ecosystems. In contrast, application of the multidimensional stability framework that we propose gives a far richer understanding of how communities respond to perturbations.     

Possible rheumatoid arthritis subtypes in terms of rheumatoid factor,depression, diagnostic delay and emotional expression: an exploratory case-control study

Introduction

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis has been implicated in the pathology of rheumatoid arthritis (RA), particularly as vulnerable personality types are exposed to chronic stress. Emotions are powerful modulators of stress responses. However, little is known about whether patients with RA process emotions differently to matched controls. In this study we: 1) assessed whether the trait emotional intelligence (trait EI) scores of patients with RA differ from healthy controls at the facet level; 2) explored any subgroups in RA, in terms of trait EI and common risk factors.

Methods

A total of 637 patients with RA were compared to 496 controls on the trait EI Questionnaire (TEIQue). RA subgroups were explored in terms of trait EI, rheumatoid factor status (RF+/-), depression and time from onset of symptoms until diagnosis (diagnostic delay).

Results

The RA group rated themselves lower on Adaptability, Stress-management, Emotion management, Self-esteem, Sociability, Assertiveness, Impulsiveness and Well-being, and higher on Empathy and Relationships than healthy controls. The RF- subtype reported more time with depression (25.2 vs. 11.3 months), a longer diagnostic delay (3.0 vs. 1.7 years), and greater emotional expression (5.15 vs. 4.72), than the RF+ subtype. These differences were significant at the P <0.05 level, but not following strict Bonferroni corrections and should therefore be treated as indicative only. RF- patients with a longer diagnostic delay reported depression lasting three times longer (42.7 months), when compared to three other subtypes (11.0 to 12.7 months).

Conclusions

RA patients and controls differ in their emotion-related personality traits, as operationalized by trait EI. These differences may make people with RA more susceptible to chronic stress and HPA-axis dysregulation. RA may be a highly heterogeneous illness where at least two subtypes may be characterized by personality, psychiatric and immunological differences. RF- status, as well as diagnostic delay and emotional expression, may predict future risk of depression. Research on the causes of RA could benefit from a systems science approach.     

Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells

Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells. This enhancement depends on ethanol oxidation. Here, we used parental (nonmetabolizing) and recombinant (ethanol-metabolizing) Hep G2 cells to identify the ethanol metabolite that causes AV enhancement by quantifying AVs or their marker protein, microtubule-associated protein 1 light chain 3-II (LC3-II). The ethanol-elicited rise in LC3-II was dependent on ethanol dose, was seen only in cells that expressed alcohol dehydrogenase (ADH) and was augmented in cells that coexpressed cytochrome CYP2E1 (P450 2E1). Furthermore, the rise in LC3-II was inversely related to a decline in proteasome activity. AV flux measurements and colocalization of AVs with lysosomes or their marker protein Lysosomal-Associated Membrane Protein 1 (LAMP1) in ethanol-metabolizing VL-17A cells (ADH⁺/CYP2E1⁺) revealed that ethanol exposure not only enhanced LC3-II synthesis but also decreased its degradation. Ethanol-induced accumulation of LC3-II in these cells was similar to that induced by the microtubule inhibitor, nocodazole. After we treated cells with either 4-methylpyrazole to block ethanol oxidation or GSH-EE to scavenge reactive species, there was no enhancement of LC3-II by ethanol. Furthermore, regardless of their ethanol-metabolizing capacity, direct exposure of cells to acetaldehyde enhanced LC3-II content. We conclude that both ADH-generated acetaldehyde and CYP2E1-generated primary and secondary oxidants caused LC3-II accumulation, which rose not only from enhanced AV biogenesis, but also from decreased LC3 degradation by the proteasome and by lysosomes.     

Molecular classification of commercial Spirulina strains and identification of their sulfolipid biosynthesis genes

Cyanobacterial strains of the genus Spirulina have recently been identified as an excellent source of sulfolipids, some of which possess anti-HIV properties. Thus, to investigate the distribution of sufolipid biosynthesis pathways in Spirulina, a genetic screening/phylogentic study was performed. Five different strains of Spirulina [Spirulina (Jiangmen), Spirulina sp., S. platensis, S. maxima, and Spirulina seawater] sourced from different locations were initially classified via 16S rDNA sequencing, and then screened for the presence of the sulfolipid biosynthesis genes sqdB and sqdX via a PCR. To assess the suitability of these strains for human consumption and safe therapeutic use, the strains were also screened for the presence of genes encoding nonribosomal peptide synthases (NRPSs) and polyketide synthases (PKSs), which are often associated with toxin pathways in cyanobacteria. The results of the 16S rDNA analysis and phylogenetic study indicated that Spirulina sp. is closely related to Halospirulina, whereas the other four Spirulina strains are closely related to Arthrospira. Homologs of sqdB and sqdX were identified in Spirulina (Jiangmen), Spirulina sp., S. platensis, and the Spirulina seawater. None of the Spirulina strains screened in this study tested positive for NRPS or PKS genes, suggesting that these strains do not produce NRP or PK toxins.     

Benzoyl Coenzyme A Pathway-Mediated Metabolism of meta-Hydroxy-Aromatic Acids in Rhodopseudomonas palustris

Photoheterotrophic metabolism of two meta-hydroxy-aromatic acids, meta-, para-dihydroxybenzoate (protocatechuate) and meta-hydroxybenzoate, was investigated in Rhodopseudomonas palustris. When protocatechuate was the sole organic carbon source, photoheterotrophic growth in R. palustris was slow relative to cells using compounds known to be metabolized by the benzoyl coenzyme A (benzoyl-CoA) pathway. R. palustris was unable to grow when meta-hydroxybenzoate was provided as a sole source of organic carbon under photoheterotrophic growth conditions. However, in cultures supplemented with known benzoyl-CoA pathway inducers (para-hydroxybenzoate, benzoate, or cyclohexanoate), protocatechuate and meta-hydroxybenzoate were taken up from the culture medium. Further, protocatechuate and meta-hydroxybenzoate were each removed from cultures containing both meta-hydroxy-aromatic acids at equimolar concentrations in the absence of other organic compounds. Analysis of changes in culture optical density and in the concentration of soluble organic compounds indicated that the loss of these meta-hydroxy-aromatic acids was accompanied by biomass production. Additional experiments with defined mutants demonstrated that enzymes known to participate in the dehydroxylation of para-hydroxybenzoyl-CoA (HbaBCD) and reductive dearomatization of benzoyl-CoA (BadDEFG) were required for metabolism of protocatechuate and meta-hydroxybenzoate. These findings indicate that, under photoheterotrophic growth conditions, R. palustris can degrade meta-hydroxy-aromatic acids via the benzoyl-CoA pathway, apparently due to the promiscuity of the enzymes involved.