The Arabidopsis pex12 and pex13 mutants are defective in both PTS1- and PTS2-dependent protein transport to peroxisomes

Peroxisome biogenesis requires various complex processes including organelle division, enlargement and protein transport. We have been studying a number of Arabidopsis apm mutants that display aberrant peroxisome morphology. Two of these mutants, apm2 and apm4, showed green fluorescent protein fluorescence in the cytosol as well as in peroxisomes, indicating a decrease of efficiency of peroxisome targeting signal 1 (PTS1)-dependent protein transport to peroxisomes. Interestingly, both mutants were defective in PTS2-dependent protein transport. Plant growth was more inhibited in apm4 than apm2 mutants, apparently because protein transport was more severely decreased in apm4 than in apm2 mutants. APM2 and APM4 were found to encode proteins homologous to the peroxins PEX13 and PEX12, respectively, which are thought to be involved in transporting matrix proteins into peroxisomes in yeasts and mammals. We show that APM2/PEX13 and APM4/PEX12 are localized on peroxisomal membranes, and that APM2/PEX13 interacts with PEX7, a cytosolic PTS2 receptor. Additionally, a PTS1 receptor, PEX5, was found to stall on peroxisomal membranes in both mutants, suggesting that PEX12 and PEX13 are components that are involved in protein transport on peroxisomal membranes in higher plants. Proteins homologous to PEX12 and PEX13 have previously been found in Arabidopsis but it is not known whether they are involved in protein transport to peroxisomes. Our findings reveal that APM2/PEX13 and APM4/PEX12 are responsible for matrix protein import to peroxisomes in planta.     

NMR‐based characterization of a refolding intermediate of β2‐microglobulin labeled using a wheat germ cell‐free system

In patients with dialysis‐related amyloidosis, β2‐microglobulin (β2‐m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of β2‐m is a prerequisite to the formation of amyloid fibrils, and that the folding intermediate trapped by the non‐native trans‐Pro32 isomer leads to the formation of amyloid fibrils. Although clarifying the structure of this refolding intermediate by high resolution NMR spectroscopy is important, this has been made difficult by the limited lifetime of the intermediate. Here, we studied the structure of the refolding intermediate using a combination of amino acid selective labeling with wheat germ cell‐free protein synthesis and NMR techniques. The HSQC spectra of β2‐ms labeled selectively at either phenylalanine, leucine, or valine enabled us to monitor the structures of the refolding intermediate. The results suggested that the refolding intermediate has an overall fold and cores similar to the native structure, but contains disordered structures around Pro32. The fluctuation of the β‐sheet regions especially the last half of the βB strand and the first half of the βE strand, both suggested to be important for amyloidogenicity, may transform β2‐m into an amyloidogenic structure.     

Destruction of amyloid fibrils of keratoepithelin peptides by laser irradiation coupled with amyloid-specific thioflavin T

Mutations in keratoepithelin are associated with blinding ocular diseases, including lattice corneal dystrophy type 1 and granular corneal dystrophy type 2. These diseases are characterized by deposits of amyloid fibrils and/or granular non-amyloid aggregates in the cornea. Removing the deposits in the cornea is important for treatment. Previously, we reported the destruction of amyloid fibrils of β(2)-microglobulin K3 fragments and amyloid β by laser irradiation coupled with the binding of an amyloid-specific thioflavin T. Here, we studied the effects of this combination on the amyloid fibrils of two 22-residue fragments of keratoepithelin. The direct observation of individual amyloid fibrils was performed in real time using total internal reflection fluorescence microscopy. Both types of amyloid fibrils were broken up by the laser irradiation, dependent on the laser power. The results suggest the laser-induced destruction of amyloid fibrils to be a useful strategy for the treatment of these corneal dystrophies.     

Apathy/depression, but not subjective fatigue, is related with cognitive dysfunction in patients with multiple sclerosis

Background

Cognitive impairment could affect quality of life for patients with multiple sclerosis (MS), and cognitive function may be correlated with several factors such as depression and fatigue. This study aimed to evaluate cognitive function in Japanese patients with MS and the association between cognitive function and apathy, fatigue, and depression.

Methods

The Brief Repeatable Battery of Neuropsychological tests (BRB-N) was performed in 184 Japanese patients with MS and 163 healthy controls matched for age, gender, and education. The Apathy Scale (AS), Fatigue Questionnaire (FQ), and Beck Depression Inventory Second Edition (BDI-II) were used to evaluate apathy, fatigue, and depression, respectively. Student’s t-test was used to compare MS patients and healthy controls. Correlations between two factors were assessed using the Pearson correlation test, and multiple regression analysis was used to evaluate how much each factor affected the BRB-N score.

Results

In all BRB-N tests, patients with MS scored significantly lower than controls, and the effect size of symbol digit modalities test was the highest among the 9 tests of the BRB-N. Patients with MS had higher AS (p?<?0.001), FQ (p?<?0.0001), and BDI-II (p?<?0.0001) scores than controls. In patients with MS, scores on most of the BRB-N tests correlated with scores on the AS and BDI-II; however, there was little correlation between scores on the BRB-N tests and those on the FQ.

Conclusions

Cognitive function was impaired, particularly information-processing speed, and decreased cognitive function was correlated with apathy and depression in Japanese patients with MS. Despite the association between cognitive variables and depression/apathy, cognitive function was impaired beyond the effect of depression and apathy. However, subjective fatigue is not related with cognitive impairment. Taken together, this suggests that different therapeutic approaches are needed to improve subjective fatigue and cognition, and thereby quality of life, in patients with MS.     

Activation of the Prereplication Complex Is Blocked by Mimosine through Reactive Oxygen Species-activated Ataxia Telangiectasia Mutated (ATM) Protein without DNA Damage

Mimosine is an effective cell synchronization reagent used for arresting cells in late G₁ phase. However, the mechanism underlying mimosine-induced G₁ cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mm mimosine for a further 15 h (thymidine → mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G₁-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.     

Vertical and temporal shifts in microbial communities in the water column and sediment of saline meromictic Lake Kaiike (Japan), as determined by a 16S rDNA-based analysis, and related to physicochemical gradients

The vertical and temporal changes in microbial communities were investigated throughout the water column and sediment of the saline meromictic Lake Kaiike by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Marked depth-related changes in microbial communities were observed at the chemocline and the sediment-water interface. However, no major temporal changes in the microbial community below the chemocline were observed during the sampling period, suggesting that the ecosystem in the anoxic zone of Lake Kaiike was nearly stable. Although the sequence of the most conspicuous DGGE band throughout the anoxic water and in the top of the microbial mat was most similar to that of an anoxic, photosynthetic, green sulphur bacterium, Pelodyction luteolum DSM273 (97% similarity), it represented a new phylotype. A comparison of DGGE banding patterns of the water column and sediment samples demonstrated that specific bacteria accumulated on the bottom from the anoxic water layers, and that indigenous microbial populations were present in the sediment. The measurements of bicarbonate assimilation rates showed significant phototrophic assimilation in the chemocline and lithoautotrophic assimilation throughout the anoxic water, but were not clearly linked with net sulphide turnover rates, indicating that sulphur and carbon metabolisms were not directly correlated.     

Purification, characterization, and molecular cloning of lectin from winter buds of Lysichiton camtschatcensis (L.) Schott

A novel lectin was purified to homogeneity from winter buds of Lysichiton camtschatcensis (L.) Schott of the Araceae family. It was a tetramer composed of two non-covalently associated polypeptides with small subunits (11 kDa) and large subunits (12 kDa). Sequencing of both subunits yielded unique N-terminal sequences. A cDNA encoding the lectin was cloned. The isolated cDNA contained an open reading frame that encoded 267 amino acids. It encoded both subunits, indicating that the lectin is synthesized as a single precursor protein that is post-translationally processed into two different subunits with 45% sequence identity. Each subunit contained a mannose-binding motif known to be conserved in monocot mannose-binding lectins, but its activity was not inhibited by monosaccharides, including methyl α-mannoside. Asialofetuin and yeast invertase were potent inhibitors. Lectin activity was detected in the buds formed during the winter season but not in the expanded leaves.     

Cysteine catabolism and cysteine desulfhydrase (CdsH/STM0458) in Salmonella enterica serovar typhimurium

Cysteine is potentially toxic and can affect diverse functions such as oxidative stress, antibiotic resistance, and swarming motility. The contribution of cysteine catabolism in modulating responses to cysteine has not been examined, in part because the genes have not been identified and mutants lacking these genes have not been isolated or characterized. We identified the gene for a previously described cysteine desulfhydrase, which we designated cdsH (formerly STM0458). We also identified a divergently transcribed gene that regulates cdsH expression, which we designated cutR (formerly ybaO, or STM0459). CdsH appears to be the major cysteine-degrading and sulfide-producing enzyme aerobically but not anaerobically. Mutants with deletions of cdsH and ybaO exhibited increased sensitivity to cysteine toxicity and altered swarming motility but unaltered cysteine-enhanced antibiotic resistance and survival in macrophages.