Utilization of n-Paraffins and Vitamin B6 Production by Pichia guilliermondii Wickerham

The accumulation of vitamin B₆ by Pichia guilliermondii Wickerham NK–2 strain grown on hydrocarbon was investigated. Ammonium acetate was more effective than other nitrogen sources tested. Satisfactory utilization by the yeast strain was observed in n-alkanes of C₁₀–C₁₈, and n-pentadecane was the best for vitamin B₆ production. Vitamin B₆ was excreted in the cultural broth mainly in the form of pyridoxal, The maximal vitamin B₆ production was approximately 25 mg per liter of the culture broth.     

Peroxisomal membrane protein Pmp47 is essential in the metabolism of middle-chain fatty acid in yeast peroxisomes and Is associated with peroxisome proliferation

Pmp47 of the methylotrophic yeast Candida boidinii belongs to a mitochondrial family of solute transporters and is localized in peroxisomal membranes. Its human homolog, Pmp34, is also known. In this study, we characterized the role of Pmp47 in fatty acid metabolism and peroxisome proliferation using the PMP47-deleted strain of C. boidinii (strain pmp47Delta). The wild-type strain grew well on a middle-chain fatty acid, laureate, as the single carbon source, and mild peroxisome proliferation was observed during its growth. The pmp47Delta strain could not grow on laureate but could grow on long-chain fatty acids including palmitate, myristate, and oleate. The levels of laureate oxidation activity in intact cells and in semi-permeabilized cells of strain pmp47Delta were lower than the respective level in the wild-type strain, although the level of laureate oxidation activity in the cell lysate and the level of lauroyl-CoA oxidation in semi-permeabilized cells of strain pmp47Delta were indistinguishable from the respective level in the wild-type strain. When lauroyl-CoA was provided in the cytosol of strain pmp47Delta through expression of Saccharomyces cerevisiae Faa2p (lauroyl-CoA synthetase) in which its peroxisome targeting signal was deleted, the growth of strain pmp47Delta on laureate was recovered to the level of growth of the wild-type strain. Laureate is converted to its CoA form in peroxisomes by the action of lauroyl-CoA synthetase. These results suggested that Pmp47 is involved in the transport of a small molecule (possibly ATP) required in the conversion of laureate to its CoA form in peroxisomes and that the absence of Pmp47 causes impairment of laureate metabolism, which results in the inability of pmp47Delta cells to grow on laureate. In addition, Pmp47 may be involved in peroxisome proliferation, because the pmp47Delta strain contained a reduced number of peroxisomes, as judged from the fluorescence analysis of cells expressing green fluorescent protein tagged with the peroxisome targeting signal 1 (GFP-AKL).     

A Pichia pastoris single-cell biosensor for detection of enzymatically produced methanol

Applied Microbiology and Biotechnology - We conducted single-cell analyses of the methylotrophic yeast Pichia pastoris to develop a biosensor for the detection of methanol produced by heterologous...     

Serum melatonin levels and insomnia in patients with Machado-Joseph Disease

We previously reported a patient with Machado-Joseph Disease (MJD) who had severe insomnia and a low serum melatonin (MLT) level, and whose insomnia was alleviated by oral MLT replacement therapy. The aims of this study were to examine whether patients with MJD are likely to have insomnia, and whether there is a relationship between the degree of insomnia and the serum MLT level among patients with MJD. This study included 8 patients with MJD. A 58-year-old-patient with cervical spondylosis was also included in this study to check the condition of the test room for sleeping. All patients filled out the Japanese version of Pittsburgh Sleep Quality Index (PSQI-J) questionnaire. We obtained blood samples at 12:00 and 24:00 hours to measure the MLT level. We checked the sleep condition of the patient once an hour and recorded the grade in sleep-logs: the grades of sleep condition were asleep, sleepy, or awake. Statistical analyses were performed to search for correlations between the PSQI score and the serum MLT level or actual sleep time using Spearman’s rank correlation coefficient. Seven of the 8 MJD patients had a total PSQI score of above 5.5 (cut-off level). The daytime MLT level (at 12:00 hours) was below 2.8 pg/mL in all 8 patients, whereas the mean night-time MLT level (at 24:00 hours) of the MJD patients (23.6 ± 17.5 pg/mL) was lower than that of the control patient (43.0 pg/mL) and also lower than the reported cut-off level among healthy people aged 30–50 years (55.5 pg/mL). There was a negative correlation between the total PSQI score and the serum MLT level among the MJD patients (P < 0.05). Our results show that a low serum MLT level may contribute to insomnia in patients with MJD.

     

Microtubules inside the plasma membrane of squid giant axons and their possible physiological function

Summary The effects of application of the microtubule-disassembling reagents to squid giant axons upon resting potential, the height of the propagated action potential, and the threshold to evoke action potential were studied using colchicine, podophyllotoxin, vinblastine, griseofulvin, sulfhydryl reagents including NEM, diamide, DTNB and PCMB, and Ca²⁺ ions. At the same time, the effects of concentrations of K halides and K glutamate on the above physiological properties were studied in comparison within vitro characteristics of microtubule assembly from purified axoplasmic tubulin.It was found that there was good correlation between conditions supporting maintenance of membrane excitability and microtubule assembly. The experiments suggest that associated with the internal surface of the plasma membrane there are microtubules which regulate in part both resting and action potentials.     

Dynamics of methane in mesotrophic Lake Biwa,Japan

As a part of a core project of IGBP (International Geosphere-Biosphere Programme), distribution, production, oxidation and transport processes of methane in bottom sediments and lake water in a mesotrophic lake (Lake Biwa) have been studied with special reference to the spatial heterogeneity of each process. In this study, we attempted to synthesize previously reported results with newly obtained ones to depict the methane dynamics in the entire lake. The pelagic water column exhibited subsurface maxima of dissolved methane during a stratified period. Transect observation at the littoral zone suggested that horizontal transportation may be a reason for the high methane concentration in epilimnion and thermocline at the offshore area. Tributary rivers and littoral sediments were suggested to be the source. Observations also showed that the internal wave caused resuspension of the bottom sediment and release of methane from the sediment into the lake water. The impact of the internal waves was pronounced in the late stage of a stratified period. The littoral sediment showed much higher methanogenic activity than the profundal sediments, and the bottom water of the littoral sediments had little methanotrophic activity. In the profundal sediment, most of the methane that diffused up from the deeper part was oxidized when it passed through the oxic layer. Active methane oxidation was also observed in the hypolimnetic water, while the lake water in the epilimnion and thermocline showed very low methane oxidation, probably due to the inhibitory effect of light. These results mean a longer residence time for methane in the epilimnion than in the hypolimnion. Horizontal inflow of dissolved methane from the river and/or littoral sediment, together with the longer residence time in the surface water, may cause the subsurface maxima, which have also been observed in other lakes and in the ocean.     

Crystal structure of an archaeal peroxiredoxin from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1

Peroxiredoxins (Prxs) are thiol-dependent peroxidases that catalyze the detoxification of various peroxide substrates such as H2O2, peroxinitrite, and hydroperoxides, and control some signal transduction in eukaryotic cells. Prxs are found in all cellular organisms and represent an enormous superfamily. Recent genome sequencing projects and biochemical studies have identified a novel subfamily, the archaeal Prxs. Their primary sequences are similar to those of the 1-Cys Prxs, which use only one cysteine residue in catalysis, while their catalytic properties resemble those of the typical 2-Cys Prxs, which utilize two cysteine residues from adjacent monomers within a dimer in catalysis. We present here the X-ray crystal structure of an archaeal Prx from the aerobic hyperthermophilic crenarchaeon, Aeropyrum pernix K1, determined at 2.3 A resolution (Rwork of 17.8% and Rfree of 23.0%). The overall subunit arrangement of the A.pernix archaeal Prx is a toroid-shaped pentamer of homodimers, or an (alpha2)5 decamer, as observed in the previously reported crystal structures of decameric Prxs. The basic folding topology and the peroxidatic active site structure are essentially the same as those of the 1-Cys Prx, hORF6, except that the C-terminal extension of the A.pernix archaeal Prx forms a unique helix with its flanking loops. The thiol group of the peroxidatic cysteine C50 is overoxidized to sulfonic acid. Notably, the resolving cysteine C213 forms the intra-monomer disulfide bond with the third cysteine, C207, which should be a unique structural characteristic in the many archaeal Prxs that retain two conserved cysteine residues in the C-terminal region. The conformational flexibility near the intra-monomer disulfide linkage might be necessary for the dramatic structural rearrangements that occur in the catalytic cycle.     

Comparative Analysis of argK-tox Clusters and Their Flanking Regions in Phaseolotoxin-Producing Pseudomonas syringae Pathovars

DNA fragments containing argK-tox clusters and their flanking regions were cloned from the chromosomes of Pseudomonas syringae pathovar (pv.) actinidiae strain KW-11 (ACT) and P. syringae pv. phaseolicola strain MAFF 302282 (PHA), and then their sequences were determined. Comparative analysis of these sequences and the sequences of P. syringae pv. tomato DC3000 (TOM) (Buell et al., Proc Natl Acad Sci USA 100:10181–10186, 2003) and pv. syringae B728a (SYR) (Feil et al., Proc Natl Acad Sci USA 102:11064–11069, 2005) revealed that the chromosomal backbone regions of ACT and TOM shared a high similarity to each other but presented a low similarity to those of PHA and SYR. Nevertheless, almost-identical DNA regions of about 38 kb were confirmed to be present on the chromosomes of both ACT and PHA, which we named “tox islands.” The facts that the GC content of such tox islands was 6% lower than that of the chromosomal backbone regions of P. syringae, and that argK-tox clusters, which are considered to be of exogenous origin based on our previous studies (Sawada et al., J Mol Evol 54:437–457, 2002), were confirmed to be contained within the tox islands, suggested that the tox islands were an exogenous, mobile genetic element inserted into the chromosomes of P. syringae strains. It was also predicted that the tox islands integrated site-specifically into the homologous sites of the chromosomes of ACT and PHA in the same direction, respectively, wherein 34 common gene coding sequences (CDSs) existed. Furthermore, at the left end of the tox islands were three CDSs, which encoded polypeptides and had similarities to the members of the tyrosine recombinase family, suggesting that these putative site-specific recombinases were involved in the recent horizontal transfer of tox islands. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.