Thermophilic Biodesulfurization of Various Heterocyclic Sulfur Compounds and Crude Straight-Run Light Gas Oil Fraction by a Newly Isolated Strain Mycobacterium phlei WU-0103

Various heterocyclic sulfur compounds such as naphtho[2,1-b]thiophene (NTH) and benzo[b]thiophene (BTH) derivatives can be detected in diesel oil, in addition to dibenzothiophene (DBT) derivatives. Mycobacterium phlei WU-0103 was newly isolated as a bacterial strain capable of growing in a medium with NTH as the sulfur source at 50°C. M. phlei WU-0103 could degrade various heterocyclic sulfur compounds, not only NTH and its derivatives but also DBT, BTH, and their derivatives at 45°C. When M. phlei WU-0103 was cultivated with the heterocyclic sulfur compounds such as NTH, NTH 3,3-dioxide, DBT, BTH, and 4,6-dialkylDBTs as sulfur sources, monohydroxy compounds and sulfone compounds corresponding to starting heterocyclic sulfur compounds were detected by gas chromatography–mass spectrometry analysis, suggesting the sulfur-specific desulfurization pathways for heterocyclic sulfur compounds. Moreover, total sulfur content in 12-fold-diluted crude straight-run light gas oil fraction was reduced from 1000 to 475 ppm S, with 52% reduction, by the biodesulfurization treatment at 45°C with growing cells of M. phlei WU-0103. Gas chromatography analysis with a flame photometric detector revealed that most of the resolvable peaks, such as those corresponding to alkylated derivatives of NTH, DBT, and BTH, disappeared after the biodesulfurization treatment. These results indicated that M. phlei WU-0103 may have a good potential as a biocatalyst for practical biodesulfurization of diesel oil.     

Cirp protects against tumor necrosis factor-alpha-induced apoptosis via activation of extracellular signal-regulated kinase

Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we analyzed the effects of low culture temperature (32 degrees C) and cold-inducible RNA-binding protein (Cirp) expression on apoptosis in vitro. In BALB/3T3 cells treated with tumor necrosis factor (TNF)-alpha and cycloheximide, the down-shift in temperature from 37 degrees C to 32 degrees C increased the expression of Cirp and suppressed the apoptosis. Activation of caspase-8 was suppressed, and the level of phosphorylated extracellular signal-regulated kinase (ERK) was increased. Transduction of Cirp into the Cirp-deficient mouse fibroblasts increased the level of phosphorylated ERK and suppressed the TNF-alpha-induced apoptosis both at 37 degrees C and 32 degrees C. The ERK-specific inhibitor PD98059 decreased the cytoprotective effect of Cirp as well as that of low culture temperature. These data suggest that mild hypothermia protects cells from TNF-alpha-induced apoptosis, at least partly, via induction of Cirp, and that Cirp protects cells by activating the ERK pathway.     

ALS-linked P56S-VAPB, an aggregated loss-of-function mutant of VAPB, predisposes motor neurons to ER stress-related death by inducing aggregation of co-expressed wild-type VAPB

A point mutation (P56S) in the vapb gene encoding an endoplasmic reticulum (ER)-integrated membrane protein [vesicle-associated membrane protein-associated protein B (VAPB)] causes autosomal-dominant amyotrophic lateral sclerosis. In our earlier study, we showed that VAPB may be involved in the IRE1/XBP1 signaling of the unfolded protein response, an ER reaction to inhibit accumulation of unfolded/misfolded proteins, while P56S-VAPB formed insoluble aggregates and lost the ability to mediate the pathway (loss-of-function), and suggested that P56S-VAPB promoted the aggregation of co-expressed wild-type (wt)-VAPB. In this study, a yeast inositol-auxotrophy assay has confirmed that P56S-VAPB is functionally a null mutant in vivo . The interaction between P56S-VAPB and wt-VAPB takes place with a high affinity through the major sperm protein domain in addition to the interaction through the C-terminal transmembrane domain. Consequently, wt-VAPB is speculated to preferentially interact with co-expressed P56S-VAPB, leading to the recruitment of wt-VAPB into cytosolic aggregates and the attenuation of its normal function. We have also found that expression of P56S-VAPB increases the vulnerability of NSC34 motoneuronal cells to ER stress-induced death. These results lead us to hypothesize that the total loss of VAPB function in unfolded protein response, induced by one P56S mutant allele, may contribute to the development of P56S-VAPB-induced amyotrophic lateral sclerosis.     

Quantitative analysis of CUG-BP1 binding to RNA repeats

CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-3-like factors (CELF) family of RNA-binding proteins, and is involved in myotonic dystrophy type 1 (DM1). Several mRNA targets of CUG-BP1 have been identified, including the insulin receptor, muscle chloride channel, and cardiac troponin T. On the other hand, CUG-BP1 has only a weak affinity for CUG repeats. We conducted quantitative-binding assays to assess CUG-BP1 affinities for several repeat RNAs by surface plasmon resonance (SPR). Although we detected interactions between CUG-BP1 and CUG repeats, other UG-rich sequences actually showed stronger interactions. Binding constants of CUG-BP1 for RNAs indicated that the affinity for UG repeats was far stronger than for CUG repeats. We also found that N-terminal deletion mutant of CUG-BP1 has UG repeat-binding activity in a yeast three-hybrid system, although C-terminal deletion mutant does not. Our data indicates that CUG-BP1 specifically recognized UG repeats, probably through cooperative binding of RNA recognition motifs at both ends of the protein. This is the first report of a binding constant for CUG-BP1 calculated in vitro.     

Utilization of hydrophobic bacterium <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> B-4 as whole-cell catalyst in anhydrous organic solvents

Rhodococcus opacus strain B-4, which has recently been isolated as an organic solvent-tolerant bacterium, has a high hydrophobicity and exhibits a high affinity for hydrocarbons. This bacterium was able to survive for at least 5 days in organic solvents, including n-tetradecane, oleyl alcohol, and bis(2-ethylhexyl) phthalate (BEHP), which contained water less than 1% (w/v). The biocatalytic ability of R. opacus B-4 was demonstrated in the essentially nonaqueous BEHP using indigo production from indole as a model conversion. By the catabolism of oleic acid for NADH regeneration, indigo production increased up to 71.6 μg ml⁻¹ by 24 h.     

Chromosomal mapping of host resistance loci to Trichinella spiralis nematode infection in rats

The differences in host response among strains of rats to intestinal nematode parasite Trichinella spiralis infection could provide a powerful benefit for further elucidation of molecular interactions between the host and the parasite. Using several strains of rats, we previously observed that DA strain is a strong responder and F344 strain is a weak responder with respect to expulsion of the adult worm. To identify the host resistance loci, quantitative trait loci (QTLs) analysis in F2 population from crosses between DA and F344 strains was performed. One significant QTL (designated as Tspe) was mapped to the middle region of chromosome 9. In addition, the effect of DA allele at Tspe locus could act recessively and lead to the rejection of more adult worms from the gut. The results from the present study provide more insights on host–parasite interactions, which may be useful in facilitating the development of novel approaches for treatment and control of intestinal parasites in human and domestic livestock.     

Involvement of ASK1 in Ca2+-induced p38 MAP kinase activation

The mammalian mitogen-activated protein (MAP) kinase kinase kinase apoptosis signal-regulating kinase 1 (ASK1) is a pivotal component in cytokine- and stress-induced apoptosis. It also regulates cell differentiation and survival through p38 MAP kinase activation. Here we show that Ca²⁺ signalling regulates the ASK1–p38 MAP kinase cascade. Ca²⁺ influx evoked by membrane depolarization in primary neurons and synaptosomes induced activation of p38, which was impaired in those derived from ASK1-deficient mice. Ca²⁺/calmodulin-dependent protein kinase type II (CaMKII) activated ASK1 by phosphorylation. Moreover, p38 activation induced by the expression of constitutively active CaMKII required endogenous ASK1. Thus, ASK1 is a critical intermediate of Ca²⁺ signalling between CaMKII and p38 MAP kinase.     

Preparation and characterization of recombinant murine p65/L-plastin expressed in Escherichia coli and high-titer antibodies against the protein

We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages. It has turned out to be a murine homologue of human L-plastin, which was identified as a novel protein in human cancer cells. p65/L-plastin is characterized by a series of Ca(2+)-, calmodulin-, and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells. We have expressed a recombinant (r) p65/L-plastin in Escherichia coli that binds to beta-actin and prepared high-titer antibodies using large amounts of the protein as immunogen. Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect the fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components. We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies, including autoantibodies, against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes.     

Plasmodium berghei: lack of antimalarial activity of an analogue of folate precursor, 2,4-diamino-6-hydroxymethylpteridine in a mouse model

It was earlier hypothesized that the malarial parasite may convert precursors of folate analogues to synthesize de novo inhibitors toxic to itself, but not to the mammalian cell. It was suggested that one such analogue, 2,4-diamino-6-hydroxymethylpteridine (DAP) may be converted to aminopterin (AMP), a known dihydrofolate reductase inhibitor. In the present study, we evaluated the ability of DAP to inhibit proliferation of Plasmodium berghei NK65 in mice, with(out) folinic acid rescue. Cumulative dosages of DAP ranging from 0.1 to 20 mg/kg bw. administered either orally or intraperitoneally showed no suppression of parasite growth, or gave mild activities that were not statistically significant (P > 0.05). Our findings do not seem to support the hypothesis of selective de novo metabolism of DAP to AMP by the malarial parasite.     

Biochemical rust removal by Thiobacillus ferrooxidans

Summary Biochemical removal of rust from iron surfaces has been investigated. By immersing a rusted iron plate in the culture medium of an iron-oxidizing bacterium, Thiobacillus ferrooxidans, iron adjacent to the rust was dissolved and the rust was peeled off. Since the amount of dissolved iron per unit iron plate surface area correlated with the concentration of ferric iron in the culture medium, the formation of ferric iron is probably involved in dissolving the iron as is the case for bacterial leaching. In the present study, rust removal in a “continuous” system in which the culture medium was circulated from the fermentor to the rust removal vessel and back again to the fermentor, has also been investigated. Although growth inhibition was observed with the formation of ferric iron precipitates during the operation in this system, it was possible to prevent this precipitation by lowering the pH of the medium during the mixed cultivation of T. ferrooxidans and a sulfur-oxidizing bacterium, T. thiooxidans.