DHMEQ, a new NF-kappaB inhibitor, induces apoptosis and enhances fludarabine effects on chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (CLL) is a low-grade lymphoid malignancy incurable with conventional modalities of chemotherapy. Strong and constitutive nuclear factor kappa B (NF-kappaB) activation is a characteristic of CLL cells. We examined the effects of a new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on CLL cells. Dehydroxymethylepoxyquinomicin completely abrogated constitutive NF-kappaB activity and induced apoptosis of CLL cells. Apoptosis induced by DHMEQ was accompanied by downregulation of NF-kappaB-dependent antiapoptotic genes: c-IAP, Bfl-1, Bcl-X(L) and c-FLIP. Dehydroxymethylepoxyquinomicin also inhibited NF-kappaB induced by CD40 and enhanced fludarabine-mediated apoptosis of CLL cells. Results of this study suggest that inhibition of constitutive and inducible NF-kappaB by DHMEQ in combination with fludarabine is a promising strategy for the treatment of CLL.

Enhancement of styrene removal by Pseudomonas sp. SR-5 in mixed culture with a benzoic acid-degrading bacterium in biofilter.

The growth of a styrene-degrading bacterium, Pseudomonas sp. SR-5, was inhibited by benzoic acid (BA), one of the styrene degradation intermediates, in liquid culture. A benzoic acid-degrading microorganism, Raoultella sp. strain A, was isolated from a peat biofilter inoculated with a wastewater. The styrene removal efficiencies of the two laboratory-scale biofilters inoculated with only strain SR-5 and a mixed culture of strains SR-5 and A were compared using a mixed packing material of peat and ceramic (1:1) for 45 days. The biofilter with the mixed culture showed a higher removal efficiency than that with a single culture of SR-5. The maximum elimination capacities of the biofilters with the mixed culture and the single culture were 141 g m(-3)h(-1) and 106 g m(-3)h(-1), respectively. In the biofilter with the single culture, 136 g of benzoic acid (m3 of dry packing material)(-1) was accumulated at the end of the experiment. However, no accumulation of benzoic acid was observed in the biofilter with the mixed culture.

Plasmid transformation in Bacillus subtilis NB22, an antifungal-antibiotic iturin producer.

A transformation system with plasmids was developed for Bacillus subtilis NB22, an antibiotic iturin producing strain. Treatment of B. subtilis NB22 with 4 M KCl was effective for the induction of competence, followed by uptake of plasmid DNA in the presence of polyethylene glycol. The efficiency of transformation of this bacterium with pC194 and pUB110 was 4.1 X 10(3) and 1.5 X 10(3) transformants per micrograms DNA, respectively and the transformation frequency was 3.3 X 10(-3) and 7.2 X 10(-4), transformants per viable cell, respectively. This method was much faster and three orders of magnitude more efficient in transformation efficiency than protoplast transformation methods.

Effect of addition of water-soluble polysaccharides on bacterial cellulose production in a 50-L airlift reactor.

Bacterial cellulose (BC) production was carried out in a batch cultivation of Acetobacter xylinum in a 50-L internal loop airlift reactor by addition of water-soluble polysaccharides into the medium. When 0.1% (w/w) agar was added, BC production reached 8.7 g/L compared with 6.3 g/L in the control, and duration of the cultivation period to reach the maximum concentration of BC was almost half of that without addition of polysaccharides. During cultivation, BC was formed into pellets whose size was smaller when the productivity of BC was higher, indicating that increase in the relative viscosity by addition of polysaccharides hindered formation of large clumps of BC and increase in the volumetric oxygen transfer coefficient at high flow rate led to increase in BC productivity.

Pathogenicity of Rhodococcus equi expressing a virulence-associated 20 kDa protein (VapB) in foals.

Rhodococcus equi strains of intermediate virulence (IMV) for mice possess a 20kDa protein designated Virulence Associated Protein B (VapB) and a virulence plasmid of 79-100kb, and can be recovered from the submaxillary lymph nodes of pigs. The pathogenicity of such R. equi strains for foals is unknown. In this study, two foals, 42 and 43 days of age, were infected intratracheally with 10(6) and 10(9) cells of R. equi IMV strain A5, respectively. The foal infected with 10(9) cells of strain A5 became clinically ill, with the onset of illness (pyrexia and depression) occurring 21 days after inoculation. R. equi was isolated from the feces and tracheal washings of the foal from 14 to 28 days after inoculation. The foal infected with 10(6) cells of A5 showed no clinical signs, and no R. equi was isolated from any of the samples of feces or tracheal washings during the 28 days of observation. Two foals of 45 and 50 days of age were infected with 10(5) or 10(6) of virulent R. equi ATCC 33701 having 15-17kDa surface proteins designated VapA. Both exhibited severe clinical signs (pyrexia, depression and anorexia) at 12 and 13 days after inoculation. Histopathological examination revealed that strain A5 caused focal granulomatous pneumonia in the foals. R. equi IMV strain A5 was isolated from lung lesions of both foals and from the contents of the intestinal tracts of the foal infected with 10(9) bacteria. These results suggest that IMV R. equi having VapB is less virulent than virulent R. equi having VapA in foals. This finding supports our previous results on the pathogenicities of R. equi strains having these virulence-associated antigens assessed by mouse pathogenicity tests.

Disappearance of growth advantage in stationary phase (GASP) phenomenon under a high magnetic field.

The phenomenon called growth advantage in stationary phase (GASP) originally discovered by Kolter et al. was confirmed using two bacterial strains, Escherichia coli ZK126Nalr and ZK126Smr, under the geomagnetic field. However, when the same experiment was conducted in an inhomogeneous high magnetic field of 5.2-6.1 T, almost no death of ZK126Smr was observed and the GASP phenomenon disappeared. When the same experiment was conducted in a homogeneous magnetic field of 7 T, the GASP was significantly delayed due to the much slower death rate of ZK126Smr than that in the geomagnetic field. These data are consistent with our previous finding that a high magnetic field reduces the death rate of bacteria and enhances their survivability in a stationary phase.

Twelve hours exposure to inhomogeneous high magnetic field after logarithmic growth phase is sufficient for drastic suppression of Escherichia coli death.

When Escherichia coli B was aerobically grown at 43 degrees C in a medium whose concentration was one-fourth that of the Luria-Bertani (LB) medium supplemented with 1.5 g/l of glutamic acid, drastic cell death was observed after the end of the logarithmic growth phase. However, when the same experiment was conducted under inhomogeneous 5.2-6.1 T magnetic field, cell death was extremely suppressed and the ratio of viable cell number under high magnetic field to that under geomagnetic field reached as much as 100,000. When the magnetic field exposure was restricted to 12 h after the logarithmic growth phase, a similar high degree of suppressive effect on the death was observed. The findings that the amount of sigma S protein encoded by the rpoS gene under the high magnetic field was larger than that under the geomagnetic field, and that the magnetic field effect disappeared when the rpoS gene-deficient strain was cultivated under the high magnetic field, suggest the interaction of magnetic field with a stationary phase specific gene.

Drastic high magnetic field effect on suppression of Escherichia coli death.

When Escherichia coli B was aerobically grown in a medium containing one-fourth the concentration of the LB medium supplemented with glutamic acid at 43 degrees C under an inhomogeneous 5.2-6.1 T magnetic field, the number of cells in the stationary phase under the high magnetic field was 100,000 times higher than that under a geomagnetic field. The finding that the amount of sigma S factor encoded by the rpoS gene under the high magnetic field was larger than that under the control geomagnetic field indicated that the activity of the rpoS gene was affected by the high magnetic field.

Bacterial control of plant diseases.

This article focuses on the effective biocontrol of plant diseases by microorganisms, which is attracting attention as an alternative to chemical control methods. As most research has so far been concentrated on fluorescent Pseudomonas species, the use of Bacillus species which has been considered to be less effective compared to that of pseudomonads, has been mainly introduced to demonstrate the effectiveness of the bacteria.

Integrated biological and chemical control of damping-off caused by Rhizoctonia solani using Bacillus subtilis RB14-C and flutolanil.

Bacillus subtilis RB14-C was isolated as a potential biological agent to control the occurrence of various plant diseases. Integrated control of damping-off in tomato plants caused by Rhizoctonia solani was carried out in pots using B. subtilis RB14-C and chemical pesticide, flutolanil. The growth of RB14-C was the same in both the flutolanil-containing and the flutolanil-free media, indicating the resistance of RB14-C to flutolanil. Although the productivity of surfactin decreased to one-third in the flutolanil-containing medium, compared with that in the flutolanil-free medium, the productivity of iturin A which is mainly associated with the suppressive ability of RB14-C against plant pathogens was unaffected. The integration of RB14-C and flutolanil reduced the amount of flutolanil used to one-fourth of that of the single use of flutolanil, with the same efficacy of reducing disease occurrence.

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005.

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27 degrees C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.

cDNA cloning and genetic analysis of a novel decolorizing enzyme, peroxidase gene dyp from Geotrichum candidum Dec 1.

A novel decolorizing peroxidase gene (dyp) was cloned from a cDNA library of a newly isolated strain of fungus Geotrichum candidum Dec 1. The open reading frame of 1494 nucleotides which corresponds to dyp predicts a primary translation product of 498 amino acids, M(r) 53,306. The deduced amino acid sequence of DyP does not contain the typical conserved motif which is characteristic of heme-containing peroxidases in the plant peroxidase superfamily. Comparison of the deduced amino acid sequence of DyP with that of a peroxidase from Polyporaceae sp. suggests that these proteins share highly homologous regions.

Batch decolorization of molasses by suspended and immobilized fungus of Geotrichum candidum Dec 1.

Geotrichum candidum Dec 1, which exhibits a broad dye-decolorizing spectrum, was used to decolorize molasses during semi-batch cultivation. An eighty % decolorization of molasses and a stable peroxidase activity were maintained for approximately four weeks, after which, both activities deteriorated significantly. Subsequently, repeated batch cultivation of fungal cells immobilized on polyurethane foam was employed to solve the aforementioned problems and stable decolorization of molasses as well as stable peroxidase activity was realized for more than eight weeks.

Comparison of the biological H2S removal characteristics among four inorganic packing materials.

Four inorganic packing materials were evaluated in terms of their availability as packing materials of a packed tower deodorization apparatus (biofilter) from the viewpoints of biological H2S removal characteristics and some physical properties. Among porous ceramics (A), calcinated cristobalite (B), calcinated and formed obsidian (C), granulated and calcinated soil (D), the superiority of these packing materials determined based on the values of non-biological removal per unit weight or unit volume of packing material, complete removal capacity of H2S per unit weight of packing material per day or unit volume of packing material per day and pressure drop of the packed bed was in the order of A approximately equal to C > D approximately equal to B, which is correlated with the maximum water content, porosity, and mean pore diameter.

Comparison of the biological NH3 removal characteristics among four inorganic packing materials.

Four inorganic packing materials were evaluated in terms of their availability as a packing material of a packed tower deodorization apparatus (biofilter) from the viewpoints of biological NH3 removal characteristics and some physical properties. Porous ceramics (A), calcinated cristobalite (B), calcinated and formed obsidian (C), granulated and calculated soil (D) were used. The superiority of these packing materials determined based on the values of non-biological removal per unit weight or unit volume of packing material, complete removal capacity of NH3 per unit weight of packing material per day or unit volume of packing material per day and pressure drop of the packed bed was in the order of A approximately = C > B > or = D. Packing materials A and C with high porosity, maximum water content, and suitable mean pore diameter showed excellent removal capacity.

Effect of molasses on the production and activity of dye-decolorizing peroxidase from Geotrichum candidum Dec1.

The production of dye-decolorizing peroxidase (DyP) was investigated by cultivating Geotrichum candidum Dec1 using molasses as a carbon source. Molasses at concentrations greater than 10 g.l(-1) was found to increase the decolorization activity of the culture broth toward dye, reactive blue 5 mainly because the amount of enzyme produced was enhanced. However, complete inhibition of DyP activity by molasses was observed at the concentration of 20 g.l(-1), indicating that the inhibitory effect of molasses on the culture broth activity to decolorize the dye was involved. When the culture broth was diluted 25 times, the dye-decolorizing activity was 7 times as much as that of non-diluted culture broth. The molasses fractions separated by gel chromatography (300-400 ml and 400-500 ml fractions) completely inhibited the purified DyP. On the basis of these results, we propose a scheme to control both positive and negative effects of molasses on the dye decolorization process.

Removal of a high load of ammonia gas by a marine bacterium, Vibrio alginolyticus.

A newly isolated marine bacterium, Vibrio alginolyticus was used to remove a high load of ammonia gas. By a stepwise increase in ammonia supply over the concentration range of 120-2000 ppm (v/v), complete removal of ammonia was observed from the start of the experiment in a suspended culture of the bacterium in basal medium containing 3% NaCl. When cells were inoculated onto an inorganic packing material in a biofilter, and a high load of ammonia was introduced continuously under nonsterile conditions, the average percentage of gas removed exceeded 85% for a 61-d operation. The maximum removal capacity and the complete removal capacity were 22.8 g-N/kg-dry packing material/d and 18.6 g-N/kg-dry packing material/d, respectively, which were about four times larger than those obtained in nitrifying sludge inoculated onto the same packing material. During this operation, the nonsterile air supply had no adverse effect on the removability of ammonia by V. alginolyticus.

Characterization of aryl alcohol oxidase produced by dye-decolorizing fungus, Geotrichum candidum Decl.

Aryl alcohol oxidase (AAO) produced by Geotrichum candidum Dec1 (Dec1), a newly isolated decolorizing fungus, was purified by ultrafiltration and by using diethylaminoethyl (DEAE) Sephacel, Butyl-Toyopearl and Mono-Q columns. H2O2 produced by concomitant AAO oxidation of veratryl alcohol (VA) to veratraldehyde was consumed by a peroxidase (DyP) purified from Dec1 culture, leading to the decolorization of a dye (in vitro). In the liquid culture of Dec1, the existence of H2O2 and veratraldehyde was confirmed during cultivation, when dye-decolorization and AAO activities were maintained. This indicates that VA produced by Dec1 was oxidized by AAO to veratraldehyde, generating H2O2, which supported dye-decolorizing activity of Decl in vivo. The prevention of polymerization of DyP oxidation products of a dye in the presence of AAO was shown.

A pitfall in the interhemispheric translamina terminalis approach for the removal of a craniopharyngioma. Significance of preserving draining veins. Part I. Clinical study.

The interhemispheric translamina terminalis approach has made total removal of a craniopharyngioma less risky. Nevertheless, one of the pitfalls of this approach is cutting of the draining veins combined with retraction. A case of craniopharyngioma in which a subcortical hematoma developed is reported. The divided draining veins and the 60-minute retraction period were thought to have caused this complication. We therefore determined the clinical effect of cutting the draining veins and retraction on the regional cerebral blood flow and the motor nerve action potential. There were marked changes and a hematoma occurred mostly when the veins were divided and retraction was applied over the area.

Functional electrical stimulation (FES) for spinal cord injury.

Restoration of respiratory motion by stimulation of the phrenic nerve was investigated. Respiratory motion was restored successfully by introducing a breathing pacemaker to a patient with respiratory disturbance due to upper cervical spinal cord injury. Breathing pacemakers are considered to be more similar to physiological conditions compared to mechanical ventilators. Although the system is very expensive, its cost effectiveness may be excellent, provided that it can be used for long hours each day over an extended period. The system is effective in improving patient QOL because it dramatically increases patient mobility. From these findings, it is concluded that breathing pacemakers should be used more frequently in Japan, and that various forms of support are necessary to cope with economic and other concerns.