Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY((R)) FL-labeled probe or primer.

We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY((R)) FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY((R)) FL-modified cytosine at its 5'-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples.

Microbial degradation and treatment of polycyclic aromatic hydrocarbons and plasticizers.

Rhodococcus erytropolis and Pseudomonas sp. rapidly degrade many kinds of polycyclic aromatic hydrocarbon (PAH) compounds such as phenanthrene and phthalate esters such as di(2-ethylhexyl) phthalate, used as plasticizers. These compounds were efficiently removed from wastewater by inoculating viable cells of Rhodococcus erythropolis and Pseudomonas sp. into activated sludge as a biological treatment system. The rapid PCR method and fluorescent antibody techniques were successfully applied for tracing the specified microorganisms, which were inoculated into a mixed culture system. The relationship of microflora to the removal rate of these compounds such as phthalate esters in inoculated biological treatment systems was examined. The metabolic pathway was investigated and enzymes were purified.

o-, m- and p-hydroxybenzoate degradative pathways in Rhodococcus erythropolis.

Rhodococcus erythropolis strain S1 uses the gentisate pathway to metabolize salicylate and m-hydroxybenzoate and the protocatechuate pathway to degrade p-hydroxybenzoate. m-Hydroxybenzoate 6-hydroxylase was induced by growth on m-hydroxybenzoate or gentisate, and salicylate 5-hydroxylase only by growth on salicylate. p-Hydroxybenzoate 3-hydroxylase could be induced only by growth on p-hydroxybenzoate. m-Hydroxybenzoate or p-hydroxybenzoate could repress the induction of salicylate 5-hydroxylase. Maleylpyruvate isomerase in the gentisate pathway did not require reduced glutathione.

Application of gel microdroplet and flow cytometry techniques to selective enrichment of non-growing bacterial cells.

We describe an application of gel microdroplet (GMD) and flow cytometry techniques to selective enrichment of non-growing Leuconostoc mesenteroides cells, which are well culturable on other media, from a mixture with Bacillus subtilis cells in nutrient broth. After encapsulating cells of the mixed population within GMDs and a brief incubation in nutrient broth, the inability of L. mesenteroides cells to form microcolonies within GMDs allowed their discrimination from B. subtilis cells. After staining the GMD mixture with 6-carboxyfluorescein diacetate, which showed no influence on cell viability, the GMDs containing single cells of L. mesenteroides were selectively collected using flow cytometry sorting based on differences in fluorescence intensity. The cells of L. mesenteroides retained viability during the process.

A new isolation method for labyrinthulids using a bacterium, Psychrobacter phenylpyruvicus.

A new isolation method for labyrinthulids, marine microbes with spindle-shaped vegetative cells and gliding movement, is presented. The method for isolating labyrinthulids has been found to be more difficult and less reproducible than that for thraustochytrids, classified in the same order. So far serum seawater agar fortified with antibiotics has been proposed to be the best for isolation of labyrinthulids. The method presented here involves placing plant samples on an agar medium on which a marine bacterium, Psychrobacter phenylpyruvicus, has been grown. The new method, which utilizes fallen mangrove leaves as source material, was more than twice as effective as isolation agar medium without the bacterium. The increased effectiveness appears to derive partly from the bacterial colonies' delaying extension of fungal mycelium. The bacterium was more effective for the isolation of labyrinthulids than either the bacterium Shewanella sp. or the yeast Rhodotorula rubra.

Production of eicosapentaenoic acid by a recombinant marine cyanobacterium, Synechococcus sp.

The eicosapentaenoic acid (EPA) synthesis gene cluster from an EPA-producing bacterium, Shewanella sp. SCRC-2738, was cloned into a broad-host range vector, pJRD215, and then introduced into a marine cyanobacterium, Synechococcus sp. NKBG15041c, by conjugation. The transconjugant cyanobacteria produced 3.7 +/- 0.2% (2.24 +/- 0.13 mg/L) EPA (n-3) and 2.5 +/- 0.2% (1.49 +/- 0.06 mg/L) eicosatetraenoic acid (n-3) of the total fatty acids when the cells were cultured at 23 degrees C at a light intensity of 1,000-1,500 Lux. The EPA and eico-satetraenoic acid contents of the cells were increased to 4.6 +/- 0.6% (3.86 +/- 1.11 mg/L) and 4.7 +/- 0.3% (3.86 +/- 0.82 mg/L), and 7.5 +/- 0.3% (1.76 +/- 0.10 mg/L) and 5.1 +/- 0.2% (1.19 +/- 0.06 mg/L) when they were cultured at low temperature (18 degrees C) and at lower light intensity (40 Lux), respectively.

Preparation of biopolymers with different viscosities by heat treatment under alkaline conditions.

Alcaligenes latus B-16 produces an extracellular biopolymer that exhibits high viscosity. The biopolymer (B-16 biopolymer) was recovered by adding an organic solvent into A. latus B-16 culture broth. B-16 biopolymers with different viscosities were prepared by heat treatment under alkaline conditions.

Photosynthetic accumulation of poly-(hydroxybutyrate) by cyanobacteria--the metabolism and potential for CO2 recycling.

Regulatory mechanism in PHB [poly-(hydroxybutyrate)] accumulation by cyanobacteria, especially by a thermophilic isolate, Synechococcus MA19 was reviewed in comparison with a genetically engineered strain. The strain, MA19 accumulates PHB under nitrogen starved and photoautotrophic conditions (MA19-N). Little PHB synthase activity was detected in crude extracts from the cells grown in nitrogen sufficient conditions (MA19 + N). The activity was detected exclusively in membrane fractions from MA19 + N. The change of the enzyme activity was insensitive to chloramphenicol, which suggests post-translational activation. In vitro, acetyl phosphate activated PHB synthase in membrane fractions from MA19 + N, and the extent of activation depended on the concentration of acetyl phosphate. Phosphotransacetylase which catalyzes the conversion of acetyl-CoA to acetyl phosphate was detected in crude extracts from MA19-N but not in those from MA19 + N. These results suggested that intracellular acetyl phosphate concentration could be controlled, depending on C-N balance and intracellular acetyl-CoA concentration. On the contrary, in genetically-engineered cyanobacterium (transformant with PHB synthesizing genes from Ralstonia eutropha), it did not seem to be PHB synthase but acetyl-CoA flux that limits PHB synthesis. The closer association of PHB granules with thylakoid membranes in MA19 is suggested than that in the genetically-engineered cyanobacterium, which may reflect the difference of distribution of PHB synthase. Transposon-mutagenesis was used to acquire mutants of its altered PHB regulatory mechanism. PHA production by cyanobacteria was considered from the aspects of photobioreactors.

Fluorescence-quenching phenomenon by photoinduced electron transfer between a fluorescent dye and a nucleotide base.

Fluorescently labeled oligonucleotide probes have been widely used in biotechnology, and fluorescence quenching by the interaction between the dyes and a nucleobase has been pointed out. This quenching causes big problem in analytical methods, but is useful in some other cases. Therefore, it is necessary to estimate the fluorescence quenching intensity under various conditions. We focused on the redox properties of some commercially available fluorescent dyes, and investigated dye-nucleotide interactions between a free dye and a nucleotide in aqueous solution by electrochemical and spectroscopic techniques. Our results suggested that the quenching was accompanied by photoinduced electron transfer between a thermodynamically quenchable excited dye and a specific base. Several kinds of fluorescent dyes labeled to the 5'-end of oligonucleotide C10T6 were prepared, and their quenching ratios compared upon hybridization with the complementary oligonucleotide A6G10. The quenching was completely reversible and their efficiencies depended on the attached fluorophore types. The fluorescence of 5-FAM, BODIPY FL or TAMRA-modified probe was strongly quenched by hybridization.

Phosphotransacetylase as a key factor in biological production of polyhydroxybutyrate.

Phosphotransacetylase (Pta) catalyzes the reversible conversion of acetyl-coenzyme A (CoA) to acetyl phosphate. Polyhydroxybutyrate (PHB) synthase and accumulation were compared between a Pta-deficient mutant and the wild-type Escherichia coli, which were transformed with pAE100, coding for 3-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHB synthase from Ralstonia eutropha. During the growth period, PHB synthase activity in the Pta-deficient mutant was lower than that in the wild type. PHB accumulation in the Pta-deficient mutant, however, was higher than that in wild-type cells grown in Luria-Bertani (LB) medium containing 1% glucose (high C:N ratio). The Pta-deficient mutant showed PHB accumulation even in LB medium (low C:N ratio), whereas wild-type cells showed no PHB accumulation. These data suggest the activation of PHB synthase by acetyl phosphate that is synthesized by Pta. A decrease in Pta activity probably causes some increase in acetyl-CoA as substrate for the PHB synthesis pathway, resulting in increased PHB accumulation.

A high-copy-number plasmid capable of replication in thermophilic cyanobacteria.

A 2.5 kb high-copy-number plasmid, pMA4 in thermophilic cyanobacterium Synechococcus sp. MA4 was isolated and characterized to develop a genetic engineering system for thermophilic cyanobacteria. The copy number of pMA4 was determined to be by densitometry about 350/cell. The pMA4 may be a type of rolling-circle plasmid, because a possible rep gene encoding 34 kD-protein and a consensus sequence of a double-stranded origin nick site of rolling circle plasmids were found in the pMA4 sequence. The pMA4 was electro-introduced into another thermophile, Synechococcus sp. MA19, which is the strongest poly-beta-hydroxybutyrate (PHB) accumulator in photoautotrophic organisms. The pMA4 was incorporated and retained in MA19. These results indicate that pMA4 could be developed as a useful vector for thermophilic cyanobacteria.

Polyhydroxybutyrate production from carbon dioxide by cyanobacteria.

Genetic characterization and enhancement of polyhydroxybutyrate (PHB) accumulation in cyanobacteria were investigated for efficient PHB production from CO2. The genome DNAs in the PHB-accumulating strains Synechococcus sp. MA19 and Spirulina platensis NIES46 retained the highly homologous region to phaC of Synechocystis PCC6803, whereas low homology was detected in the nonaccumulating strains Synechococcus sp. PCC7942 and Anabaena cylindrica NIES19. Synechococcus sp. MA19, which accumulates PHB up to 30% of dry cell weight from CO2 as the sole carbon source, was mutated by insertion of transposon Tn5 to enhance the PHB accumulation. Genetic and physiological analysis of the mutant indicated that decreased phosphotransacetylase activity could trigger an increase of acetyl coenzyme A leading to enhancement of PHB accumulation. PHB synthase in Synechococcus sp. MA19 was probably attached to thylakoid membrane since PHB granules were associated with pigments. A genetically engineered cyanobacteria retaining soluble PHB synthase from Ralstonia eutropha accumulated pigment-free PHB granules, which is an advantage for the purification of PHB.

[Empiric therapy with fluconazole in granulocytopenic patients with carcinoma or leukemia].

We performed a randomized clinical trial in granulocytopenic patients with carcinoma or leukemia. Patients with persistent fever for more than 2 days despite antibiotic therapy were randomized to antibiotic plus fluconazole therapy group (FLCZ group) or antibiotic therapy only group (antibiotic group) by the envelope method. It was possible to evaluate clinical efficacies in 62 patients (37 patients in FLCZ group and 25 patients in antibiotics group). In patients whose neutrophil counts were less than 100/microliters on the initial day of therapy, clinical efficacy rates were 72.0% (18/25) in FLCZ group and 57.1% (8/14) in antibiotics group. In patients whose neutrophil counts continued to be less than 100/microliters during therapy, clinical efficacy rates were 64.3% (9/14) and 50.0% (3/6), respectively. Further, in patients whose neutrophil counts continued to be less than 500/microliters during therapy, they were 76.9% (20/26) and 53.3% (8/15), respectively. No severe side effects nor severe case of abnormal change in laboratory test values due to fluconazole were observed in this trial. These data suggest that empiric antifungal therapy with fluconazole is effective for fungal infections in granulocytopenic patients with carcinoma and leukemia.

[A pharmacokinetic study of cefoperazone during percutaneous transhepatic cholangial catheterization].

The metabolic fate of cefoperazone (CPZ) was studied in 19 cases which underwent percutaneous transhepatic cholangial catheterization (PTC-catheterization, PTCC) and were under various conditions of the liver function. The peak of bile levels of CPZ immediately after PTCC differed greatly from one case to another at 12.6-7,260 micrograms/ml with 1 g intravenous injection and 23.0-5,800 micrograms/ml with 2 g intravenous injection. The ratio of the peak of bile level to the serum level immediately after PTCC showed the highest negative correlation with the serum total bilirubin level. It also showed a significant negative correlation with GOT, GPT, Al-P and LAP. The serum CPZ level and half-life showed no significant trend except half-life showed a significant correlation with LAP. The recovery rate in urine up to 12 hours was in the range of 14.8-93.6%, showing a significant correlation with the ratio of the peak of bile levels to the serum level and the date of liver function tests. The bile level, serum level and recovery rate in urine at the time the bile outflow from the catheter has become constant after PTCC (during the course of PTCC) showed a trend almost similar to that immediately after PTCC, there being no significant difference as to each parameter during the course of PTCC and immediately after PTCC. In the cases in which the sample was collected by the cross-over technique, the ratio of the peak of bile levels to the serum level from immediately after PTCC to during the course of PTCC increased in 2 cases and decreased in 6 cases. The 2 cases that showed the increase in the ratio were the case in which the serum total bilirubin level improved almost to normal. Findings above suggest that sufficient biliary decompression can improve the movement of CPZ into bile, despite the fact that the pharmacokinetics of CPZ is affected by the liver function, particularly serum total bilirubin level, that a decrease in the movement to bile and a compensatory increase in urinary excretion are observed in jaundice and disturbance of the liver function and that the ratio of the peak of bile level to the serum level decreases during the course of PTCC rather than immediately after PTCC in some cases.

[Acute myelomonocytic leukemia with Inv(16) (p13 q22) relapsed in transverse myelopathy].

We report a case of AML (M 4) with eosinophilia who developed meningeal relapse and transverse myelopathy. A 37-year-old woman was admitted to our hospital because of lymphadenopathy and ecchymosis. One week prior to admission, she noticed swelling of the cervical lymph nodes and bleeding tendency. On admission, low-grade fever, gingival swelling, generalized lymphadenopathy, and ecchymosis on the lower legs were found. A white blood cell count was 93,900/microliters with 82% blast cells, and a platelet count was 24,000/microliters. A bone marrow was composed of 45.3% myeloblasts, 27% monocytes and 7.1% eosinophils. Chromosome analysis revealed inv(16). The diagnosis of M4Eo was made. About one year after she gained complete remission, she was readmitted because of disturbance of urination. There was a sign of transverse myelopathy at the seventh vertebral level, and blast cells were detected in the cerebrospinal fluid. Despite of radiation and chemotherapy, paresis of lower extremities and sensory disturbance were persistent, and the patient died on 52th hospital day.

[Acute myelofibrosis terminating in acute myelomegakaryoblastic leukemia].

We report a case of acute myelofibrosis (AMF) developing into acute myelomegakaryoblastic leukemia. A 33-year-old woman was admitted to our hospital because of fever and chest pain. On physical examination, hepatosplenomegaly was not noticed. Pancytopenia and a small number of blast cells were observed in the peripheral blood. Poikilocytosis was not detected. Bone marrow examination revealed dry tap on aspiration, and moderate increase in reticulin fiber on biopsy. The diagnosis of AMF was made. Eight months later, blast cells markedly increased. Surface marker was investigated and MCS-2 (CD13), C17 (CDw41) and P2 (CDw41) were found to be positive. Electron microscopic examination revealed that blast cells were composed of PPO-positive cells and MPO-positive cells. Based on these findings, it was considered that the patient developed acute myelomegakaryoblastic leukemia. Recently AMF is thought to be a state to have the ability to develop into various types of acute leukemia. Adequate therapy may be required before the development of leukemia.