Pleiotropic phenotypes of fission yeast defective in ubiquinone-10 production. A study from the abc1Sp (coq8Sp) mutant

We previously constructed two Schizosaccahromyces pombe ubiquinone-10 (or Coenzyme Q10) less mutants, which are either defective for decaprenyl diphosphate synthase or p-hydroxybenzoate polyprenyl diphosphate transferase. To further confirm the roles of ubiquinone in S. pombe, we examined the phenotype of the abc1Sp (coq8Sp) mutant, which is highly speculated to be defective in ubiquinone biosynthesis. We show here that the abc1Sp defective strain did not produce UQ-10 and could not grow on minimal medium. The abc1Sp-deficient strain required supplementation with antioxidants such as cysteine or glutathione to grow on minimal medium. In support of the antioxidant function of ubiquinone, the abc1Sp-deficient strain is sensitive to H2O2 and Cu2+. In addition, expression of the stress inducible ctt1 gene was much induced in the ubiquinone less mutant than wild type. Interestingly, we also found that the abc1-deficient strain as well as other ubiquinone less mutants produced a significant amount of H2S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Thus, analysis of the phenotypes of S. pombe ubiquinone less mutants clearly demonstrate that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.     

Human EVI9, a homologue of the mouse myeloid leukemia gene, is expressed in the hematopoietic progenitors and down-regulated during myeloid differentiation of HL60 cells

Evi9, a common site of retroviral integration in BXH2 murine myeloid leukemias, encodes a C2H2 zinc finger protein and is overexpressed in these leukemic cells. To investigate a possible role of EVI9 in the human hematopoietic system, we isolated the cDNA clone of the human homologue. Human EVI9, located on the chromosome 2p13 region, contains an open reading frame of 797 amino acids that is 98.7% identical to the mouse protein. RT-PCR analysis of purified human hematopoietic cells showed that EVI9 is expressed in CD34-positive myeloid precursors, B cells, monocytes, and megakaryocytes, but only weakly in T lymphocytes, suggesting that EVI9 may play an important role in hematopoiesis. Furthermore, EVI9 was down-regulated during myeloid differentiation of HL60 cells induced by all-trans-retinoic acid, whereas the expression remained during monocytic differentiation induced by phorbol 12-myristate 13-acetate. These results indicate a distinct role for EVI9 in human hematopoietic cells and suggest that EVI9 may cause leukemia through inhibition of myeloid differentiation.     

Nitrogen removal reactor using packed gel envelopes containing Nitrosomonas europaea and Paracoccus denitrificans

Packed gel envelopes were constructed as simple, compact reactors for removing nitrogen from wastewater. Each packed gel envelope consisted of two plate gels with a spacer in between. Nitrosomonas europaea and Paracoccus denitrificans were co-immobilized in the plate gels, and ethanol, serving as an electron donor for denitrification, was injected into the internal spaces of the envelopes. The external surfaces of the envelopes were in contact with ammonia-containing wastewater; the N. europaea present in the gels oxidized the ammonia to nitrite aerobically. On the other hand, the internal surfaces of the envelopes were in contact with the ethanol solution, which P. denitrificans used to reduce the nitrite to nitrogen gas anaerobically. In this way, the reactor using the packed gel envelopes removed ammonia from wastewater in a single step. When artificial wastewater containing 200 mg-N/L was treated using the reactor using eight envelopes, the ammonia was removed by the reactor without accumulating nitrite or ethanol. This simple system exhibited high rates of nitrification (ammonia to nitrite; 1.9 kg-N/day for 1m(3) of reactor volume) and nitrogen removal (ammonia to nitrogen gas; 1.6 kg-N/day). It is presumed that these high rates were achieved as a consequence of cooperation between the N. europaea and P. denitrificans present in the gels and the efficient uptake and exhaust of gases leading to the smooth conversion of ammonia to nitrogen gas.     

High density cultivation of two strains of iron-oxidizing bacteria through reduction of ferric iron by intermittent electrolysis

Electrolytic cultivation was applied to Leptospirillum ferrooxidans strains P3A and CF27, which use ferrous iron to respire aerobically. Ferrous iron was supplied to the bacteria by intermittent electrolytic reduction of ferric iron as electron shuttle using an electrode. The yield of L. ferrooxidans and strain CF27 reached 20- and 50-fold, respectively, higher density than were achievable yields without electrolysis. The time required to obtain high density depended not on the growth ratio, but rather on the original growth rate of each strain.     

Evi9 encodes a novel zinc finger protein that physically interacts with BCL6, a known human B-cell proto-oncogene product

Evi9 is a common site of retroviral integration in BXH2 murine myeloid leukemias. Here we show that Evi9 encodes a novel zinc finger protein with three tissue-specific isoforms: Evi9a (773 amino acids [aa]) contains two C(2)H(2)-type zinc finger motifs, a proline-rich region, and an acidic domain; Evi9b (486 aa) lacks the first zinc finger motif and part of the proline-rich region; Evi9c (239 aa) lacks all but the first zinc finger motif. Proviral integration sites are located in the first intron of the gene and lead to increased gene expression. Evi9a and Evi9c, but not Evi9b, show transforming activity for NIH 3T3 cells, suggesting that Evi9 is a dominantly acting proto-oncogene. Immunolocalization studies show that Evi9c is restricted to the cytoplasm whereas Evi9a and Evi9b are located in the nucleus, where they form a speckled localization pattern identical to that observed for BCL6, a human B-cell proto-oncogene product. Coimmunoprecipitation and glutathione S-transferase pull-down experiments show that Evi9a and Evi9b, but not Evi9c, physically interact with BCL6, while deletion mutagenesis localized the interaction domains in or near the second zinc finger and POZ domains of Evi9 and BCL6, respectively. These results suggest that Evi9 is a leukemia disease gene that functions, in part, through its interaction with BCL6.     

Cytotoxic cycloartane-type triterpenes from Combretum quadrangulare

Seven novel cycloartane-type triterpenes were isolated from Combretum quadrangulare, and their structures were elucidated on the basis of spectral analysis. All these compounds were tested for their cytotoxicity against murine colon 26-L5 carcinoma cells. Methyl quadrangularate B (2) and methyl quadrangularate D (4) exhibited potent cytotoxicity having ED₅₀ values 9.54 and 5.42 μM, respectively.     

Iron accumulation in tobacco plants expressing soyabean ferritin gene

High iron-content transgenic tobacco plants have been produced by transfer via Agrobacterium tumefaciens of soyabean ferritin cDNA under the control of a CaMV 35S promoter. Immunoblot analysis of protein from transgenic tobacco plants suggested mature ferritin subunits are produced by cleavage of transit peptides. The expressed ferritin was observed in the tissues of leaves and stems. The maximal iron content of transformant leaves was approximately 30% higher than leaves from non-transformants. The increased iron content of each transformant was correlated with increases in ferritin content. These results demonstrate the potential of breeding high iron content crops by introduction of the ferritin gene     

Biogas production using anaerobic groundwater containing a subterranean microbial community associated with the accretionary prism

In a deep aquifer associated with an accretionary prism, significant methane (CH₄) is produced by a subterranean microbial community. Here, we developed bioreactors for producing CH₄ and hydrogen (H₂) using anaerobic groundwater collected from the deep aquifer. To generate CH₄, the anaerobic groundwater amended with organic substrates was incubated in the bioreactor. At first, H₂ was detected and accumulated in the gas phase of the bioreactor. After the H₂ decreased, rapid CH₄ production was observed. Phylogenetic analysis targeting 16S rRNA genes revealed that the H₂-producing fermentative bacterium and hydrogenotrophic methanogen were predominant in the reactor. The results suggested that syntrophic biodegradation of organic substrates by the H₂-producing fermentative bacterium and the hydrogenotrophic methanogen contributed to the CH₄ production. For H₂ production, the anaerobic groundwater, amended with organic substrates and an inhibitor of methanogens (2-bromoethanesulfonate), was incubated in a bioreactor. After incubation for 24 h, H₂ was detected from the gas phase of the bioreactor and accumulated. Bacterial 16S rRNA gene analysis suggested the dominance of the H₂-producing fermentative bacterium in the reactor. Our study demonstrated a simple and rapid CH₄ and H₂ production utilizing anaerobic groundwater containing an active subterranean microbial community.