Involvement of the Glu724-Pro760 region of the dihydropyridine receptor II-III loop in skeletal muscle-type excitation-contraction coupling

Our previous study (El-Hayek, R., Antoniu, B., Wang, J. P., Hamilton, S. L., and Ikemoto, N. (1995) J. Biol. Chem. 270, 22116-22118) suggested the hypothesis that skeletal muscle-type excitation-contraction coupling is regulated by two domains (activating and blocking) of the II-III loop of the dihydropyridine receptor alpha1 subunit. We investigated this hypothesis by examining conformational changes in the ryanodine receptor induced by synthetic peptides and by transverse tubular system (T-tubule) depolarization. Peptide A, corresponding to the Thr671-Leu690 region, rapidly changed the ryanodine receptor conformation from a blocked state (low fluorescence of the conformational probe, methyl coumarin acetamide, attached specifically to the ryanodine receptor) to an activated state (high methyl coumarin acetamide fluorescence) as T-tubule depolarization did. Peptide C, corresponding to the Glu724-Pro760 region, blocked both conformational changes induced by peptide A and T-tubule depolarization. Its ability to block peptide A-induced and depolarization-induced activation was considerably impaired by replacing the portion of peptide C corresponding to the Phe725-Pro742 region of the loop with cardiac muscle-type sequence. These results are consistent with the model that depolarization-induced activation of excitation-contraction coupling and blocking/repriming are mediated by the peptide A region and the peptide C region (containing the critical Phe725-Pro742 sequence) of the II-III loop, respectively.     

A new Bennettitalean trunk with unilacunar five-trace nodal structure from the Upper Cretaceous of Hokkaido, Japan

A new species of Bucklandia is described based on a permineralized fossil trunk that was obtained from sediments of Upper Cretaceous age from Hokkaido, Japan. The quality of preservation is exquisite, and anatomical and morphological features are preserved at the cellular level. The specimen is clearly bennettitalean because of the cycadeoidean-type arrangement of vascular bundles in the petiole. However, this specimen is unique among previously described progymnosperms and gymnosperms in possessing a unilacunar five-trace type of nodal structure. The fossil thus contributes to a more complete understanding of the variation of nodal structure in bennettitalean trunks.     

Numerical simulation for electrochemical cultivation of iron oxidizing bacteria

A numerical simulation model was constructed for electrochemical cultivation of iron oxidizing bacterium, Thiobacillus ferrooxidans, based on Monod's dual limitation equation. In this model, two limiting factors were examined, low supply of Fe(II) ion and dissolved oxygen, from empirical viewpoints. The simulation model was constructed taking into consideration the energy balance based on the amount of the electronic flow from the electrode to bacteria via an iron ion, and then to oxygen. The model consisted of a logarithmic bacterial growth phase during the first three days, followed by a plateau and growth limitation thereafter. The predicted results were in agreement with the actual growth under electrochemical cultivation. It was predicted the growth limiting factor would be changed from insufficient supply of Fe(II) ions to that of oxygen by decreasing the value of oxygen transfer constant K, which correlated with the aeration rate. The optimum aeration rate was determined for the ideal electrochemical cultivation. The algorithm described here can be used in any electrochemical cultivation by modifying the parameters for each system.     

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.