Enhancing cellulase production by overexpression of xylanase regulator protein gene,xlnR, in Talaromyces cellulolyticus cellulase hyperproducing mutant strain

We obtained strains with the xylanase regulator gene, xlnR, overexpressed (HXlnR) and disrupted (DXlnR) derived from Talaromyces cellulolyticus strain C-1, which is a cellulase hyperproducing mutant. Filter paper degrading enzyme activity and cellobiohydrolase I gene expression was the highest in HXlnR, followed by C-1 and DXlnR. These results indicate that the enhancement of cellulase productivity was succeeded by xlnR overexpression.     

Characterization of the reaction of decoupling ubiquinone with bovine mitochondrial respiratory complex I

We previously produced the unique ubiquinone QT (“decoupling” quinone), the catalytic reduction of which in NADH-quinone oxidoreduction with bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) is completely decoupled from proton translocation across the membrane domain. This feature is markedly distinct from those of typical short-chain quinones such as ubiquinone-1. To further characterize the features of the QT reaction with complex I, we herein synthesized three QT analogs, QT2–QT4, and characterized their electron transfer reactions. We found that all aspects of electron transfer (e.g. electron-accepting activity and membrane potential formation) vary significantly among these analogs. The features of QT2 as decoupling quinone were slightly superior to those of original QT. Based on these results, we conclude that the bound positions of QTs within the quinone binding cavity susceptibly change depending on their side-chain structures, and the positions, in turn, govern the behavior of QTs as electron acceptors.     

Cloning and characterization of elastase structural gene from Pseudomonas aeruginosa IFO 3455

An 8.3 Kb DNA fragment was cloned from Pseudomonas aeruginosa IFO 3455. This fragment-containing Escherichia clone, pEL2, produced a high level of elastase activity. A smaller EcoRI-KpnI fragment was subcloned into pUC118 and E. coli HB101 was transformed with the plasmid. A deletion mutant clone was also constructed in the same bacteria. These deletion mutants were tested for elastase activity and it became clear that the full length of the elastase gene was 1.0-1.3 Kb. DNA sequencing analysis revealed that this DNA fragment contains the DNA sequence coding N-terminal amino acid sequence of the elastase protein.     

Metabolic responses of chicken embryos to graded, prolonged alterations in ambient temperature

1. Chicken embryos aged 12, 16, 18 and 20 (externally pipped) days of incubation were exposed to graded reductions (2 degrees C) in ambient temperature from 38 to 28 degrees C, exposure to each temperature lasting up to 9 hr. 2. Oxygen uptake was measured first at 38 degrees C and then in the quasi-equilibrium state at lowered temperatures. The temperature coefficient (Q10) was calculated for each egg. 3. For mild cooling (32 degrees C), the Q10 in 18-day-old embryos was about 1.5, while 12- and 16-day-old embryos had a Q10 value of about 2, indicating that a feeble homeothermic metabolic response to cooling appears in late prenatal embryos. It became more marked in externally pipped embryos and further augmented in hatchlings.     

Effect of IL-12 encoding plasmid administration on tight-skin mouse

The tight-skin (Tsk/+) mutant mice, a putative murine model of scleroderma, are characterized by the excessive deposition of collagen and the presence of antinuclear antibodies. Type 2 cytokines, such as IL-4 and IL-6, are capable of regulating the synthesis of various matrix molecules, including type I collagen, by fibroblasts. IL-12 is well known to induce type 1 cytokine production and to reduce type 2 activity. Here, we examined the effect of IL-12 encoding plasmid (pCAGGSIL-12) on the disease progression of Tsk/+ mice. pCAGGSIL-12 plasmid or pCAGGS parental vector was injected intramuscularly 7 times at 3 week intervals into Tsk/+ mice. One week after the last injection, pCAGGSIL-12 administered Tsk/+ mice exhibited a marked decrease in the skin thickness compared with the mice treated with pCAGGS vector. The serum levels of antinuclear antibodies were diminished in pCAGGSIL-12 treated mice. IL-4 production by spleen cells from pCAGGSIL-12 plasmid treated mice was significantly lower than that from vector treated mice. These results indicate that pCAGGSIL-12 administration into Tsk/+ mice had beneficial effects in preventing the collagen accumulation in the skin and suppressing the autoimmunity via improvement of Th1/Th2 balance. The present study suggests that the IL-12 encoding plasmid administration might have a therapeutic effect on systemic sclerosis.     

Selective isolation and distribution of the genus Planomonospora in soils

For the screening of bioactive compounds and study of global distribution, a selective isolation method for Planomonospora strains by centrifugation from soil is examined. Planomonospora strains produced characteristic sporangia on the humic acid-trace salts gellan gum medium (pH 9.0) so that this genus was readily recognized on the isolation plate. High yields of motile spores were obtained by using a flooding solution containing 0.1% skim milk in 5 mM N-cyclohexyl-2-amino-ethanesulfonic acid (pH 9.0) followed by incubating the preparation at 32 degreesC for 90 min, centrifuging it at 1000 x g for 10 min, and further incubation at 32 degreesC for 60 min after centrifugation. By combining the techniques described above, we isolated 246 Planomonospora strains from 137 of the 1200 soil samples examined. Ninety-four percent of these strains were recovered from neutral to slightly alkaline soils (pH 7.0 to 9.0). Strains of P. venezuelensis group were obtained from 13 soil samples (1.1%), which were collected in Bolivia, Cyprus, Egypt, Greece, India, Japan, New Caledonia, and Turkey. Strains of this group appear widely distributed in the soil of tropical to temperate regions. To our knowledge, this is the first record that strains of this group have been isolated from a location other than Venezuela.     

Identification of new genes ndr2 and ndr3 which are related to Ndr1/RTP/Drg1 but show distinct tissue specificity and response to N-myc

Ndr1 was isolated as a gene upregulated in N-myc mutant mouse embryos and is repressed by N-myc and c-myc. Consistent with Myc regulation, the same gene was also isolated as one sensitive to transformation (Drg1), and in addition as one induced under a few stress conditions (RTP). Two new genes, Ndr2 and Ndr3, were identified which encode proteins highly related to Ndr1/RTP/Drg1 and constitute the Ndr gene family. Ndr2 and Ndr3 are under spatio-temporal regulations distinct from Ndr1, and are not activated in N-myc mutants. When whole embryo RNA was analyzed, Ndr3 expression was already high at 9.5 days postcoitus (dpc), while expression of Ndr2 and Ndr1 became significant after 12.5 dpc and 13. 5 dpc, respectively. At 14.5 dpc, expression of these genes partially overlaps, but many tissues are unique to one of them. For instance, Ndr1 is strongly expressed in the liver and gut epithelium, Ndr2 in the ventricular zone throughout the CNS, and Ndr3 in the spinal cord and the thymus rudiment. Genes of the Ndr family probably have tissue-dependent allotments of the possibly related functions.