• BMB Reports
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• v.40 no.5
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• pp.697-707
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• 2007

Cytochrome $cbb_3$ oxidase is a member of the heme-copper oxidase superfamily that catalyses the reduction of molecular oxygen to the water and conserves the liberated energy in the form of a proton gradient. Comparison of the amino acid sequences of subunit I from different classes of heme-copper oxidases showed that transmembrane helix VIII and the loop between transmembrane helices IX and X contain five highly conserved polar residues; Ser333, Ser340, Thr350, Asn390 and Thr394. To determine the relationship between these conserved amino acids and the activity and assembly of the $cbb_3$ oxidase in Rhodobacter capsulatus, each of these five conserved amino acids was substituted for alanine by site-directed mutagenesis. The effects of these mutations on catalytic activity were determined using a NADI plate assay and by measurements of the rate of oxygen consumption. The consequence of these mutations for the structural integrity of the $cbb_3$ oxidase was determined by SDS-PAGE analysis of chromatophore membranes followed by TMBZ staining. The results indicate that the Asn390Ala mutation led to a complete loss of enzyme activity and that the Ser333Ala mutation decreased the activity significantly. The remaining mutants cause a partial loss of catalytic activity. All of the mutant enzymes, except Asn390Ala, were apparently correctly assembled and stable in the membrane of the R. capsulatus.

• Journal of Life Science
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• v.19 no.7
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• pp.852-858
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• 2009

The homeostasis of the pyridine nucleotide pool [NAD(P)H and NAD(P)$^+$] is maintained in Rhodobacter sphaeroides mutant strains defective in the cytochrome bci complex or the cytochrome c oxidases in terms of its concentration and redox state. Aerobic derepression of the puf operon, which is under the control of the PrrBA two-component system, in the CBB3 mutant strain of R. sphaeroides was shown to be not the result of changes in the redox state of the pyridine nucleotides and the ubiquinone/ubiquinol pool. Using the bc$_1$ complex knock-out mutant strain of R. sphaeroides, we clearly demonstrated that the inhibitory effect of cbb$_3$, oxidase on spectral complex formation is not caused indirectly by the redox change of the ubiquinone/ubiquinol pool.

• Proceedings of the KAIS Fall Conference
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• 2005.05a
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• pp.292-294
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• 2005

본 연구에서는 전기영동 겔에 대한 음극전리수의 침투력과 녹차성분에 대한 음극전리수의 용해력을 일반 물과 서로 비교하였다. 음극전리수와 증류수로 제조한 CBB-R 염색시약으로 polyacrylamide 겔 상에서 단백질을 다양한 시간 동안 염색한 후 염색강도를 서로 비교하였다. 그 결과 음극전리수로 제조한 CBB-R 염색시약은 증류수로 제조한 CBB-R 염색시약보다 같은 반응 시간 동안에 먼저 단백질을 강하게 염색시켰다. 뿐만 아니라 $25^{\circ}C$에서 음극전리수는 일반 물에 비하여 녹차성분에 대해 극히 탁월한 용해력을 나타내었다.

• Journal of Life Science
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• v.22 no.8
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• pp.1009-1017
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• 2012

The orf282 gene of Rhodobacter sphaeroides is located between the ccoNOQP operon encoding $cbb_3$ terminal oxidase and the fnrL gene encoding an anaerobic activator, FnrL. Its function remains unknown. In an attempt to reveal the function of the orf282 gene, we disrupted the gene by deleting a portion of the orf282 gene and constructed an orf282-knockout mutant. Two FnrL binding sites were found to be located upstream of orf282, and it was demonstrated that orf282 is positively regulated by FnrL. The orf282 gene is not involved in the regulation of spectral complex formation. The $cbb_3$ oxidase activity detected in the orf282 mutant was comparable to that in the wild-type sample, indicating that the orf282 gene is not involved in the regulation of the ccoNOQP operon and the biosynthesis of the cbb3 cytochrome c oxidase. The elevated promoter activity of the nifH and nifA genes, which are the structural genes of nitrogenase and its regulator, respectively, in the orf282 mutant, suggests that the orf282 gene product acts as a negative effector for nifH and nifA expression.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.9
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• pp.57-63
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• 2020

To develop simple acrosome staining of horse spermatozoa, this study tested the binding properties of Coomassie brilliant blue G or R on the sperm smears after 3.7% paraformaldehyde (PF) or 35% methanol (MT) fixation. After being fixed with PF and stained with 0.05, 0.1, or 0.2 % of CBB G or R for 2 min, horse spermatozoa were examined for their intact acrosome status. The intact acrosome of fresh horse spermatozoa were 62.6% and 61.5% with 0.05% of the G and R CBB solution, but 80.2 and 79.7% with G type and 78.1 and 76.0% with R type. On the other hand, when MT was used for fixation, the acrosome reacting sperm ratio was 3.5%, but was 9.0% in the case of PF. These results show that the intact acrosome of horse sperm could be judged using a 0.1~0.2% CBB G or R staining technique. PF would be an essential fixative for examining acrosome reacting horse spermatozoa. This method could be used to identify sperm with a damaged acrosome during low-temperature storage or cryopreservation for artificial insemination of horses.

• Proceedings of the Korean Society of Potoscience Conference
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• 2005.06a
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• pp.49-49
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• 2005

• Proceedings of the Korean Society of Life Science Conference
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• 2003.05a
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• pp.13-18
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• 2003

• Korean Journal of Animal Reproduction
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• v.16 no.4
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• pp.289-299
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• 1993

The polypeptide patterns of cellular and follicular components were analysed by SDS-PAGE and two dimensional(2-D)electrophoresis combined with isoelectric focusing (IEF) to establish protein profiles in each of the components in porcine follicles. Oocyte-cumulus complexes were cultured in M16+FCS+Gn at 39 in an atmosphere of 5% CO$_2$, in air for 35 h. At the end of the culture, the zona-free oocyte, ZP alone and cumulus cells were prepared and analysed either on 10% SDS-PAGE for the protein profile at the first dimensional gel or 2-D protein pattern. The amounts of each samples were determined for the visualization with Coomasie brilliant blue (CBB) or silver staining, thus giving useful information for the identification of specific proteins in the components or appropriate amount of samples for proper visualization. Oocyte showed 25 and 114 kd major protein band. Other minor components were additionally visualized with CBB on the same gel after silver staining procedure. Cumulus cells also showed specific proteins which is not present in the oocytes. The number of cumulus cell was proper to give major bands with CBB and additional minor bands with silver staining. To establish the degree of contamination from the remnant of the corona radiata to the ZP, zonae were differently prepared or analysed by SDS-PAGE.The preparation of the ZP in this study did not showed any contamination judged by the protein profile of the components. Also follicular fluid showed its specific protein profile without any significant differences among the different sizes of follicles. The established protein profile of each follicular component should be helpful for the identification and elimination of contaminated components, i. e., antigen preparation or immunological studies. The results also suggest that the preparation of each components in the study was appropriate and can be used for a further sensitive biochemical analysis in mammalian oocytes and early embryos.

• Nuclear Engineering and Technology
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• v.52 no.11
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• pp.2552-2564
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• 2020

The crevice stress corrosion cracking (SCC) susceptibility of austenitic stainless steels was evaluated in simulated pressurized water reactor (PWR) environments. To simulate the abnormal condition in temporary clamping devices on leaking small bore pipes, crevice bent beam (CBB) tests were performed in the oxygenated as well as hydrogenated conditions. No SCC cracks were found for SS316 in both conditions. SS304 also showed good resistance in the hydrogenated condition. However, all SS304 specimens showed SCC cracks in the oxygenated condition, indicating poor crevice SCC resistance. It was found that residual ferrites were selectively dissolved because of the galvanic corrosion coupled with the neigh-bouring austenite phase, resulting in SCC initiation in SS304. Crack morphologies were mostly transgranular assisted by the damaged δ-ferrite and deformation-induced slip bands.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.5
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• pp.580-586
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• 2000

Data collected between 1981 and 1991 at the Philippine Carabao Center at Central Luzon State University (PCC-CLSU) were used for the comparison of growth, milk yield and draughtability of Murrah-Philippine crossbred and Philippine native buffaloes. Body weights and body measurements were available at 3-month intervals from birth to 36 months of age for a total of 34 $Murrah{\times}Philippine$ native buffalo F1 crossbreds (CBB; 21 cows, 13 bulls) and 32 Philippine native buffaloes (PNB; 16 cows, 16 bulls). Lactation records were available for 14 CBB and 19 PNB cows. Data for draughtability under wet and dry ploughing conditions were available for 4 CBB and 4 PNB steers. The results indicate that crossbreds grow faster (0-9 months of age: cows $442{\pm}19$ vs. $301{\pm}21g/day$, bulls $305{\pm}23$ vs. $296{\pm}21g/day$; 9-36 months of age: cows $227{\pm}10$ vs. $147{\pm}12g/day$, bulls $282{\pm}13$ vs. $138{\pm}12g/day$), mature earlier and produce more milk (1st lactation: $1139{\pm}153$ vs. $450{\pm}112kg$; 2nd lactation: $1115{\pm}132$ vs. $488{\pm}136kg$) than native buffaloes, but have a poorer draughtability (wet ploughing; force as % of body weight $8.8{\pm}0.2$ vs. $12.2{\pm}0.6$; dry ploughing: cut depth $10.98{\pm}0.25$ vs. $11.92{\pm}0.13cm$, velocity $0.50{\pm}0.03$ vs. $0.60{\pm}0.02m/sec$, force as % of body weight $9.0{\pm}0.6$ vs. $11.3{\pm}0.7$). The correlation coefficients between body weight and body measurements at birth and at 3-month intervals indicate that heart girth has a relatively high correlation with body weight, especially in crossbreds. It is concluded that in Philippine smallholder farming systems in which meat and milk production are secondary to draught power, the native buffalo is preferable from the point of view of input needed to maintain the number of animals kept for a required draught force.