Intraspecific Relationship of Sheep Based on Mitochondrial Cytochrome b Gene

[ Objective] To preliminarily explore the intraspecific relationship of sheep based on cytochrome b （ Cyt b） gene of mitochondrial DNA （mtDNA）. [Method] The Cyt b gene sequences in 112 sheep individuals of two local sheep breeds were amplified by PCR. Then the amplified products were digested with EcoR I and analyzed by restriction fragment length polymorphism （RFLP）. [ Result] As many as 56 samples from Tan sheep and 56 samples from Wadi sheep were detected. The results showed that the amplified Cyt b gene in 51 individuals of Tan sheep had one EcoR I restriction site and no EcoR I restriction site in other five individuals, thus the Cyt b gene in Tan sheep showed two restriction morphs; the Cyt b gene in all 56 individuals of Wadi sheep had one EcoR I restriction site and showed one restriction morph. [ Conclusion] The polymorphism of mitochondrial Cyt b gene in Tan sheep and Wadi sheep is poor, and the Cyt b gene in sheep breeds is very conservative. Therefore, using Cyt b gene as gene marker to study the intraspecific relationship of sheep has some limitations.

The thylakoid membrane protein NTA1 is an assembly factor of the cytochrome b6f complex essential for chloroplast development in Arabidopsis

The cytochrome b_(6f)(Cyt b_(6f))complex is a multisubunit protein complex in chloroplast thylakoid membranes required for photosynthetic electron transport.Here we report the isolation and characterization of the new tiny albino 1(nta1)mutant in Arabidopsis,which has severe defects in Cyt b_(6f) accumulation and chloroplast development.Gene cloning revealed that the nta1 phenotype was caused by disruption of a single nuclear gene,NTA1,which encodes an integral thylakoid membrane protein conserved across green algae and plants.Overexpression of NTA1 completely rescued the nta1 phenotype,and knockout of NTA1 in wild-type plants recapitulated the mutant phenotype.Loss of NTA1 function severely impaired the accumulation of multiprotein complexes related to photosynthesis in thylakoid membranes,particularly the components of Cyt b_(6f).NTA1 was shown to directly interact with four subunits(Cyt b6/PetB,PetD,PetG,and PetN)of Cyt b_(6f) through the DUF1279 domain and C-terminal sequence to mediate their assembly.Taken together,our results identify NTA1 as a new and key regulator of chloroplast development that plays essential roles in assembly of the Cyt b_(6f) complex by interacting with multiple Cyt b_(6f) subunits.

Arabidopsis Sucrose Transporter SUT4 Interacts with Cytochrome b5-2 to Regulate Seed Germination in Response to Sucrose and Glucose

It remains unknown whether a sucrose transporter mediates sugar signaling. Here, we report that the Arabidopsis （Arabidopsis thaliana） sucrose transporter SUT4 interacts with five members of the Arabidopsis cytochrome b5 （Cyb5） family, and sucrose represses the interaction between SUT4 and a Cyb5 member Cyb5-2/A. We observed that down- regulation of SUT4 and three cytochrome b5 members （Cyb5-2, Cyb5-4, and Cyb5-6） confers the sucrose- and glucose- insensitive phenotypes in the sucrose/glucose-induced inhibition of seed germination. The sut4 cybS-2 double mutant displays slightly stronger sucrose/glucose-insensitive phenotypes than either the sut4 or cyb5-2 single mutant. We showed that the SUT4/Cyb5-2-mediated signaling in the sucrose/glucose-induced inhibition of seed germination does not require ABA or the currently known ABI2/ABI4/ABI5-mediated signaling pathway（s）. These data provide evidence that the sucrose transporter SUT4 interacts with Cyb5 to positively mediate sucrose and glucose signaling in the sucrose/ glucose-induced inhibition of seed germination.

Mutation of Residue Arginine^18 of Cytochrome b559 α-Subunit and its Effects on Photosystem Ⅱ Activities in Chlamydomonas reinhardtii

It has been known that arginine is used as the basic amino acid in the α-subunit of cytochrome bsss （Cyt bsss） except histidine. However, previous studies have focused on the function of histidine in the activities of photosystem （PS） Ⅱ and there are no reports regarding the structural and/or functional roles of arginine in PSll complexes. In the present study, two arginine18 （R18） mutants of Chlamydomonas reinhardtii were constructed using site-directed mutagenesis, in which R18 was replaced by glutamic acid （E） and glycine （G）. The results show that the oxygen evolution of the PSII complex in the R18G and R18E mutants was approximately 60% of wild-type （WT） levels and that, after irradiation at high light intensity, oxygen evolution for the PSll of mutants was reduced to zero compared with 40% in WT cells. The efficiency of light capture by PSll （Fv/Fm） of R18G and R18E mutants was approximately 42%-46% that of WT cells. Furthermore, levels of the α-subunit of Cyt bsss and PsbO proteins were reduced in thylakoid membranes compared with WT. Overall, these data suggest that R18 plays a significant role in helping Cyt bss9 maintain the structure of the PSll complex and its activity, although it is not directly bound to the heme group.

Genetic diversity and the biogeographical process of Acheilognathus macropterus revealed by sequence variations of mitochondrial cytochrome b gene

In this study,thirty-six individuals of Acheilognathus macropterus were collected from the Heilongjiang River,the Yangtze River,and the Nandujiang River.Partial mitochondrial cytochrome b gene region(636 base pair)was sequenced to these samples and 22 haplo-types were found.With A.chankaensis and A.tokinensis as outgroups,their relationships were analyzed.The p-distances were calculated with Mega software and a molecular phylogenetic tree was constructed using the neighbor-joining(NJ)method.The proportions of main morphological characters were compared as well.P-distances showed that the genetic differences in A.macropterus samples were far smaller than those between these samples and the outgroups.The molecular phylogenetic tree shows that samples with barbels and those without barbels were intermingled.There was no distinctive difference in proportions of morphological characteristics among them.These results suggested that samples with barbels and those without barbels(formally identified as A.taenianalis)are the same species;A.taenianalis is synonymous with A.macropterus.The thirty-six individuals were grouped into five clades and the positions of the samples in the clades were correspondingly grouped within their geographical distributions.Among the five clades,clades 1 and 5 included samples from the Heilongjiang River and Nandujiang River respectively.The samples from the Yangtze River scattered into clades 2,3,and 4.There were distinctive genetic differences(>5%)among them.Interestingly,the distributions of the 21 samples in these three clades were not correlated to their geographical distributions.It is postulated that these genetic differences were due to the bitterlings’mating choice mechanism,the prozygotic isolation.The genetic differences between the fish from Nandujiang River and those from the mainland indicated that they were separated early.However,the small genetic differences among the samples and the positions of the fish from the Heilonjiang River in the molecular phylogenetic tree indicate that fish in Heilongjiang River might have dispersed from the Yangtze River to that area much later.

Cytochrome b5 Reductase 1 Triggers Serial Reactions that Lead to Iron Uptake in Plants

Rhizosphere acidification is essential for iron （Fe） uptake into plant roots. Plasma membrane （PM） H＊-ATPases play key roles in rhizosphere acidification. However, it is not fully understood how PM H＋-ATPase activity is regulated to enhance root Fe uptake under Fe-deficient conditions. Here, we present evidence that cytochrome b5 reductase 1 （CBR1） increases the levels of unsaturated fatty acids, which stimulate PM H＋-ATPase activity and thus lead to rhizosphere acidification. CBRl-overexpressing （CBRI-OX） Arabidopsis thaliana plants had higher levels of unsaturated fatty acids （18：2 and 18：3）, higher PM H＊-ATPase activity, and lower rhizosphere pH than wild-type plants. By contrast, cbrl loss-of-function mutant plants showed lower levels of unsaturated fatty acids and lower PM H＊-ATPase activity but higher rhizosphere pH. Reduced PM H＊-ATPase activity in cbrl could be restored in vitro by addition of unsatu- rated fatty acids. Transcript levels of CBR1, fatty acids desaturase 2 （FAD2）, and fatty acids desaturase 3 （FAD3） were increased under Fe-deficient conditions. We propose that CBR1 has a crucial role in increasing the levels of unsaturated fatty acids, which activate the PM H＊-ATPase and thus reduce rhizosphere pH. This reaction cascade ultimately promotes root Fe uptake.

Induction of Apoptosis in Purified Nuclei from Tobacco-Suspension Cells by Cytochrome b_6/f Complex

An apoptotic cell-free system containing cytosol and nuclei from normally cultured tobacco sus-pension cells was used to show that a spinach chloroplast preparation can induce apoptosis in nuclei, evi-denced by DNA electrophoresis and fluorescence microscopy observations. Further study showed that the chloroplast preparation or its pellet (thylakoid membrane) after hypoosmotic or supersonic treatment still ex-hibited the apoptosis-inducing activity, but the supernatant had no effect, which indicates that the apoptosis-inducing effector in the chloroplast preparation is water-insoluble. The induction of apoptosis by chloroplast preparation could be attenuated by Ac-DEVD-CHO, the specific inhibitor of Caspase-3, implying involve-ment of a Caspase-3-like protease during the process. Furthermore, extensive apoptosis in nuclei was in-duced by cytochrome b6/f on the thylakoid membrane, indicating that this important cytochrome complex may have an important role in the chloroplast-related apoptotic pathway.

Influences of the Hydrophobicity of the Heme-binding Pocket on the Properties and Functions of Cytochrome b_5 Mutants

The mutation sites of the four mutants F35Y, P40V, V45E and V45Y of cytochrome b 5 are located at the edge of the heme binding pocket. The solvent accessible areas of the “pocket interior” of the four mutants and the wild type cytochrome b 5 have been calculated based on their crystal structures at high resolution. The change in the hydrophobicity of the heme binding pocket resulting from the mutation can be quantitatively described using the difference of the solvent accessible area of the “pocket interior” of each mutant from that of the wild type cytochrome b 5. The influences of the hydrophobicity of the heme binding pocket on the protein stability and redox potential are discussed.

Structures of Cytochrome b_5 Mutated at the Charged Surface-Residues and Their Interactions with Cytochrome c

Glu44, Glu48, Glu56 and Asp60 are the negatively charged residues located at the molecular surface of cytochrome b 5. Two mutants of cytochrome b 5 were prepared, in which two or all of these four residues were mutated to alanines. The mutations give rise to slightly positive shifts of the redox potentials of cytochrome b 5 and obvious decrease of the cytochrome b 5-cytochrome c binding constants and electron transfer rates. The crystal structures of the two mutants were determined at 0.18 nm resolution, showing no alteration in overall structures and exhibiting slight changes in the local conformations around the mutation sites as compared with the wild-type protein. Based on the crystal structure of the quadruple-site mutant, a model for the binding of this mutant with cytochrome c is proposed, which involves the salt bridges from Glu37, Glu38 and heme propionate of cytochrome b 5 to three lysines of cytochrome c and can well account for the properties and behaviors of this mutant.

4-Hydroxycinnamic acid attenuates neuronal cell death by inducing expression of plasma membrane redox enzymes and improving mitochondrial functions

Many approaches to neurodegenerative diseases that focus on amyloid-βclearance and gene therapy have not been successful.Some therapeutic applications focus on enhancing neuronal cell survival during the pathogenesis of neurodegenerative diseases,including mitochondrial dysfunction.Plasma membrane(PM)redox enzymes are crucial in maintaining cellular physiology and redox homeostasis in response to mitochondrial dysfunction.Neurohormetic phytochemicals are known to induce the expression of detoxifying enzymes under stress conditions.In this study,mechanisms of neuroprotective effects of 4-hydroxycinnamic acid(HCA)were examined by analyzing cell survival,levels of abnormal proteins,and mitochondrial functions in two different neuronal cells.HCA protected two neuronal cells exhibited high expression of PM redox enzymes and the consequent increase in the NAD^(+)/NADH ratio.Cells cultured with HCA showed delayed apoptosis and decreased oxidative/nitrative damage accompanied by decreased ROS production in the mitochondria.HCA increased the mitochondrial complexes I and II activities and ATP production.Also,HCA increased mitochondrial fusion and decreased mitochondrial fission.Overall,HCA maintains redox homeostasis and energy metabolism under oxidative/metabolic stress conditions.These findings suggest that HCA could be a promising therapeutic approach for neurodegenerative diseases.

Proline-40 is Essential to Maintaining Cytochrome b_5's Stability and Its Electron Transfer with Cytochrome c

In order to illustrate the roles played by Pro40 in the structure, properties and functions of Cytochrome b 5, three mutated genes, P40V, P40Y, P40G were constructed in this work. Only the P40V gene was successfully expressed into holoprotein in E. coli JM83. According to the results of X-ray crystallographic analysis and various kinds of spectroscopy studies, it is evident that substituting valine for Pro40 does not result in significant alterations in the protein's overall structure; however, local conformational perturbations in the proximity of the heme do occur. The redox potential of the P40V mutant is 40 mV lower than that of the wild type protein. Its stability towards heat, urea, acid and ethanol were significantly decreased. The mutation leads to a decrease in the hydrophobicity of the heme pocket, which is probably the major factor contributing to the above changes. Binding constants and electron transfer rates between cytochrome b 5 and cytochrome c were determined using UV-visible spectroscopy and stopped-flow techniques for both the wild type and the mutant. The results showed that the substitution of Pro40 by valine does not influence the binding constant of cytochrome b 5 to cytochrome c; however, the electron transfer rate between them decreased significantly. This indicates that proline-40 is essential to maintaining cytochrome b 5's stability and its electron transfer with cytochrome c. These studies also provided a good example that property and functional changes of a protein do not necessarily require large overall structural alterations; in most cases, only perturbations on the local conformations are sufficient to induce significant changes in protein′s properties and functions.

Effects of Hyperoxia on Mitochondrial Multienzyme Complex Ⅲ and Ⅴ in Premature Newborn Rat Lung

To investigate the effects of hyperoxia on mitochondrial multienzyme complex Ⅲ （cytochrome, Cytb） and Ⅴ （ATPase6, 8） in premature newborn rat lung, the 1-day-old preterm SD rats were randomly assigned to hyperoxia group and air group, The rats in hyperoxia group were continuously exposed to 85% oxygen and those in air group to room air. After 1, 4, 7, 10, 14 day（s） of exposure, these rats were killed, total lung RNA was extracted and Cytb, ATPase6, 8 mRNA were detected by reverse transcription polymerase chain reaction （RT-PCR）. Western blotting was used to detect the expression of Cytb protein in lung tissue. The results showed that compared with air group, Cytb mRNA expression was significantly increased （P〉0,05） after 1, 4 day（s） of exposure. The general tendency decreased after 7 days, and its expression became weak but difference in mRNA expression between the two groups was not significant （P〉0.05）. ATPase6 mRNA expression was significantly increased 1 day after the exposure （P〈0.05） and did not show any significant change 4, 7, 10 days after the exposure （P〉0.05）. At the 14th day, ATPase6 mRNA expression was significantly increased （P〈0.05）, ATPase8 mRNA expression did not show any significant change 1, 4, 10 day（s） after the exposure （P〉0.05）, At the 7th and 14th day, ATPase8 mRNA expression was significantly increased （P〈0.05）. Western blotting showed that Cytb protein expression was increased 1,4 day（s） after the exposure, but the difference between the two groups was not significant （P〉0.05）. The general tendency was decreased after 7 days, and its expression became weak but difference was not significant 7, 10 days after the exposure （P〉0.05）. At day 14 its expression became significantly weak （P〈0.05）. We are led to conclude that exposure to high concentrations of oxygen can significantly change the expression of Cytb and ATPase6, 8, which results in uncoupling of oxidative phosphorylation in mitochondrial respiration chain, and plays an important role in the mechanism of hyperoxia-induced lung injury.

Phlebotomus(Adlerius) kabulensis(Diptera: Psychodidae) a new record sand fly species from Iran: Morphological and molecular aspects

Objective: To represent a new geographical record, Phlebotomus(Adlerius) kabulensis(P.kabulensis), which is suspected to be a potential vector of visceral leishmaniasis.Methods: For the first time, P.kabulensis specimens were collected using the sticky paper traps method in outdoor places in mountainous areas with vegetation coverage of three provinces in Iran.Identification of males was based on ecological, morphological, morphometric and molecular(mtDNA cytochrome b gene sequences) criteria.Generally, males have two ascoids on the 8^(th)antennal segment and one ascoid on segments 9^(th)to 15^(th), aedeagus with normal obtuseangled sub-terminal notch and coxite with 27–50 groups of hairs on the distal half of its body.Results: Morphometric measurement revealed that P.kabulensis specimens were the same as compared with seven other morphological characters in three provinces of the country but lengths of the coxite were significantly different.The PCR result of the cytochrome b(Cyt b)-mtDNA fragment shows 550-bp length, with its special nucleotide arrangement.The male and female of P.kabulensis were newly discovered members of the subgenus Adlerius from Iran.Initial DNA analysis indicated how distinct this species is.Conclusions: The results show that the P.kabulensis female will be identified by comparing with other Adlerius female groups regarding its morphometric characters and molecular sequencing.

红鮊属三种鱼类线粒体细胞色素b基因片段序列分析

本文测定了红珀属的翘嘴红约、青梢红铂和蒙古红铂细胞色素b基因片段序列（1091bp），对其碱基组成变异情况及核苷酸序列差异进行了分析。并以红鳍珀为外类群，用NJ法和UPGMA法构建系统关系树。结果表明：（1）四种鱼（包括外类群）Cytb基因片段序列碱基平均差异为6．2％，变异位点184个，具有简约性信息位点141个，平均转换颠换比为7．4；（2）本文测定的红鲐属三种鱼形成一个单系群，其中翘嘴红珀与青梢红珀亲缘关系较近。

Genetic Diversity and Structure in Japanese Populations of the Osprey (<i>Pandion haliaetus</i>), Based on mtDNA

Osprey is a type of bird of prey that lives almost all over the world. In Japan, it is designated as a near-threatened species because it has less than 1000 individuals. In recent years, it inhabits more inland than in coastal areas. In this study, we conducted a population genetic analysis focusing on what kind of genetic structure Japanese Osprey retains and whether there are differences between coastal and inland populations. We also performed genetic diversity assessments. We sequenced about 2.3 kb of mtDNA for 27 individuals in Japan, and phylogenetic analysis, network analysis, neutrality test and mismatch distribution analysis were performed. Eighteen haplotypes were detected in 27 individuals, indicating that genetic diversity was sufficiently high. Both unique and common haplotypes were detected between inland and coastal populations, suggesting gene flow between the two populations. Phylogenetic analysis results show no genetic differentiation in the Japanese Osprey population. From the results of network analysis, neutrality test and mismatch distribution analysis, it was inferred that the Japanese Osprey had a population expansion in the past. This study indicated that the dispersion of Japanese Osprey was random and there were no restrictions on the breeding area. The information presented here can be used towards implementing future conservation actions.

A preliminary investigation on genetic diversity of Sousa chinensis in the Pearl River Estuary and Xiamen of Chinese waters

In this study, the mitochondrial DNA （mtDNA） control region and the mitochondrial cytochrome b gene of stranded Indo-Pacific humpback dolphin （Sousa chinensis） samples from the Pearl River Estuary and Xiamen waters were sequenced and analyzed. The result of mtDNA control region revealed 34 variable sites and four unique haplotypes （named as A, B, C and D） identified among the total samples from these two water areas, and the most common haplotype （A） was shared by 75% of the dolphins sampled from the two water areas. The haplotypic diversity （h） was 0.455 and the nucleodde diversity （π） was 0.0088. The phylogenetic analysis showed that the haplotype A, C, and D were closely related, but the haplotype B （unique for XM01 from Xiamen） was far from the other three. By scanning cytochrome b fragments, two haplotypes （A and B） were identified in these two water areas, and the most common haplotype （A） was shared by 91.67% individuals, while XM01 from Xiamen as the only exception. The data suggest that there is a possibility of gene exchange between the two populations in the Pearl River Estuary and Xiamen waters, and there possibly exists a unique maternal lineage in Xiamen waters.

New insights into the role of cytochrome P450 reductase (POR) in microsomal redox biology

Cytochrome P450 reductase(POR)is an essential electron transfer protein located on the endoplasmic reticulum of most cell types,and has long been appreciated for its role in cytochrome P450-mediated drug metabolism.Additional roles and electron acceptors for POR have been described,but it is largely with the recent availability of POR-null tissues that these supplemental roles for POR have been able to be explored.These studies have confirmed POR as the principal redox partner for the microsomal P450s responsible for drug and xenobiotic metabolism as well as cholesterol and bile acid synthesis,and for heme oxygenase,which catalyzes the initial step in the breakdown of heme.Surprisingly,these studies have revealed that squalene monooxygenase,an enzyme essential to cholesterol synthesis,has a second unknown redox partner in addition to POR,and that 7-dehydrocholesterol reductase,previously proposed to require POR as an electron donor,functions fully independently of POR.These studies have also helped define the role of cytochrome b5 in P450 catalysis,and raise the question as to the extent to which POR contributes to b5-dependent redox pathways.

Continued surprises in the cytochrome c biogenesis story

Cytochromes c covalently bind their heme prosthetic groups through thioether bonds between the vinyl groups of the heme and the thiols of a CXXCH motif within the protein.In Gramnegative bacteria,this process is catalyzed by the Ccm(cytochrome c maturation)proteins,also called System I.The Ccm proteins are found in the bacterial inner membrane,but some(CcmE,CcmG,CcmH,and CcmI)also have soluble functional domains on the periplasmic face of the membrane.Elucidation of the mechanisms involved in the transport and relay of heme and the apo-cytochrome from the bacterial cytosol into the periplasm,and their subsequent reaction,has proved challenging due to the fact that most of the proteins involved are membrane-associated,but recent progress in understanding some key components has thrown up some surprises.In this Review,we discuss advances in our understanding of this process arising from a substrate’s point of view and from recent structural information about individual components.

Detection of food source by PCR analysis of the gut contents of Aldrichina grahami （Aldrich） （Diptera： Calliphoridae） during post-feeding period

The last meal of sarcophagous maggots may be useful in identifying the species on whose flesh they have fed （the＂host＂species）. The DNA profile of the host species may indeed be detectable in the＂last meal＂. In this paper, mitochondrial DNA analysis of gut contents was used to identify the prior host of post-feeding larvae of Aldrichina grahami （Aldrich） （Diptera： Calliphoridae）. A modified logistic equation was fitted to estimate the probability of identifying the host under five different constant temperatures （16, 20, 24, 28 and 32 ℃）. Our results shows that the detected time ranged from a maximum of 24 h at 32℃ to 42 h at 16℃ and a minimum of 12 h at 32~C to 30 h at 16℃. Furthermore, the host detection time was also calculated to give the maximal time after larval hatching from the egg. These results indicate that, in criminal cases where the maggots stray from the corpse, the last meal of the larvae should not be overlooked as potentially critical evidence.