Effects of sperm ultraviolet irradiation on the embryonic development of Rana pipiens

This investigation is concerned with the role played by irradiated sperm in the subsequent embryonic development of the frog Rana pipiens. The ultraviolet irradiation of the sperm was followed by the fertilization of untreated eggs. Determinations included mitotic index, nuclear diameter, chromosomal constitution, and percentage survival. Results show that the low-dose embryos were the most severely affected ones in spite of their diploid constitution. Embryos resulting from higher dosages of sperm irradiation were all haploid but showed less severe growth and cytological abnormalities. It is conjectured that radiation-induced damage to the sperm also involves its protamine moiety since considerable damage can occur without any alteration in the chromosomal constitution of the low-dose embryos.     

COX8, the structural gene for yeast cytochrome c oxidase subunit VIII. DNA sequence and gene disruption indicate that subunit VIII is required for maximal levels of cellular respiration and is derived from a precursor which is extended at both its NH2 and COOH termini

From the amino acid sequence of yeast cytochrome c oxidase subunit VIII published previously (Power, S. D., Lochrie, M.A., Patterson, T.E., and Poyton, R.C. (1984) J. Biol. Chem. 259, 6571-6574), we have synthesized a pair of oligonucleotide probes and used them to identify COX8, its structural gene. By genomic Southern blot analysis and disruption of the COX8 chromosomal locus, we have shown that this gene is present in one copy per haploid genome and that its product, subunit VIII, is essential for maximal levels of cellular respiration and cytochrome c oxidase activity. Alignment of the amino acid sequence predicted from the DNA sequence of COX8 with the determined amino acid sequence of subunit VIII indicates that mature subunit VIII is derived from a larger precursor that extends from both the NH2 and COOH termini of the mature polypeptide. Thus, like many other nuclear coded mitochondrial proteins, subunit VIII is derived from a precursor which carries a leader peptide. In addition, this precursor, like that for yeast cytochrome c oxidase subunit VIIa, appears to carry a four-amino acid "trailer peptide" at its COOH terminus.     

Back-transfer of late gastrula nuclei of nucleocytoplasmic hybrids

Late gastrula-early neurula endoderm nuclei from nucleocytoplasmic hybrids consisting of Rana pipiens nuclei and Rana palustris cytoplasm were back-transferred into enucleated eggs of Rana pipiens. Approximately half of the normally cleaved back-transfer embryos developed normally. A similar result was obtained with nuclear transfer controls. As expected, the development of the nucleocytoplasmic hybrid donors, at the time the back-transfers were carried out, was clearly abnormal. Consequently, a significant proportion of endoderm nuclei from these embryos undergo no irreversible changes in their developmental capacity even after the nucleocytoplasmic incompatibility between these two amphibian species is expressed.     

Biochemistry and physiological role of methionine sulfoxide residues in proteins

The cytochrome system in eggs and embryos of the sea urchin, Hemicentrotus pulcherrimus, was investigated. Difference spectra of the mitochondrial fraction demonstrated the presence of a complete cytochrome system in unfertilized eggs. Cytochrome levels and the activities of respiratory enzymes were measured in crude extracts of eggs both before and after fertilization. Unfertilized eggs contained cytochromes aa₃, b, and c + c₁ in a ratio of 1.0:1.8:0.7. Gastrulae contained almost the same amount of cytochromes aa₃and b as unfertilized eggs. However, the amount of cytochrome c + c₁ in gastrulae was 1.5 times greater than that in unfertilized eggs. The activity of cytochrome oxidase remained unchanged during development. No cytochrome oxidase inhibitor was found in unfertilized eggs. Both antimycin A-sensitive and insensitive NADH-cytochrome c reductase activities increased during development. The activity of succinate-cytochrome c reductase increased during early development, reached a temporary plateau, and then declined at the pluteus stage. These results are discussed in relation to the increase of respiration during early development.     

Coevolution of symbiotic bacteria Wolbachia and host mtDNA in Russian populations of the Culex pipiens mosquito complex

The mitochondrial DNA phylogenies of closely related forms of mosquitoes from the Culex pipiens complex and of strains of the endosymbiotic bacteria Wolbachia pipientis were compared. Based on the cytochrome oxidase subunit I gene polymorphism, six mitochondrial haplotypes and four W. pipientis groups were discovered in mosquitoes from geographically remote populations. A strict correlation between the COI type and the type of W. pipientis proves the stable coinheritance and distribution of both cytoplasmic components in the examined mosquito populations and suggests either the absence or rarity of horizontal transfer of the symbionts in the Culex pipiens complex.     

Effects of haploidy and hybridization on the activities of four dehydrogenases in frog embryos

Total activities of four enzymes, malate dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase, and 6-phosphogluconate dehydrogenase were measured in diploid and androgenetic haploid embryos resulting from crosses of Rana pipiens pipiens and Rana pipiens sphenocephala. Developmental curves of these enzyme activities were compared with the DNA content of the embryos. The results suggest that decreased total enzyme activity in abnormal androgenetic haploid hybrids is due to the effects of reduced cellular proliferation and cellular degradation caused by a general nucleocytoplasmic incompatibility, not to the factors directly affecting the synthesis of each enzyme.     

Nuclear and cytoplasmic contributions to dehydrogenase phenotypes in hybrid frog embryos

Frogs in theRana pipiens group includingR. palustris and geographic races ofR. pipiens form viable diploid hybrids which develop through metamorphosis.     

Aging-related decrease in hepatic cytochrome oxidase of the Fischer 344 rat

The effect of aging on the hepatic mitochondrial population has been determined using a rigorously defined group of Fischer 344 rats with known survivorship data. The age groups studied included mature adult controls (8.5 months; 100% survivorship), an intermediate aged group (17.5 months; 90% survivorship), and an aged group (29 months; 20% survivorship). Cytochrome oxidase activity and content were determined in homogenates and mitochondrial fractions. The mitochondrial fractions were characterized by determination of respiratory activity, and monoamine oxidase activity as well as evaluation of the polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The yield of protein in the isolated mitochondrial fraction as well as the mitochondrial specific content decreased significantly as a function of aging. Mitochondrial specific content was determined from the specific activities of cytochrome oxidase in the homogenate (per gram liver) and in the isolated mitochondrial fraction (per mg protein). Specific activity of hepatic cytochrome oxidase decreased approximately 15% (P = 0.035) in homogenates from the 17.5-month animals with a further, highly significant (P = 0.0002) decrease (29%) in the 29-month animals. In contrast, there was no statistically significant difference among the age groups in the cytochrome oxidase specific activity in the isolated hepatic mitochondrial fractions. However, the percentage of the total homogenate cytochrome oxidase activity recovered in the isolated mitochondrial fraction decreased significantly in the 29-month animals (P = 0.0063 vs the 8.5-month controls; P = 0.022 vs the 17.5-month group). Cytochrome aa₃ content of total liver homogenates from aged animals decreased (P = 0.00064) which is in agreement with the decline in cytochrome oxidase specific activity in this age group. In the mitochondrial fraction from the aged animals, cytochrome aa₃ content was essentially unchanged which is consistent with the lack of aging-related change in mitochondrial cytochrome oxidase specific activity. In freshly isolated mitochondrial fractions, no aging-related alterations were observed in respiratory control and $\text{ADP}\text{O}$ ratios. The addition of exogenous NADH and cytochrome c did not change significantly the respiratory rate of hepatic mitochondria from control or aged animals. These results demonstrate the integrity of freshly isolated mitochondrial preparations from both control and aged Fischer 344 rats. In addition, there was no aging-related alteration in either monoamine oxidase specific activity or polypeptide composition. The similarities observed in the specific activities of cytochrome oxidase and monoamine oxidase, as well as in the cytochrome aa₃ content and polypeptide composition of the isolated mitochondrial fraction, suggest a generalized decrease in hepatic mitochondrial content as a function of aging rather than a selective loss of mitochondrial components.     

Mitochondrial biogenesis during fungal spore germination. Development of cytochrome c oxidase activity.

Mitochondria from dormant spores of the fungus Botryodiplodia theobromae did not contain extractable cyctochrome c oxidase (EC 1.9.3.1) activity; however, this enzyme activity was elaborated rapidly after 150 min of the 240-min germination sequence. The absence of cytochrome c oxidase activity in the dormant spores apparently is not an artifact caused by spore disruption and fractionation procedures, transient enzyme instability, or insensitivity of the enzyme assay. Mitochondria from dormant spores of three other phylogenetically diverse genera of fungi were observed to contain readily detectable quantities of cytochrome c oxidase, suggesting that the absence of the enzyme in B. theobromae may be relatively novel. The elaboration of cytochrome c oxidase activity in germinating spores was abolished by cycloheximide if the drug was added at or before 95 min of germination, but development of enzyme activity was initially insensitive to inhibitors of the mitochondrial genetic system, chloramphenicol or ethidium bromide. Incubation of spores in both ethionine and S-2-aminoethyl-l-cysteine reduced the amount of extracted cytochrome c oxidase activity. Elaboration of enzyme activity was severely retarded by cerulenin, an inhibitor of fatty acid biosynthesis and of spore germination. This enzyme activity developed in water-incubated or 1% Tween 80-incubated spores in which only the cytoplasmic ribosomes are functional in translation of a stored nuclear messenger RNA. The results of this study show that cytoplasmic (but not mitochondrial) ribosome function is required for development of this enzyme activity during spore germination, and they suggest that a portion of the cytochrome c oxidase enzyme or some other protein required for its activity is synthesized de novo upon germination.     

The Complete Mitochondrial Genome of the Foodborne Parasitic Pathogen Cyclospora cayetanensis

Cyclospora cayetanensis is a human-specific coccidian parasite responsible for several food and water-related outbreaks around the world, including the most recent ones involving over 900 persons in 2013 and 2014 outbreaks in the USA. Multicopy organellar DNA such as mitochondrion genomes have been particularly informative for detection and genetic traceback analysis in other parasites. We sequenced the C. cayetanensis genomic DNA obtained from stool samples from patients infected with Cyclospora in Nepal using the Illumina MiSeq platform. By bioinformatically filtering out the metagenomic reads of non-coccidian origin sequences and concentrating the reads by targeted alignment, we were able to obtain contigs containing Eimeria-like mitochondrial, apicoplastic and some chromosomal genomic fragments. A mitochondrial genomic sequence was assembled and confirmed by cloning and sequencing targeted PCR products amplified from Cyclospora DNA using primers based on our draft assembly sequence. The results show that the C. cayetanensis mitochondrion genome is 6274 bp in length, with 33% GC content, and likely exists in concatemeric arrays as in Eimeria mitochondrial genomes. Phylogenetic analysis of the C. cayetanensis mitochondrial genome places this organism in a tight cluster with Eimeria species. The mitochondrial genome of C. cayetanensis contains three protein coding genes, cytochrome (cytb), cytochrome C oxidase subunit 1 (cox1), and cytochrome C oxidase subunit 3 (cox3), in addition to 14 large subunit (LSU) and nine small subunit (SSU) fragmented rRNA genes.     

Formamide probes a role for water in the catalytic cycle of cytochrome c oxidase

Formamide is a slow-onset inhibitor of mitochondrial cytochrome c oxidase that is proposed to act by blocking water movement through the protein. In the presence of formamide the redox level of mitochondrial cytochrome c oxidase evolves over the steady state as the apparent electron transfer rate from cytochrome a to cytochrome a₃ slows. At maximal inhibition cytochrome a and cytochrome c are fully reduced, whereas cytochrome a₃ and Cu_B remain fully oxidized consistent with the idea that formamide interferes with electron transfer between cytochrome a and the oxygen reaction site. However, transient kinetic studies show that intrinsic rates of electron transfer are unchanged in the formamide-inhibited enzyme. Formamide inhibition is demonstrated for another member of the heme-oxidase family, cytochrome c oxidase from Bacillus subtilis, but the onset of inhibition is much quicker than for mitochondrial oxidase. If formamide inhibition arises from a steric blockade of water exchange during catalysis then water exchange in the smaller bacterial oxidase is more open. Subunit III removal from the mitochondrial oxidase hastens the onset of formamide inhibition suggesting a role for subunit III in controlling water exchange during the cytochrome c oxidase reaction.     

Enhanced cytochrome oxidase activity and modification of lipids in heart mitochondria from hyperthyroid rats

In order to further investigate the mechanism regulating the control of mitochondrial respiration by thyroid hormones, the effect of the hyperthyroidism on the kinetic characteristics of cytocrome c oxidase in rat heart mitochondria was studied. Mitochondrial preparations from both control and hyperthyroid rats had equivalent K_m values for cytochrome c, while the maximal activity of cytochrome oxidase was significantly increased (by around 30%) in mitochondrial rats. This enhanced activity of cytochrome oxidase was associated to a parallel increases in mitochondrial State 3 respiration. The hormone treatment resulted in a decrease in the flux control coefficient of the oxidase. The enhanced activity of cytochrome oxidase in hyperthyroid rats does not appear to be dependent on an increases in the mass of this enzyme complex in that the heme aa₃ content was equivalent in both hyperthyroid and control preparations. The Arrhenius plot characteristics differ for cytochrome oxidase activity in mitochondria from hyperthyroid rats as compared with control rats in the breakpoint of the biphasic plot is shifted to a lower temperature. Cardiolipin content was significantly increased in mitochondrial preparations from hyperthyroid rats, while there were no significant alterations in the fatty acid composition of cardiolipin of control and hyperthyroid preparations. The results support the conclusion that the enhanced cytochrome oxidase activity in heart mitochondrial preparations from hyperthyroid rats is due to a specific increase in the content of cardiolipin.     

Transcriptome Analysis of Honeybee (Apis Mellifera) Haploid and Diploid Embryos Reveals Early Zygotic Transcription during Cleavage

In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development.     

Increase of Mitochondrial Fraction in Sweet Potato Root Tissue after Wounding or Infection with Ceratocystis fimbriata

The acid-insoluble nitrogen content, lipid content, and cytochrome oxidase activity in the mitochondrial fraction are found to increase during incubation of slices of sweet potato (Ipomoea batatas) root tissue. These increases appear to be related to an increase in the number of the mitochondrial particles. The increase in the mitochondrial fraction is not accompanied by an increase in cell number. The nitrogen content in the mitochondrial fraction increases prior to the changes in the activity of cytochrome oxidase and lipid content. The increase in the numbers of the mitochondrial particles lags behind the increase in the cytochrome oxidase activity. Such findings are also found in the tissue infected by Ceratocystis fimbriata.     

Studies of mitochondrial energy systems during embryogenesis in the rat

Mitochondria isolated from rat embryos of Day 11 to Day 14 of gestation showed an equal capacity to carry out oxidative phosphorylation with P:O ratios of approximately 3 for pyruvate-malate and 2 for succinate as substrates. Activities of succinic dehydrogenase, cytochrome oxidase, and mitochondrial ATPase remained at nearly constant levels in the preparations during this period of gestation, but DPNH oxidase activity increased over 2-fold. The increase in DPNH oxidase activity could be prematurely induced at Day 11 of gestation of the embryos by prior treatment of the pregnant rats with an atmosphere containing 85% oxygen and 0.4% CO₂, but no changes in the growth and development of the treated embryos were found by gross examination.     

The biochemistry of HEXA and HEXB gene mutations causing GM2 gangliosidosis

Immuno-flow cytometry was tested as a tool to estimate the cellular concentration of mitochondrial proteins in cultured cells, using cytochrome c oxidase as a model enzyme. Cells labelled with antibodies against cytochrome c oxidase, in which the amount of the enzyme was reduced by various extents, showed a linear relationship between the size of the signal obtained by immuno-flow cytometry and the amount of the enzyme. The determination by immuno-flow cytometry resulted in data comparable to the results obtained by immunoprecipitation and activity measurement. Since immuno-flow cytometry requires only limited numbers of cells, the method could especially be of value for diagnostic purposes. This is illustrated by the results obtained by comparing activity measurements and immuno-flow cytometry in the initial screening of cell lines derived from patients with deficiencies in the activity of cytochrome c oxidase.     

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity – a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.     

Mitochondrial bioenergetics linked to the manifestation of programmed cell death during somatic embryogenesis of <Emphasis Type="Italic">Abies alba</Emphasis>

The present work reports changes in bioenergetic parameters and mitochondrial activities during the manifestation of two events of programmed cell death (PCD), linked to Abies alba somatic embryogenesis. PCD, evidenced by in situ nuclear DNA fragmentation (TUNEL assay), DNA laddering and cytochrome c release, was decreased in maturing embryogenic tissue with respect to the proliferation stage. In addition, the major cellular energetic metabolites (ATP, NAD(P)H and glucose-6-phosphate) were highered during maturation. The main mitochondrial activities changed during two developmental stages. Mitochondria, isolated from maturing, with respect to proliferating cell masses, showed an increased activity of the alternative oxidase, external NADH dehydrogenase and fatty-acid mediated uncoupling. Conversely, a significant decrease of the mitochondrial K_ATP⁺ channel activity was observed. These results suggest a correlation between mitochondrial activities and the manifestation of PCD during the development of somatic embryos. In particular, it is suggested that the K_ATP⁺ channel activity could induce an entry of K⁺ into the matrix, followed by swelling and a release of cytochrome c during proliferation, whereas the alternative pathways, acting as anti-apoptotic factors, may partially counteract PCD events occurring during maturation of somatic embryos.     

Succinic Dehydrogenase and Cytochrome Oxidase Activities in Cell Cultures

Succinic dehydrogenase and cytochrome oxidase have been assayed in permanent cell lines (HEP 1, HEP 2, and HLM), in short-term cultures of chick embryo heart cells, and in various tissues. Their activities in different cells are compared by relating them to deoxyribonucleic acid. They are very low in HEP 1, HEP 2, and HLM cells by comparison with the activities in any normal tissues examined. All the succinic dehydrogenase was shown to be located in the mitochondria of the permanent cell lines by staining with tetrazolium derivatives. Both enzymes were more active in tissues of 19-day chick embryos than in those of 11- or 14-day embryos. The increasing activities found during normal development were quickly curtailed or reversed when heart cells were grown as monolayer cultures. Digitonin-treated mitochondria produced preparations with much higher activities of cytochrome oxidase than untreated samples. Activities measured in this way were again very much lower in HEP 1, HEP 2, and HLM cells than in the normal tissues. From the derived ratio of cytochrome oxidase:succinic dehydrogenase, it was apparent that cytochrome oxidase is diminished to a greater extent than succinic dehydrogenase in both permanent cell lines and short-term cultures, by comparison with the corresponding activities in embryonic and adult tissues. The features common to the metabolism of proliferating cells in vitro and malignant cells are discussed.