Specific mitochondrial proteins in pollen: presence of an additional ATP synthase beta subunit.

A protocol was designed to obtain a pure fraction of pollen mitochondria from the diploid species Nicotiana sylvestris, the female parent of the allotetraploid Nicotiana tabacum. Most organelles were morphologically intact and able to perform in organello mitochondrial (mt) protein synthesis. As revealed by two-dimensional protein electrophoresis, numerous quantitative differences exist between leaf and pollen mt proteins. Moreover, additional mt polypeptides, named R (for reproductive), encoded by either nuclear or mitochondrial genes, are found in pollen. The most abundant R polypeptide, R1 (M(r) 53,000, pI 5.6), is nuclearly encoded, is membrane bound, and cross-reacts with an antibody directed against the beta subunit of the mt ATP synthase (ATPase). N-terminal microsequence analysis showed that the two ATPase beta subunits present in leaves (beta 1 and beta 2) and the R1 pollen-specific subunit are encoded by distinct genes. A similar additional ATPase beta subunit was observed in pollen mitochondria from Petunia, suggesting that this polypeptide is of general importance for male gametophytic development in Solanaceaes.     

Specific cleavage analysis of mammalian mitochondrial DNA.

Mitochondrial DNA from several mammalian species has been digested with a site-specific restriction endonuclease (HaeIII) from Haemophilus aegyptius. A quantitative analysis of the resulting specific fragments indicates that the mtDNA of any individual mammal is predominantly a single molecular clone. Gel analysis of specific cleavage products has proven quite sensitive in detecting differences in mtDNA: mtDNAs from the more distantly related mammals studied (e.g., donkey and dog) are found to have few bands in common and very closely related mammals (e.g., donkey and horse) share only about 50% of their bands. This procedure has detected several intraspecies mtDNA differences. Six distinct human patterns have been found, with one pattern usually differing from another in two or three bands. mtDNAs from different organs of single individuals have also been analyzed, and no differences have been found.     

The myosin step size: measurement of the unit displacement per ATP hydrolyzed in an in vitro assay.

Chemomechanical coupling in muscle contraction may be due to "swinging crossbridges," such that a change in the angle at which the myosin head binds to the actin filament is tightly coupled to release of products of ATP hydrolysis. This model would limit the step size, the unit displacement of actin produced by a single ATP hydrolysis, to less than twice the chord length of the myosin head. Recent measurements have found the step size to be significantly larger than this geometric limit, bringing into question any direct correspondence between the crossbridge and ATP-hydrolysis cycles. We have measured the rate of ATP hydrolysis due to actin sliding movement in an in vitro motility assay consisting of purified actin and purified myosin. We have calculated an apparent myosin step size well within the geometric limit set by the size of the myosin head. These data are consistent with tight coupling between myosin crossbridge movement and ATP hydrolysis.     

Evidence for mitochondrial K ATP channels as effectors of human myocardial preconditioning

BACKGROUND: Sublethal periods of ischemia preceding a prolonged interval of ischaemia protect the myocardium. This myocardial preconditioning (PC) appears to be effected by KATP channels. These channels occur both in the sarcolemma and the mitochondrial membrane. We investigated whether mitochondrial KATP channels are the end-effector of PC in the human myocardium. METHODS: Right atrium specimens obtained from patients undergoing cardiac surgery were prepared and incubated in buffer solution at 37 degrees C. After 30-min stabilisation, the muscles were made ischemic for 90 min and then reperfused for 120 min. The preparations were randomised into eight experimental groups (n = 6/group): (1) Aerobic control--incubated in oxygenated buffer for 210 min, (2) ischemia alone--90 min ischemia followed by 120 min reperfusion, (3) PC--preconditioned with 5 min ischemia/5 min reperfusion, (4) Glibenclamide (10 microM) in the incubation media for 10 min before PC, (5) 5-hydroxydecanoate (5-HD, MitoKATP blocker, 1 mM) in the incubation media for 10 min before PC, (6) HMR 1883 (SarcKATP blocker, 10 microM) in the incubation media for 10 min before PC, (7) Pinacidil (0.5 mM) in the incubation media for 10 min before ischemia, and (8) Diazoxide (MitoKATP opener, 0.1 mM) in the incubation media for 10 min before ischemia. Creatinine kinase leakage into the medium (CK, IU/g wet wt) and MTT reduction (OD/mg wet wt.), an index of cell viability, were assessed at the end of the experiment. RESULTS: Ischemia alone resulted in a significant increase in CK leakage (8.01 +/- 0.35) and decrease in MTT (0.15 +/- 0.01) from the values seen in the aerobic control (2.24 +/- 0.52 and 0.78 +/- 0.10 respectively, P < 0.05 in both instances). PC fully reversed the effect of ischemia (CK = 2.97 +/- 0.31 and MTT = 0.61 +/- 0.05; P < 0.05 vs. ischemia alone group but P = NS vs. aerobic control group). Both Glibenclamide and 5-HD abolished the protection induced by PC (CK = 6.23 +/- 0.5 and 7.84 +/- 0.64; MTT = 0.18 +/- 0.03 and 0.13 +/- 0.02, respectively, P < 0.05 vs. PC), but interestingly, the protective effect of PC was not abolished by HMR 1883 (CK = 2.85 +/- 0.24 and MTT = 0.58 +/- 0.05, P = NS vs. PC). Diazoxide mimicked the protective effect of PC (CK = 3.56 +/- 0.32 and MTT = 0.58 +/- 0.02, P = NS vs. PC), however pinacidil exhibited less protection than PC (CK = 4.02 +/- 0.16 and MTT = 0.30 +/- 0.02, P < 0.05 vs. PC). CONCLUSIONS: These studies demonstrate that KATP channels are the end-effectors of ischemic preconditioning and that protection is mediated by mitochondrial KATP channels in human right atrial myocardium.     

Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein

P-glycoprotein (Pgp) is an ATP-dependent hydrophobic natural product anticancer drug efflux pump whose overexpression confers multidrug resistance to tumor cells. The work reported here deals with the elucidation of the energy requirement for substrate interaction with Pgp during the catalytic cycle. We show that the K(d) (412 nM) of the substrate analogue [(125)I]iodoarylazidoprazoin for Pgp is not altered by the presence of the nonhydrolyzable nucleotide 5'-adenylylimididiphosphate and vanadate (K(d) = 403 nM). Though binding of nucleotide per se does not affect interactions with the substrate, ATP hydrolysis results in a dramatic conformational change where the affinity of [(125)I]iodoarylazidoprazoin for Pgp trapped in transition-state conformation (Pgp x ADP x vanadate) is reduced >30-fold. To transform Pgp from this intermediate state of low affinity for substrate to the next catalytic cycle, i.e., a conformation that binds substrate with high affinity, requires conditions that permit ATP hydrolysis. Additionally, there is an inverse correlation (R(2) = 0.96) between 8AzidoADP (or ADP) release and the recovery of substrate binding. These results suggest that the release of nucleotide is necessary for reactivation but not sufficient. The hydrolysis of additional molecule(s) of ATP (or 8AzidoATP) is obligatory for the catalytic cycle to advance to completion. These data are consistent with the observed stoichiometry of two ATP molecules hydrolyzed for the transport of every substrate molecule. Our data demonstrate two distinct roles for ATP hydrolysis in a single turnover of the catalytic cycle of Pgp, one in the transport of substrate and the other in effecting conformational changes to reset the pump for the next catalytic cycle.     

Specific mitochondrial DNA mutation in mice regulates diabetes and lymphoma development

It has been hypothesized that respiration defects caused by accumulation of pathogenic mitochondrial DNA (mtDNA) mutations and the resultant overproduction of reactive oxygen species (ROS) or lactates are responsible for aging and age-associated disorders, including diabetes and tumor development. However, there is no direct evidence to prove the involvement of mtDNA mutations in these processes, because it is difficult to exclude the possible involvement of nuclear DNA mutations. Our previous studies resolved this issue by using an mtDNA exchange technology and showed that a G13997A mtDNA mutation found in mouse tumor cells induces metastasis via ROS overproduction. Here, using transmitochondrial mice (mito-mice), which we had generated previously by introducing G13997A mtDNA from mouse tumor cells into mouse embryonic stem cells, we provide convincing evidence supporting part of the abovementioned hypothesis by showing that G13997A mtDNA regulates diabetes development, lymphoma formation, and metastasis--but not aging--in this model.     

Nucleus-driven mutations of human mitochondrial DNA

Summary Neuromuscular disorders due to abnormalities of mitochondrial energy supply have become an important area of human pathology. In particular, lesions of the mitochondrial genome (mtDNA), a small extra-nuclear chromosome which encodes 13 subunits of the respiratory chain complexes, are responsible for a steadily increasing number of neuromuscular syndromes. In addition to sporadic or maternally-inherited mutations, either qualitative or quantitative abnormalities of mtDNA can be transmitted as Mendelian traits, leading to well-defined mitochondrial encephalomyopathies. The latter are presumably caused by mutations in still unknown nucleus-encoded genes which deleteriously interact with the mitochondrial genome. These observations are of importance from both clinical and theoretical points of view, because they are the first examples of diseases produced by abnormalities of the nuclear control over mitochondrial biogenesis.     

Restriction assay for integrative recombination of bacteriophage lambda DNA in vitro: requirement for closed circular DNA substrate.

A novel assay has been developed for in vitro genetic recombination of DNA. Substrate and product DNAs are cleaved with a restriction endonuclease and the resulting fragments are separated by electrophoresis in agarose gels. The substrate DNA has been chosen so that the recombination to be studied deletes a segment of DNA. The remaining DNA gives rise to a unique restriciton fragment, as does the DNA segment that has been removed. The method provides a convenient and physical, rather than genetic, assessment of the conversion of parental to recombinant DNA. This method has been applied to an in vitro system that carries out integrative recombination of bacteriophage lambda. We find that, different molecular forms of DNA tested, closed circular DNA is the only efficient substrate. Linear DNA and three kinds of circular DNA containing interruptions are at best very poor substrates. The implications of this surprising result are discussed. In addition, we show that the in vitro recombination system completes the breaking and rejoining steps of recombination. No stable DNA intermediates involving chiasmata or broken end structures are found.     

Replication of plasmid pT181 DNA in vitro: requirement for a plasmid-encoded product.

PT181 is a naturally occurring 4.5-kilobase Staphylococcus aureus plasmid encoding resistance to tetracycline. The plasmid has a copy number of about 20 per cell; a mutant, cop-608, that has a copy number of 800-1000 has been isolated. A cell-free extract has been developed that carries out complete replication of this plasmid. Extracts made from a strain containing the mutant have much greater replication activity than do extracts of strains containing pT181. In an extract from which endogenous DNA has been removed, DNA synthesis is dependent upon the addition of exogenous plasmid DNA. The replication system is specific for pT181 and related plasmids but it is inactive with other S. aureus plasmids. Furthermore, pT181 DNA does not replicate in extracts made from plasmid-negative strains or strains containing other plasmids. The results suggest that a specific plasmid-encoded substance is required for the replication of pT181 DNA.     

Maternal inheritance of human mitochondrial DNA.

Human mitochondrial DNA was obtained from peripheral blood platelets donated by the members of several independent families. The samples were screened for nucleotide sequence polymorphisms between individuals within these families. In each family in which we were able to detect a distinctly different restriction endonuclease cleavage pattern between the parents, the progeny exhibited the maternal cleavage pattern. Informative polymorphisms were detected for Hae II (PuGCGCPy) in a three-generation family composed of 33 members, for HincII (GTPyPuAC) in a two-generation family composed of four members, and for Hae III(GGCC) in a two-generation family composed of four members. The Hae II polymorphism was analyzed through all three generations in both the maternal and paternal lines. The results of this study demonstrate that human mitochondrial DNA is maternally inherited. The techniques described for using peripheral blood platelets as a source of human mitochondrial DNA represent a convenient way to obtain data on mitochondrial DNA variation in both individuals and populations.     

Branching pattern in the evolutionary tree for human mitochondrial DNA.

Eighty-eight types of mitochondrial (mt) DNA were found by sequencing the most variable part of the control region from 117 Caucasians. In the tree relating those types, most of the branching events occur about two-thirds of the way from the root of the tree to the tips of the branches. Moreover, the distribution of sequence differences between all possible pairs of individuals is approximately Poisson. Other non-African populations show a similar pattern. Assuming a neutral model, these findings imply that the probability of survival of new lineages has undergone dramatic changes, probably due to population expansion. Conversely, African populations show multimodal distributions fitting with a model of constant population size.     

Conditional requirement for the Flk-1 receptor in the in vitro generation of early hematopoietic cells.

Genetic studies in mice have previously demonstrated an intrinsic requirement for the vascular endothelial growth factor (VEGF) receptor Flk-1 in the early development of both the hematopoietic and endothelial cell lineages. In this study, embryonic stem (ES) cells homozygous for a targeted null mutation in flk-1 (flk-1 (-/-)) were examined for their hematopoietic potential in vitro during embryoid body (EB) formation or when cultured on the stromal cell line OP9. Surprisingly, in EB cultures flk-1 (-/-) ES cells were able to differentiate into all myeloid-erythroid lineages, albeit at half the frequency of heterozygous lines. In contrast, although flk-1 (-/-) ES cells formed mesodermal-like colonies on OP9 monolayers, they failed to generate hematopoietic clusters even in the presence of exogenous cytokines. However, flk-1 (-/-) OP9 cultures did contain myeloid precursors, albeit at greatly reduced percentages. This defect was rescued by first allowing flk-1 (-/-) ES cells to differentiate into EBs and then passaging these cells onto OP9 stroma. Thus, the requirement for Flk-1 in early hematopoietic development can be abrogated by alterations in the microenvironment. This finding is consistent with a role for Flk-1 in regulating the migration of early mesodermally derived precursors into a microenvironment that is permissive for hematopoiesis.