Effect of 2',3'-dideoxycytidine on oxidative phosphorylation in the PC12 cell, a neuronal model.

Peripheral neuropathy induced by 2',3'-dideoxycytidine (ddC) could result from the previously shown inhibition of mtDNA replication by the action of ddC on the mitochondrial enzyme DNA polymerase gamma. Such inhibition would be expected to impair oxidative phosphorylation, and this was demonstrated in the present study for the PC12 cell, a model of a peripheral neuron. The dramatic rise in lactate formation upon exposure of the cell to ddC indicated that increased glycolysis was needed to produce ATP. A concomitant rise in O2 uptake indicated that oxidative phosphorylation had become uncoupled. When tested in a standard respiratory control system (isolated rat liver mitochondria), however, we found ddC not to be an uncoupler. Rather, the uncoupling most likely resulted from the failure of synthesis of one or more mitochondrial gene products necessary for oxidative phosphorylation. We also observed an important distinction between the manner in which ddC and 3'-azido-3'-deoxythymidine (AZT) act. ddC-exerted inhibition of oxidative phosphorylation was delayed for several days. This is consistent with the inhibition occurring indirectly, most likely as a result of the prior destruction of the mitochondrial genome, which encodes many of the components of the oxidative phosphorylation system. In contrast, we have shown previously that although AZT also impairs replication of the mitochondrial genome (in the Friend murine erythroleukemic cell), it also attacks directly an additional primary target leading to impairment of oxidative phosphorylation; its initial inhibition of this process is immediate, not occurring via inhibition of mitochondrial DNA replication.

Cellular targets of 3'-azido-3'-deoxythymidine: an early (non-delayed) effect on oxidative phosphorylation.

Previous results demonstrated that incubation of the Friend murine erythroleukemic cell with 5 microM AZT for several days leads to a decrease in the rate of cell growth, inhibition of mtDNA replication, reduction of mtDNA per cell and per mitochondrion, and an increase in mitochondria per cell. As shown here, such treatment also leads to changes in lactate and ATP synthesis and in O2 uptake, suggesting impairment of oxidative phosphorylation. Direct measurement of ATP synthesis in mitochondria isolated from AZT-treated cells confirmed this view. The most significant new finding in this paper, however, is that in addition to these delayed effects of AZT, similar but very rapidly appearing effects on oxidative phosphorylation were noted, with changes observed in the above parameters including mitochondrial proliferation. Some of these occurred as early as 3 hr, only 7% of the doubling time, after exposure of the cells to 5 microM AZT, a period too short for initiation of appreciable mtDNA-mediated effects. Studies on isolated mitochondria provided no evidence of the identity of the immediate target of AZT: AZT does not act as an uncoupler or inhibitor of respiratory control, and previous results failed to implicate adenylate kinase. We have also begun to address the question of the mechanism of AZT-induced mitochondrial proliferation. Initial experiments showed that AZT inhibited synthesis of total cytosolic protein but stimulated synthesis of those proteins imported into mitochondria from the cytoplasm. We also report that aminothymidine, a catabolite of AZT in liver capable of inhibiting cell growth, was not generated by Friend cells.

Challenge Studies with Clostridium botulinum in a Sous-Vide Spaghetti and Meat-Sauce Product.

Challenge studies were carried out to evaluate the safety of a reformulated sous-vide processed spaghetti and meat-sauce product (aw 0.992-0.972, pH 4.5-6) inoculated with Clostridium botulinum types A and B spores. Following processing at 75°C for 36 min (equivalent to a 13 D process for Streptococcus faecium ), samples were stored at 15°C and analyzed at selected time intervals for toxin production and visible signs of spoilage. Toxin was detected in samples of pH > 5.5 after 14-21 days and in products of pH 5.25 after 35 days. Toxin was not detected in any samples of pH < 5.25 within 42 days storage at 15°C. All products of pH 5.75 and 6 were visibly spoiled, i.e., swollen due to CO2 production, prior to toxigenesis. However, for products of pH 5.5 and 5.25, toxigenesis preceded spoilage. Subsequent studies were done to test the effect of additional salt (1-3% w/w) on the safety of a product of pH 5.5 at mild temperature-abuse storage conditions (i.e., 15°C). Toxin production was not delayed in samples containing < 1.5% added salt, while > 1.5% salt (w/w) prevented toxin production throughout the 42-day storage period at 15°C. Microwave heating of products for 5-10 min at full or half power in a domestic microwave (800 watts) inactivated the preformed toxin in all samples.

Mechanisms of toxicity of 3'-azido-3'-deoxythymidine. Its interaction with adenylate kinase.

Recent experiments from our laboratory have indicated that the inhibitory effect of 3'-azido-3'-deoxythymidine (AZT) on oxidative phosphorylation may occur directly, in addition to being brought about by its inhibition of mtDNA replication. We report here studies on the effect of AZT on adenylate kinase, an enzyme crucial to oxidative phosphorylation. AZT decreased the aromatic residues fluorescence of rabbit muscle adenylate kinase, indicating binding of AZT to the enzyme. Of three other enzymes studied as controls, AZT bound only to those that possessed ATP/ADP binding sites. Up to concentrations of 15 microM, AZT was a more potent effector of fluorescence quenching than were ATP, ADP, AMP, and the AZT control, deoxythymidine. AZT strongly inhibited adenylate kinase in the direction of ATP synthesis (Ki, 8 microM), the inhibition being of the partial competitive type, whereas deoxythymidine inhibition, also partially competitive, was much weaker (Ki, 90 microM). When measured in the direction of ADP synthesis, AZT failed to demonstrate any inhibition at concentrations up to 10 microM. Experiments on isolated intact rat liver mitochondria with the enzyme activity measured in both directions confirmed the isolated enzyme results. Respiratory control by these mitochondria was not affected by AZT. The finding of AZT affinity for ATP/ADP binding sites may open new avenues of approach to the study of AZT toxicity.

Anti-human immunodeficiency virus type 1 therapy and peripheral neuropathy: prevention of 2',3'-dideoxycytidine toxicity in PC12 cells, a neuronal model, by uridine and pyruvate.

A strategy for preventing or delaying the peripheral neuropathy induced by 2',3'-dideoxycytidine (ddC) therapy in patients with acquired immunodeficiency syndrome was suggested by findings, in two laboratories, that cultured avian and mammalian cells devoid of mitochondrial DNA continue to replicate at virtually normal rates, provided that the medium is supplemented with uridine and pyruvate. Inasmuch as it is likely that a depletion of mitochondrial DNA also takes place in neuronal cells exposed to ddC, we used PC12 cells, the neuronal model we have reported on previously, in an attempt to rescue these cells from the deleterious effects of ddC. We first show, using undifferentiated PC12 cells, that DNA replication is impaired in mitochondria isolated from cells grown in the presence of ddC. Then, using growth rate as a criterion of the well-being of the cells, we show that the addition of uridine and pyruvate to uninduced cells growing in the presence of ddC results in an average rescue efficiency of 51%, based on the uridine/pyruvate-treated control. This value increases considerably at substantially higher concentrations of uridine alone. Rescue efficiencies of differentiated cells, which do not proliferate, were assessed using neurite outgrowth and neurite survival as criteria. Here the rescue efficiency is 56%, based on the uridine/pyruvate-treated control. In addition, uridine and pyruvate prolong the viability of ddC-treated cells and maintain their healthy appearance; without these compounds, the ddC-treated cells have an abnormal morphology and die off quite rapidly.

The three-dimensional structure of the Na,K-ATPase from electron microscopy.

The structure of Na,K-ATPase has been studied by electron microscopy and image reconstruction. A three-dimensional structure of this enzyme has been obtained to an overall resolution of 2.5 nm using data from specimens of negatively stained dimer sheets tilted through a range of angles +/- 60 degrees. The reconstruction shows a complex mass distribution consisting of ribbons of paired molecules extending approximately 6.0 nm from the cytoplasmic side of the membrane. The molecular envelope consists of a massive "body" with "lobe" and "arm" structures projecting from it. The body has a columnar shape and is tilted with respect to the plane of the membrane. The region of interaction responsible for dimer formation is located between two bodies and is clearly visible in the reconstruction. It has been identified as a segment in the amino-terminal portion of the alpha subunit. The arms that interconnect the ribbons are located close to the membrane and are most probably formed by the beta subunits.

Transitions in histone variants during sea urchin spermatogenesis.

Transitions in the histone complement of nuclei during sea urchin spermatogenesis were investigated by two-dimensional gel electrophoresis. Nuclei were isolated from male gonads of individuals differing in degree of maturity. Unlike protamines, the sea urchin sperm-specific histone variants Sp H1 and Sp H2B appear early in spermatogenesis, well before spermatid differentiation, as the predominant representatives of their classes. Both proteins are phosphorylated from their first appearance until the last steps of spermiogenesis, when the highly condensed late spermatid nuclei become spermatozoan nuclei. Phosphorylation of serine occurs mostly (Sp H1) or entirely (Sp H2B) on the N-terminal portions of these molecules. We conclude that phosphorylated sperm-specific histone variants in the sea urchin function in spermatocytes during meiosis and are the major histones present during replication and transcription in some spermatogonia as well. We propose that the dephosphorylation of Sp H1 and Sp H2B in late spermatids is not primarily responsible for spermatid chromatin condensation but instead may act to stabilize the chromatin of the spermatozoon or aid in the final shaping of the sperm nucleus.

Sea urchin testicular cells evaluated by fluorescence microscopy of unfixed tissue.

A procedure is presented for rapid, quantitative evaluation of cell and nuclear types present in the male gonad of the sea urchin. Vitally stained whole mounts of tissue fragments or dissociated cells are prepared, which reveal detailed 3-dimensional chromatin patterns and enough cytoplasmic features to provide reliable markers for most of the somatic and germ line cell types. Representative cellular morphologies are described. Nuclear volume changes during spermatogenesis are quantified. Spermatid nuclei contain an apparently interconnected network of heterochromatin. Regions relatively devoid of chromatin decrease in size as nuclear condensation proceeds and spherical nuclear shape is maintained. The major decrease in nuclear volume occurs prior to the late spermatid stage. The volume of the spermatozoan nucleus is achieved by the smallest late spermatid nucleus before the change from spherical to conoid morphology. The relationship of this morphological transition to sperm histone dephosphorylation is discussed.

Effect of the bacterial DNA gyrase inhibitors, novobiocin, nalidixic acid, and oxolinic acid, on oxidative phosphorylation.

When incubated with isolated intact rat liver mitochondria, novobiocin and nalidixic acid act as uncouplers of oxidative phosphorylation; they stimulate oxygen uptake and inhibit ATP synthesis. Novobiocin is about as powerful an uncoupler as is 2,4-dinitrophenol, nalidixic acid is somewhat less powerful, and oxolinic acid exerts no inhibition whatsoever at the concentrations used. The three inhibitors are without effect on oxidative phosphorylation in Escherichia coli nor does novobiocin affect this process in a novobiocin-permeable mutant of yeast. While it would appear that oxolinic acid may be a relatively specific tool for the manipulation of the superhelicity of DNA in complex systems such as mammalian mitochondria and intact mammalian cells, the specificity of each of these inhibitors may depend upon the particular conditions and species used and such experiments require adequate controls on oxidative phosphorylation.

Fluorescence studies on a streptomycin-induced conformational change in ribosomes which correlates with misreading.

The fluorescent reagent N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-AEDANS) was employed to detect and study the previously reported conformational change in the Escherichia coli ribosome induced by streptomycin. Labeling of ribosomes with this probe, which results in the derivatization of proteins S18 and L31', described earlier, inhibits neither their ribosomal protein synthesizing nor misreading ability. To calculate the amount of streptomycin bound to the ribosome, we determined the K'D for streptomycin, which is 0.24 micron, indicating that under our conditions, bound streptomycin/ribosome molar ratios are low, not in excess of 1. Under these conditions, streptomycin addition induces fluorescence quenching by 15% but does not affect streptomycin-resistant ribosomes. Maximal misreading occurs at these same ratios. Removal of AEDANS-L31' from the ribosomes drastically reduces streptomycin-induced quenching indicating the involvement of the environment of this protein in streptomycin action. The finding that streptomycin decreases AEDANS-L31' affinity for the ribosome supports this view. Streptomycin has been shown to bind to the 30 S subunit protein S4 while the 50 S protein L31' has been shown to be localized at the subunit interface. Thus, the observation that streptomycin influences this 50 S subunit protein L31', combined with the tight correlation between the effects of streptomycin on quenching and on misreading, strongly suggests that this antibiotic induces a conformational change at the subunit interface of the ribosome, and that this results in misreading. Polyuridylic acid also induces a conformational change in the ribosome but the polynucleotide and streptomycin seem to act independently. Streptomycin-resistant ribosomes, which undergo neither streptomycin-induced fluorescence nor streptomycin-induced misreading, are resistant to misreading induced by high Mg2+ as well.

Modification of Escherichia coli ribosomes with the fluorescent reagent N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid. Identification of derivatized L31' and studies on its intraribosomal properties.

N-[[(Iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (IAEDANS) is a fluorescent reagent which reacts covalently with the free thiol groups of proteins. When the reagent is reacted with the Escherichia coli ribosome under mild conditions, gel electrophoresis shows modification of predominantly two proteins, S18 and L31', which become labeled to an equal extent. When the native (i.e., untreated) ribosome is dissociated into 30S and 50S subunits, only the 30S ribosomal protein S18 reacts with IAEDANS despite the fact that L31' is still present on the large subunit. Upon heat activation of the subunits, a procedure which alters subunit conformation, S18 plus a number of higher molecular weight proteins is modified, but not L31'; the latter reacts with IAEDANS only in the 70S ribosome or when it is free. In contrast to the relatively stable association of L31' with native or with dissociated ribosomes, dissociation of N-[(acetylamino)ethyl]-5-naphthylaminesulfonic acid (AEDANS)-treated ribosomes weakens the AEDANS-L31'/ribosome interaction, resulting, upon gel filtration analysis, in ribosomes devoid of this derivatized protein.

Studies on mitochondrial type I topoisomerase and on its function.

We have reported previously that rat liver mitochondria contain a topoisomerase and have shown it to be distinct from the nuclear enzyme by its sensitivity to Berenil and ethidium bromide. We report here some additional characterization. The enzyme differs further from its nuclear counterpart in its failure to bind to ssDNA cellulose and its chromatographic behavior on Sephadex; the latter procedure yields an Mr of 44 000 for the mitochondrial and 70 000 for the nuclear enzyme. The topoisomerase is strongly associated with mitochondrial membranes; only 10% of the activity could be extracted. The pH optimum of the enzyme falls between 6.0 and 8.5, with an NaCl optimum of 0.13 M in 0.1 M Tris (pH 8.3). Dithiothreitol is required, while N-ethylmaleimide is inhibitory. Tosylphenylalanine chloromethyl ketone, a serine proteinase inhibitor, abolishes activity; another, phenylmethanesulfonyl fluoride, has no effect. Berenil, a non-intercalating drug, and four of its analogues all inhibit with up to 100-fold differences in potency. No dependence on ATP, Mg2+, or both together could be shown. Neither novobiocin nor oxolinic acid shows any inhibitory effect. Nicked circles are generated in the presence of DMSO. These three observations are consistent with the topoisomerase being of the Type I class. Positively supercoiled pBR322 DNA, whose 6-8 positive turns were generated by altering solution conditions, is relaxed by the enzyme, indicating a lack of requirement for a negatively supercoiled substrate. We have also examined a partially purified preparation of the corresponding mitochondrial enzyme from mouse L cells. This enzyme is largely similar in properties to the rat liver enzyme. In isolated mitochondria, Berenil causes biphasic alterations in [3H]dATP incorporation into DNA, 10(-4) mM stimulating 2-fold, while higher concentrations inhibit. [3H]UTP incorporation into mitochondrial RNA also follows this pattern.

The effect of bacterial DNA gyrase inhibitors on DNA synthesis in mammalian mitochondria.

Using isolated rat liver mitochondria, which have previously been shown to carry out true replicative DNA synthesis, we have obtained results which are in accord with the presence and functioning of a DNA gyrase in this organelle. The effects of the Escherichia coli DNA gyrase inhibitors, novobiocin, coumermycin, nalidixic acid and oxolinic acid, upon mtDNA replication suggest the involvement of the putative mitochondrial enzyme in various aspects of this process. First, the preferential inhibition of [3H]dATP incorporation into highly supercoiled DNA together with the appearance of labeled, relaxed DNA are consistent with the involvement of a gyrase in the process of generating negative supercoils in mature mtDNA. Second, the overall depression of incorporation of labeled dATP into mtDNA, including the reduction of radioactivity incorporated into replicative intermediates, suggests a 'swivelase' role for the putative gyrase, and this hypothesis is further supported by results obtained on sucrose gradient centrifugation of heat-denatured, D-loop mtDNA. Here, the synthesis of the completed clean circles is inhibited while 9 S initiator strand synthesis is not, suggesting that chain elongation is blocked by the gyrase inhibitors.

Novel features of animal mtDNA evolution as shown by sequences of two rat cytochrome oxidase subunit II genes.

The sequence of the region of the mitochondrial genome that encodes cytochrome oxidase subunit II (COII) has been determined for each of two closely related rat species, Rattus norvegicus and R. rattus. Comparison of the two sequences shows that 94.4% of the nucleotide substitutions are silent. The occurrence of this high proportion of silent substitutions leads us to propose that the rapid evolution of mtDNA relative to nuclear DNA is due only to silent changes and that amino acid-altering substitutions accumulate in nuclear and mtDNA at comparable rates. Other novel features of the nucleotide substitution pattern in the rat COII gene are a high transition/transversion ratio (8.0:1) and a strong bias toward C in equilibrium T transitions in the light strand. Comparison of the R. norvegicus COII sequence with the bovine and human sequences show that there may be selective constraints on some silent positions within the gene and that its rate of evolution may be different in different mammalian lineages.

Intra- and interspecific variation of the mitochondrial genome in Rattus norvegicus and Rattus rattus: restriction enzyme analysis of variant mitochondrial DNA molecules and their evolutionary relationships.

Restriction endonuclease analysis has revealed extensive mtDNA polymorphism in two species of rats, Rattus rattus and Rattus norvegicus. Sequence divergence values for the eight detected R. norvegicus variants range from 0.2% to 1.8% and for the eight R. rattus variants, from 0.2% to 9.6%. Three of the most closely related R. norvegicus mtDNA's appear to differ by deletions/insertions of about 4 base pairs apiece. Restriction sites for seven enzymes have been mapped for 11 of these variants. The 31 intraspecific and 41 interspecific variant sites appear to be evenly distributed on the mtDNA molecule outside of the rRNA cistrons. The location of sites present in all the DNAs suggests that the rRNA genes and possibly the light strand origin of replication may be more highly evolutionarily conserved than other parts of the molecule. The sequence divergences among the mtDNAs of animals whose geographic origins are separated by major barriers, such as oceans, were significantly greater than those among animals found within large land masses, such as the continental United States. Dendrograms (phenograms), which have been constructed to depict the relationships among the various DNAs, indicate that East Asian members of the R. rattus species are more closely related to American rats of this species than to other Asian R. rattus animals from Sri Lanka. Moreover, it appears that R. norvegicus comprises a group taxonomically distinct from any of the R. rattus subspecies.

Mitochondrial DNA polymorphism: evidence that variants detected by restriction enzymes differ in nucleotide sequence rather than in methylation.

Restriction enzyme analysis of mtDNAs for the purpose of determining sequence divergence rests on the assumption that variant recognition sites differ with respect to sequence and not methylation. This assumption was tested on two mtDNAs, A and B, which are distributed throughout the laboratory rat population and which can be distinguished by a number of restriction enzymes. The mtDNAs were cloned and the nucleotide sequences of corresponding small HindIII fragments, in which a variant EcoRI site occurs, were determined. Evidence that the fragments differ in sequence and not methylation is as follows: (i) The cloned mtDNA yielded the same fragment pattern as did native mtDNA when treated with EcoRI, Hha I, HinfI, and Hae III; (ii) three nucleotide replacements were found in the 169-base pair fragment, A.T in equilibrium G.C, A.T in equilibrium G.C, T.A in equilibrium G.C; (iii) one of these replacements, A.T in equilibrium G.C at position 80, accounts for the presence of the EcoRI site in the type A and its absence in the type B mtDNA. Examination of the sequence leads to the suggestion that these three nucleotide replacements are silent; i.e., they would not lead to amino acid substitutions in a possible encoded protein.