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As part of our studies on the regulation of polyamine biosynthesis in Saccharomyces cerevisiae, we have investigated the effect of spermidine on the degradation of ornithine decarboxylase in this organism. We have found that in S. cerevisiae, as in other eukaryotic cells, the rate of degradation of ornithine decarboxylase, measured either enzymatically or immunologically, is increased by the addition of spermidine to a yeast culture. It is noteworthy that this effect of added spermidine is found even when the experiments are conducted with strains in which the ornithine decarboxylase is overexpressed several hundred-fold more than the wild-type level. The effect of added spermidine in the overexpressed SPE1 strains is best seen in spe2 mutants in which the initial intracellular spermidine is very low or absent. Experiments with cycloheximide show that new protein synthesis is required to effect the breakdown of the ornithine decarboxylase. These results indicate that S. cerevisiae contains an antizyme-like mechanism for the control of the level of ornithine decarboxylase by spermidine, even though, as contrasted with other eukaryotic cells, no specific antizyme homologue has been detected either in in vitro experiments or in the S. cerevisiae genome.

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S-adenosylmethionine decarboxylase (AdoMetDC), a key enzyme in the biosynthesis of spermidine and spermine, is first synthesized as a proenzyme, which is cleaved posttranslationally to form alpha and beta subunits. The alpha subunit contains a covalently bound pyruvoyl group derived from serine that is essential for activity. With the use of an Escherichia coli overexpression system, we have purified AdoMetDCs encoded by the E. coli, Saccharomyces cerevisiae, and Salmonella typhimurium genes. Unexpectedly we found by mass spectrometry that these enzymes had been modified posttranslationally in vivo by a mechanism-based "suicide" inactivation. A large percentage of the alpha subunit of each enzyme had been modified in vivo to give peaks with masses m/z = 57 +/- 1 and m/z = 75 +/- 1 daltons higher than the parent peak. AdoMetDC activity decreased markedly during overexpression concurrently with the increase of the additional peaks for the alpha subunit. Sequencing of a tryptic fragment by tandem mass spectrometry showed that Cys-140 was modified with a +75 +/- 1 adduct, which is probably derived from the reaction product. Comparable modification of the alpha subunit was also observed in in vitro experiments after incubation with the substrate or with the reaction product, which is consistent with the in vitro alkylation of E. coli AdoMetDC reported by Diaz and Anton [Diaz, E. & Anton, D. L. (1991) Biochemistry 30, 4078-4081].

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The dif locus is a site-specific recombination site located within the terminus region of the chromosome of Escherichia coli. Recombination at dif resolves circular dimer chromosomes to monomers, and this recombination requires the XerC, XerD and FtsK proteins, as well as cell division. In order to characterize other enzymes that interact at dif, we tested whether quinolone-induced cleavage occurs at this site. Quinolone drugs, such as norfloxacin, inhibit the type 2 topoisomerases, DNA gyrase and topoisomerase IV, and can cleave DNA at sites where these enzymes interact with the chromosome. Using strains in which either DNA gyrase or topoisomerase IV, or both, were resistant to norfloxacin, we determined that specific interactions between dif and topoisomerase IV caused cleavage at that site. This interaction required XerC and XerD, but did not require the C-terminal region of FtsK or cell division.

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Spermidine-deficient Saccharomyces cerevisiae cells are much more sensitive to paromomycin than nondeficient cells, resulting in cessation of growth and cell death.

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Anthropometry and clinical examination best evaluate the morphology of repaired cleft lip and nose. An original, accurate, and practical image analysis of the lip and nose, which takes advantage of the mathematic, geometric, and organizational capabilities of public domain NIH-Image software (http://rsb.info.nih.gov/nih-image/), has been developed and tested over the past 6 years. A modified structured physical examination form that complements this analysis is under study. Accuracy of NIH-Image-based anthropometry was compared with direct measurements of 22 linear distances on the lip and nose. Twenty-five sets of direct measurements were taken, prospectively, on 15 children with repaired cleft lip over a 6-year period. The results were submitted to regression analysis. Then, relevant lip and nasal tip aesthetics were evaluated by the measuring capabilities of NIH-Image to create a quantitative assessment tool. For each episode, 15 possible faults were weighted, according to aesthetics and deformity, to provide an adverse score. The sum of the 5 lip scores, 10 nose scores, and combination gave respective grades. The analysis was modified to stratify congenital deformity to relate severity of disease to outcome. This analysis was applied to digitized images of 19 consecutive children, immediately prior to repair of complete unilateral cleft lip and nose, at the time of palate repair, and annually from the age of 3 to 6 years. There were 19 NIH-Image-based measurements of the congenital deformity and 35 measurements of surgical results; four children had three sets of records, eight had two sets, and seven had one set Descriptive statistics were applied. Following 556 paired direct and computer-assisted measurements, exceptional linear correlation was shown with a Pearson R coefficient of 0.96. The best correlation was lines within the plane of the camera lens, with the average difference ranging between 0.025 and 0.997 mm. Visual inspection of frontal and submental photographs of excellent, good, and poor results substantiates the ability of this analysis to quantify and grade a spectrum of relevant cleft lip and nasal anatomy. For these 19 patients, there was a broad range of performance scores, approximating a normal distribution. The mean of the NIH-Image-based analysis scores, 16.91, was a (very) good grade. A single standard deviation of 6.88 extended up into excellent and down to fair. The congenital analysis indicated a range of deformity. Comparing deformity with outcome, simple regression analysis had a coefficient of determination (R2) of 0.223, indicative of a weak positive relationship. An accurate and practical morphologic computer-assisted outcome assessment of repaired cleft lip and nasal deformity has been developed. There is a weak direct correlation between severity of deformity and outcome. Testing in multiple clinics is warranted.

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We first met on a Boston streetcar in 1940, being introduced by a mutual friend. Celia was returning from research work at the Massachusetts General Hospital as part of her senior thesis at Radcliffe College, and Herb was returning from a concert by the Boston Symphony. We were married in 1946 after Celia had finished her medical training. We started working together in 1952, and we are still actively collaborating in our studies on various aspects of the biosynthesis and function of polyamines. We are honored to have been invited by the editors of the Annual Review of Biochemistry to summarize our activities in biochemical research over the past 60 years. During most of this time we have been at the National Institutes of Health in Bethesda, Md., and we have witnessed the enormous expansion of biomedical research that has occurred during this period. In addition to summarizing our research, Herb summarizes his association with the Journal of Biological Chemistry and the remarkable developments that have occurred recently in electronic publication and dissemination of scientific literature.

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Spermine, ubiquitously present in most organisms, is the final product of the biosynthetic pathway for polyamines and is synthesized from spermidine. In order to investigate the physiological roles of spermine, we identified the SPE4 gene, which codes for spermine synthase, on the right arm of chromosome XII of Saccharomyces cerevisiae and prepared a deletion mutant in this gene. This mutant has neither spermine nor spermine synthase activity. Using the spe4 deletion mutant, we show that S. cerevisiae does not require spermine for growth, even though spermine is normally present in the wild-type organism. This is in striking contrast to the absolute requirement of S. cerevisiae for spermidine for growth, which we had previously reported using a mutant lacking the SPE3 gene (spermidine synthase) [Hamasaki-Katagiri, N., Tabor, C. W., Tabor, H., 1997. Spermidine biosynthesis in Saccharomyces cerevisiae: Polyamine requirement of a null mutant of the SPE3 gene (spermidine synthase). Gene 187, 35-43].

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The Saccharomyces cerevisiae SPE3 gene, coding for spermidine synthase, was cloned, sequenced, and localized on the right arm of chromosome XVI. The deduced amino acid sequence has a high similarity to mammalian spermidine synthases, and has putative S-adenosylmethionine binding motifs. To investigate the effect of total loss of the SPE3 gene, we constructed a null mutant of this gene, spe3delta, which has no spermidine synthase activity and has an absolute requirement for spermidine or spermine for the growth. This requirement is satisfied by a very low concentration of spermidine (10(-8) M) or a higher concentration of spermine (10(-6) M).

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Saccharomyces cerevisiae cells that cannot synthesize spermidine or spermine because of a deletion in the gene coding for S-adenosylmethionine decarboxylase are very sensitive to elevated temperatures when incubated in a polyamine-deficient medium; i.e., growth is inhibited and the cells are killed. This sensitivity is very pronounced at 39 degrees C, but a moderate effect is noted even at 33 to 34 degrees C. These findings support findings from other studies from our laboratory on the importance of polyamines in protecting cell components against damage. The sensitivity of spermidine-deficient cells to the temperature 39 degrees C provides a useful method for screening for polyamine auxotrophs.

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We previously showed that a mutant of Saccharomyces cerevisiae, which cannot make spermidine as a result of a deletion in the SPE2 gene (spe2 delta), exhibits a marked elevation in +1 ribosomal frameshifting efficiency in response to the Ty1 frameshift sequence, CUU AGG C. In the present study, we found that spermidine deprivation alone does not result in increased +1 ribosomal frameshifting efficiency. The high level of +1 ribosomal frameshifting efficiency in spe2 delta cells is the result of the combined effects of both spermidine deprivation and the large increase in the level of intracellular putrescine resulting from the derepression of the gene for ornithine decarboxylase (SPE1) in spermidine-deficient strains.

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Saccharomyces cerevisiae spe1 delta SPE2 mutants (lacking ornithine decarboxylase) and spe1 delta spe2 delta mutants (lacking both ornithine decarboxylase and S-adenosylmethionine decarboxylase) are equally unable to synthesize putrescine, spermidine, and spermine and require spermidine or spermine for growth in amine-free media. The cessation of growth, however, occurs more rapidly in spe1 delta SPE2 cells than in SPE1 spe2 delta or spe1 delta spe2 delta cells. Since spe1 delta SPE2 cells can synthesize decarboxylated adenosylmethionine (dcAdoMet), these data indicate that dcAdoMet may be toxic to amine-deficient cells.

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Polyamines have been implicated in nucleic acid-related functions and in protein biosynthesis. RNA sequences that specifically direct ribosomes to shift reading frame in the -1 and +1 directions may be used to probe the mechanisms controlling translational fidelity. We examined the effects of spermidine on translational fidelity by an in vivo assay in which changes in beta-galactosidase activity are dependent on yeast retrovirus Ty +1 and yeast double-stranded RNA virus L-A -1 ribosomal frameshifting signals. In spe2 delta mutants of Saccharomyces cerevisiae, which cannot make spermidine as a result of a deletion in the SPE2 gene, there is a marked elevation in +1 but no change in -1 ribosomal frameshifting. The increase in +1 ribosomal frameshifting efficiency is accompanied by a striking decrease in Ty1 retrotransposition.

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When a mutant of Saccharomyces cerevisiae (spe2 delta) that cannot make spermidine or spermine was incubated in a polyamine-deficient medium in oxygen, there was a rapid cessation of cell growth and associated cell death. In contrast, when the mutant cells were incubated in the polyamine-deficient medium in air or anaerobically, the culture stopped growing more gradually, and there was no significant loss of cell viability. We also found that the polyamine-deficient cells grown in air, but not those grown anaerobically, showed a permanent loss of functional mitochondria ("respiratory competency"), as evidenced by their inability to grow on glycerol as the sole carbon source. These data support the postulation that polyamines act, in part, by protecting cell components from damage resulting from oxidation. However, since the mutant cells still required spermidine or spermine for growth when incubated under strictly anaerobic conditions, polyamines must also have other essential functions.

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Null mutants of Escherichia coli were constructed that cannot synthesize spermidine, because of deletions in the gene encoding S-adenosylmethionine decarboxylase. These mutants are still able to grow at near normal rates in purified media deficient in polyamines. These results in E. coli differ from recent findings that null mutants of Saccharomyces cerevisiae and of Neurospora crassa have an absolute growth requirement for spermidine.

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Fick's second law has been used to predict the time course of electrical conductance change in isolated cuticles following the rapid change in bathing solution (KCI) from concentration C to 0.1 C. The theoretical time course is dependent on the coefficient of diffusion of KCI in the cuticle and the cuticle thickness. Experimental results, obtained from cuticles isolated from sour orange (Citrus aurantium), fit with a diffusion model of an isolated cuticle in which about 90% of the conductance change following a solution change is due to salts diffusing from polar pores in the wax, and 10% of the change is due to salt diffusion from the wax. Short and long time constants for the washout of KCI were found to be 0.11 and 3.8 hours, respectively. These time constants correspond to KCI diffusion coefficients of 1 x 10(-15) and 3 x 10(-17) square meters per second, respectively. The larger coefficient is close to the diffusion coefficient for water in polar pores of Citrus reported elsewhere (M Becker, G Kerstiens, J Schönherr [1986] Trees 1: 54-60). This supports our interpretation of the washout kinetics of KCI following a change in concentration of bathing solution.

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We report a new method for measuring cation and anion permeability across cuticles of sour orange, Citrus aurantium, leaves. The method requires the measurement of two electrical parameters: the diffusion potential arising when the two sides of the cuticle are bathed in unequal concentrations of a Cl(-) salt; and the electrical conductance of the cuticle measured at a salt concentration equal to the average of that used in the diffusion-potential measurement. The permeabilities of H(+), Li(+), Na(+), K(+), and Cs(+) ranged from 2 x 10(-8) to 0.6 x 10(-8) meters per second when cuticles were bathed in 2 moles per cubic meter Cl(-) salts. The permeability of Cl(-) was 3 x 10(-9) meters per second. The permeability of Li(+), Na(+), and K(+) was about five times less when measured in 500 moles per cubic meter Cl(-) salts. We also report an asymmetry in cuticle-conductance values depending on the magnitude and the direction of current flow. The asymmetry disappears at low current-pulse magnitude and increases linearly with the magnitude of the current pulse. This phenomenon is explained in terms of transport-number effects in a bilayer model of the cuticle. Conductance is not augmented by current carried by exchangeable cations in cuticles; conductance is rate limited by the outer waxy layer of the cuticle.

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A null mutation in the SPE2 gene of Saccharomyces cerevisiae, encoding S-adenosylmethionine decarboxylase, results in cells with no detectable S-adenosylmethionine decarboxylase, spermidine, and spermine. This mutant has an absolute requirement for spermidine or spermine for growth; this requirement is not satisfied by putrescine. Polyamine-depleted cells show a number of microscopic abnormalities that are similar to those reported for several cell division cycle (cdc) and actin mutants. These include a striking increase in cell size, a marked decrease in budding, accumulation of vesicle-like bodies, absence of specific localization of chitin-like material, and abnormal distribution of actin-like material. The absolute requirement for polyamines for growth and the microscopic abnormalities are not seen if the cultures are grown under anaerobic conditions.

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We have cloned and sequenced the Saccharomyces cerevisiae gene for S-adenosylmethionine decarboxylase. This enzyme contains covalently bound pyruvate which is essential for enzymatic activity. We have shown that this enzyme is synthesized as a Mr 46,000 proenzyme which is then cleaved post-translationally to form two polypeptide chains: a beta subunit (Mr 10,000) from the amino-terminal portion and an alpha subunit (Mr 36,000) from the carboxyl-terminal portion. The protein was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme contains both the alpha and beta subunits. About half of the alpha subunits have pyruvate blocking the amino-terminal end; the remaining alpha subunits have alanine in this position. From a comparison of the amino acid sequence deduced from the nucleotide sequence with the amino acid sequence of the amino-terminal portion of each subunit (determined by Edman degradation), we have identified the cleavage site of the proenzyme as the peptide bond between glutamic acid 87 and serine 88. The pyruvate moiety, which is essential for activity, is generated from serine 88 during the cleavage. The amino acid sequence of the yeast enzyme has essentially no homology with S-adenosylmethionine decarboxylase of E. coli (Tabor, C. W., and Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040) and only a moderate degree of homology with the human and rat enzymes (Pajunen, A., Crozat, A., Jänne, O. A., Ihalainen, R., Laitinen, P. H., Stanley, B., Madhubala, R., and Pegg, A. E. (1988) J. Biol. Chem. 263, 17040-17049); all of these enzymes are pyruvoyl-containing proteins. Despite this limited overall homology the cleavage site of the yeast proenzyme is identical to the cleavage sites in the human and rat proenzymes, and seven of the eight amino acids adjacent to the cleavage site are identical in the three eukaryote enzymes.

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The gene for ornithine decarboxylase in Saccharomyces cerevisiae, SPE1, has been assigned to chromosome XI by the technique of transverse alternating pulsed field electrophoresis and DNA-DNA hybridization. Genetic mapping by tetrad analysis shows that the SPE1 gene is located on the left arm of chromosome XI, 6 cM from the LAP1 gene and 43 cM from the TRP3 gene. The spe10 mutation previously isolated in this laboratory is mapped to the N-terminal region of the SPE1 gene, and therefore should be designated as a spe1 allele.

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We examined some biophysical mechanisms of ion migration across leaf cuticles enzymatically isolated from Acer saccharum L. and Citrus aurantium L. leaves. Diffusion potential measurements were used to calculate the permeabilities of Cl(-), Li(+), Na(+), and Cs(+) ions all as a ratio with respect to the permeability of K(+) in cuticles. In 2 millimolar ionic strength solutions the permeability sequence from high to low was K = Cs > Na > Li >> Cl. When the outer and inner surfaces of cuticles were bathed in artificial precipitation and artificial apoplast, respectively, diffusion potentials ranging from -52 to -91 millivolts were measured (inside negative). The Goldman equation predicted that the measured potentials were enough to increase the driving force on the accumulation of heavy metals by a factor of 4 to 7. Other ions migrate with forces 3 to 10 times less than predicted by the Goldman equation for concentration differences alone. Our analysis showed that Ca(2+), and perhaps Mg(2+), might even be accumulated against concentration gradients under some circumstances. Their uptake was apparently driven by the diffusion potentials created by the outward migration of monovalent salts. We feel that future models predicting leaching of nutrients from trees during acid rain events must be modified to account for the probable influence of diffusion potentials on ion migration.