Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase

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., J?nne, 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.     

Molecular analysis of male-viable deletions and duplications allows ordering of 52 DNA probes on proximal Xq.

While performing a systematic search for chromosomal microdeletions in patients with clinically complex X-linked syndromes, we have observed that large male-viable deletions and duplications are clustered in heterochromatic regions of the X chromosome. Apart from the Xp21 band, where numerous deletions have been found that encompass the Duchenne muscular dystrophy gene, an increasing number of deletions and duplications have been observed that span (part of) the Xq21 segment. To refine the molecular and genetic map of this region, we have employed 52 cloned single-copy DNA sequences from the Xcen-q22 segment to characterize two partly overlapping tandem duplications and two interstitial deletions on the proximal long arm of the human X chromosome. Together with a panel of somatic cell hybrids that had been described earlier, these four rearrangements enabled us to order the 52 probes into nine different groups and to narrow the regional assignment of several genes, including those for tapetochoroidal dystrophy and anhidrotic ectodermal dysplasia.     

The concurrent expression of Griffonia simplicifolia-IB4 binding and tumor necrosis factor-alpha differs between alveolar and peritoneal macrophages.

As a corollary to their anatomic location, alveolar macrophages (AM) have a lower threshold for generating some physiologic functions than peritoneal macrophages (PM). In this study, we examined both of these populations for their ability to bind the lectin Griffonia simplicifolia-IB4 (GSIB4) and to produce tumor necrosis factor (TNF)-alpha. The results showed that these two responses were concurrently expressed in activated macrophages, although they differed in magnitude when AM and PM were compared. Following in vitro incubation, AM from lipopolysaccharide-treated rats demonstrated a higher percentage of GSIB4 positivity and TNF production when compared with their respective PM. Since prostaglandin E2 can regulate the expression of some macrophage activities, experiments were conducted to determine whether this could also affect the ability of macrophages to bind the GSIB4 lectin. Neither the administration of indomethacin nor exogenous prostaglandin E2 altered the expression of this marker. Conversely, these treatments produced significant changes in TNF-alpha production in both alveolar and peritoneal macrophages. When the concurrent expression of GSIB4 lectin binding and TNF-alpha production was analyzed, AM from lipopolysaccharide-treated rats demonstrated both superior GSIB4 positivity and TNF-alpha production compared with all other macrophages examined. The results of this work show that AM and PM differ in their expression of GSIB4 binding and TNF-alpha production. These differential responses may be important in determining the level of activity of macrophages that are participating in an immune response.     

Land use legacy effects on woody vegetation in agricultural landscapes of south‐western Ethiopia

Aim

Past land use legacy effects—extinction debts and immigration credits—might be particularly pronounced in regions characterized by complex and dynamic landscape change. The aim of this study was to evaluate how current woody plant species distribution, composition and richness related to historical and present land uses.

Location

A smallholder farming landscape in south‐western Ethiopia.

Methods

We surveyed woody plants in 72 randomly selected 1‐ha sites in farmland and grouped them into forest specialist, generalist and pioneer species. First, we investigated woody plant composition and distribution using non‐metric multidimensional scaling. Second, we modelled species richness in response to historical and current distance from the forest edge. Third, we examined diameter class distributions of trees in recently converted vs. permanent farmland.

Results

Historical distance was a primary driver of woody plant composition and distribution. Generalist and pioneer species richness increased with historical distance. Forest specialists, however, did not respond to historical distance. Only few old individuals of forest specialist species remained in both recently converted and permanent farmlands.

Main conclusions

Our findings suggest that any possible extinction debt for forest specialist species in farmland at the landscape scale was rapidly paid off, possibly because farmers cleared large remnant trees. In contrast, we found substantial evidence of immigration credits in farmland for generalist and pioneer species. This suggests that long‐established farmland may have unrecognized conservation values, although apparently not for forest specialist species. We suggest that conservation policies in south‐western Ethiopia should recognize not only forests, but also the complementary value of the agricultural mosaic—similar to the case of European cultural landscapes. A possible future priority could be to better reintegrate forest species in the farmland mosaic.

     

Alkaline Benzoquinone Aqueous Flow Battery for Large‐Scale Storage of Electrical Energy

An aqueous flow battery based on low‐cost, nonflammable, noncorrosive, and earth‐abundant elements is introduced. During charging, electrons are stored in a concentrated water solution of 2,5‐dihydroxy‐1,4‐benzoquinone, which rapidly receives electrons with inexpensive carbon electrodes without the assistance of any metal electrocatalyst. Electrons are withdrawn from a second water solution of a food additive, potassium ferrocyanide. When these two solutions flow along opposite sides of a cation‐conducting membrane, this flow battery delivers a cell potential of 1.21 V, a peak galvanic power density of 300 mW cm^?2, and a coulombic efficiency exceeding 99%. Continuous cell cycling at 100 mA cm^?2 shows a capacity retention rate of 99.76% cycle^?1 over 150 cycles. Various molecular modifications involving substitution for hydrogens on the aryl ring are implemented to block decomposition by nucleophilic attack of hydroxide ions. These modifications result in increased capacity retention rates of up to 99.96% cycle^?1 over 400 consecutive cycles, accompanied by changes in voltage, solubility, kinetics, and cell resistance. Quantum chemistry calculations of a large number of organic compounds predict a number of related structures that should have even higher performance and stability. Flow batteries based on alkaline‐soluble dihydroxybenzoquinones and derivatives are promising candidates for large‐scale, stationary storage of electrical energy.     

Primate animal models and titered inocula for the study of human hepatitis A, hepatitis B, and non-A, non-B hepatitis

Although many primate species have been inoculated with the agents of human hepatitis A, B, and non-A, non-B, only a small number of species have been shown to be susceptible, and only the chimpanzee (Pan troglodytes) has been shown to be reproducibly susceptible to all three types of human hepatitis. Infectious inocula containing each agent have been identified in different laboratories and the end-point titers of infectivity determined, in most cases by inoculation of chimpanzees. These inocula and the nonhuman primate models have permitted investigators to study the inactivation of these agents and to evaluate passive and active immunization against the agents.     

Regulatory mutations affecting ornithine decarboxylase activity in Saccharomyces cerevisiae.

We isolated several strains of Saccharomyces cerevisiae containing mutations mapping at a single chromosomal gene (spe10); these strains are defective in the decarboxylation of L-ornithine to form putrescine and consequently do not synthesize spermidine and spermine. The growth of one of these mutants was completely eliminated in a polyamine-deficient medium; the growth rate was restored to normal if putrescine, spermidine, or spermine was added. spe10 is not linked to spe2 (adenosylmethionine decarboxylase) or spe3 (putrescine aminopropyltransferase [spermidine synthease]). spe 10 is probably a regulatory gene rather than the structural gene for ornithine decarboxylase, since we isolated two different mutations which bypassed spe10 mutants; these were spe4, an unliked recessive mutation, and spe40, a dominant mutation linked to spe10. Both spe4 and spe40 mutants exhibited a deficiency of spermidine aminopropyltransferase (spermine synthase), but not of putrescine aminopropyltransferase. This suggests that ornithine decarboxylase activity is negatively controlled by the presence of spermidine aminopropyltransferase.