Distribution of δ-aminolevulinic acid biosynthetic pathways among phototrophic bacterial groups

Two biosynthetic pathways are known for the universal tetrapyrrole precursor, -aminolevulinic acid (ALA). In the ALA synthase pathway which was first described in animal and some bacterial cells, the pyridoxal phosphate-dependent enzyme ALA synthase catalyzes condensation of glycine and succinyl-CoA to form ALA with the loss of C-1 of glycine as CO₂. In the five-carbon pathway which was first described in plant and algal cells, the carbon skeleton of glutamate is converted intact to ALA in a proposed reaction sequence that requires three enzymes, tRNA^Glu, ATP, Mg²⁺, NADPH, and pyridoxal phosphate. We have examined the distribution of the two ALA biosynthetic pathways among various genera, using cell-free extracts obtained from representative organisms. Evidence for the operation of the five-carbon pathway was obtained by the measurement of RNase-sensitive label incorporation from glutamate into ALA, using 3,4-[³H]glutamate or 1-[¹⁴C]glutamate as substrate. ALA synthase activity was indicated by RNase-insensitive incorporation of label from 2-[¹⁴C]glycine into ALA. The distribution of the two pathways among the bacteria tested was in general agreement with their previously established phylogenetic relationships and clearly indicates that the five-carbon pathway is the more ancient process, whereas the pathway utilizing ALA synthase probably evolved much later. The five-carbon pathway is apparently the more widely utilized one among bacteria, while the ALA synthase pathway seems to be limited to the subgroup of purple bacteria.Abbreviations ALA -aminolevulinic acid - DTT dithiothreitol - PALP pyridoxal phosphate - SDS sodium dodecyl sulfate - tricine N-tris-(hydroxymethyl)methylglycine     

Cloning and sequencing of the hemA gene of Rhodobacter capsulatus and isolation of a δ-aminolevulinic acid-dependent mutant strain

Summary The Rhodobacter capsulatus hemA gene, coding for the enzyme -aminolevulinic acid synthase (ALAS), was isolated from a genome bank by hybridization with a hemT probe from Rhodobacter sphaeroides. Subcloning of the initial 3.9 kb HindIII fragment allowed the isolation of a 2.5 kb HindIII-BglII fragment which was able to complement the -aminolevulinic acid-requiring (ALA-requiring) Escherichia coli mutant SHSP19. DNA sequencing revealed an open reading frame coding for a protein with 401 amino acids which displayed similarity to the amino acid sequences of other known ALASs. However, no resemblance was seen to the HemA protein of E. coli K12. Based on the sequence data, an ALA-requiring mutant strain of R. capsulatus was constructed by site-directed insertion mutagenesis. Introduction of a plasmid, containing the hemA gene of R. capsulatus on the 3.9 kb HindIII fragment, restored ALA-independent growth of the mutant indicating that there is only one gene for ALA biosynthesis in R. capsulatus. Transfer of the R factor pRPS404 and hybridization analysis revealed that the ALAS gene is not located within the major photosynthetic gene cluster.Part of this research was presented at the Symposium on Molecular Biology of Membrane-Bound Complexes in Phototrophic Bacteria, Freiburg, FRG, 2–5 August 1989     

A rapid and simple method for staining of the crystal protein ofBacillus thuringiensis

Summary A rapid and simple method of staining for the crystal protein (-endotoxin or parasporal body) ofBacillus thuringiensis has been developed. Changes in colonial morphology were observed when cells lost their ability to form crystal protein or both crystal protein and spore.     

Melatonin prevents δ-aminolevulinic acid-induced oxidative DNA damage in the presence of Fe2+

-aminolevulinic acid (ALA), a heme precursor which accumulates during lead poisoning and acute intermittent porphyria, is reported to cause liver cancer. The carcinogenic mechanisms of ALA may relate to its ability to generate free radicals through metal-catalyzed oxidation which cause oxidative DNA damage. The aim of this study was to compare the efficacy of melatonin, trolox (vitamin E) and mannitol in altering DNA damage induced by ALA. Herein, we found, in the presence of Fe²⁺, that ALA-induced formation of 8-hydroxydeoxyguanosine in calf thymus DNA was dose and time-dependent. Melatonin, mannitol and trolox, all of which are free radical scavengers, inhibited the formation of 8-hydroxydeoxyguanosine in a concentration-dependent manner. The concentration of each (melatonin, mannitol and trolox) required to reduce DNA damage by 50%, i.e., the IC₅₀, was 0.52, 0.84 and 0.90 mM, respectively.     

Interpretation of sulfur cycling in two catchments in the Black Forest (Germany) using stable sulfur and oxygen isotope data

The isotopic composition of SO ₄ ^2- in bulk precipitation, canopy throughfall, seepage water at three different soil depths, stream water, and groundwater was monitored in two forested catchments in the Black Forest (Germany) between November 1989 and February 1992. Isotope measurements on aqueous sulfate were complemented by ³⁴S-analyses on SO₂ in the air, total sulfur and inorganic sulfate in the soil, and bedrock sulfur, in order to identify sources and biogeochemical processes affecting S cycling in catchments with base poor, siliceous bedrock. Stable S isotope data indicated that atmospheric deposition and not mineral weathering is the major source of S in both catchments since ³⁴S-values for sulfate in the soil, in seepage water, and in stream water were generally found to be similar to the mean ³⁴S-values of precipitation SO ₄ ^2- (+2.1. However, ¹⁸O-values of seepage water SO ₄ ^2- at 30 cm and especially at 80 cm depth were depleted by several per mil with respect to those of the atmospheric deposition (+7.5 to +13.5. This indicates that in both catchments a considerable proportion of the seepage water SO ₄ ^2- is derived from mineralization of carbon-bonded soil S and must therefore have cycled through the organic soil S pool. ³⁴S-values for different S compounds in the solid soil were found to differ markedly depending on S fraction and soil depth. Since atmospheric S deposition with rather constant ³⁴S-values was identified as the dominant S source in both catchments, this is interpreted as a result ofin situ isotope fractionation rather than admixture of isotopically different S. The differences between the ³⁴S-values of seepage water and soil sulfate and those of organic soil S compounds are consistent with a model in which SO ₄ ^2- uptake by vegetation and soil microorganisms favours³⁴SO ₄ ^2- slightly, whereas during mineralization of organic soil S to aqueous SOSO ₄ ^2- ,³²S reacts preferentially. However, the data provide evidence for negligible isotope fractionation during physico-chemical S transformations such as adsorption/desorption in aerated forest soils.     

Characterization and expression of three novel differentiation-related genes belong to the human NDRG gene family

NDRG1(N-Myc downstream regulated) is upregulated during cell differentiation, repressed by N-myc and c-myc in embryonic cells, and suppressed in several tumor cells. A nonsense mutation in the NDRG1 gene has been reported to be causative for hereditary motor and sensory neuropathy-Lom (HMSNL), indicating that NDRG1 functions in the peripheral nervous system necessary for axonal survival. Here, we cloned three human cDNAs encoding NDRG2 (371aa), NDRG3 (375aa) and NDRG4 (339aa), which are homologous to NDRG1. These three genes, together with NDRG1, constitute the NDRG gene family. The phylogenetic analysis of the family demonstrated that human NDRG1 and NDRG3 belong to a subfamily, and NDRG2 and NDRG4 to another. At amino acid (aa) level, the four members share 53–65% identity. Each of the four proteins contains an / hydrolase fold as in human lysosomal acid lipase. Expression of the fusion proteins NDRG2/GFP, NDRG3/GFP and NDRG4/GFP in COS-7 cells showed that all of them are cytosolic proteins. Based on UniGene cluster analysis, the genes NDRG2, NDRG3 and NDRG4 are located at chromosome 14q11.1–11.2, 20q12–11.23 and 16q21–22.1, respectively. Northern and dot blot analysis shows that all of the three genes are highly expressed in adult brain and almost not detected in the eight human cancer lines. In addition, in contrast to the relatively ubiquitous expression of NDRG1, NDRG2 is highly expressed in adult skeletal muscle and brain, NDRG3 highly expressed in brain and testis, and NDRG4 specifically expressed in brain and heart, suggesting that they might display different specific functions in distinct tissues.     

Evidence suggesting energy-dependent formaldehyde transport in an RuMP-type methylotroph (T15)

Formaldehyde accumulation ratios ([¹⁴CH₂O]_i/[¹⁴CH₂O]_o) as high as 12-fold were measured in anaerobic, CH₃OH-energized, whole cell suspensions of the ribulose monophosphate (RuMP)-type methylotrophic strain T15. Uptake kinetics were extremely rapid, enabling the attainment of equilibrium in only 10–30 s. Transport appears to be energy-dependent and associated with the protonmotive force (pmf). Anaerobic incubation with 5 M carbonyl p-(trifluoromethoxy)-phenylhydrazone (FCCP) led to 70%–90% reduction of the accumulation ratio. Though not as pronounced, diminished uptake was also observed in the presence of 140 M nigericin, 161 M valinomycin and 90 mM KSCN, commensurate with their effects on pmf. Accumulation of CH₂O as a function of external pH followed a trend more similar to that of pmf than either pH or . Preventing energization by incubation with 100 M N,N-dicyclohexylcarbodiimide (DCCD) led to nearly 80% inhibition of CH₂O transport. Over short time periods it was possible to chase accumulated ¹⁴CH₂O from previously loaded cells by collapsing pmf; however, this technique also indicated that significant ¹⁴CH₂O incorporation began to occur within 3 min.Abbreviations FCCP Carbonyl cyanide p-(trifluoromethyoxy)-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - RuMP ribulose monophosphate - TPP⁺ tetra[U-¹⁴C]phenylphosphonium - pmf protonmotive force     

Role of the pituitary gland in experimental hormonal induction and prevention of benign prostatic hyperplasia in the dog

Summary The antiandrogen, cyproterone acetate (CPA), prevents development of prostatic hyperplasia, induced in castrated dogs by a 6 month-treatment with 5-androstane-3,l 7-diol (A)alone or in combination with 17-oestradiol (E ₂). The immunoperoxidase technique was used to study functional cell types in the pars distalis of the pituitary gland and to detect growth hormone (GH) and prolactin (PRL) target sites in the prostate gland. Homologous radioimmunoassays for estimation of serum canine GH and PRL concentrations were also performed. Treatment with the combinations A + E ₂ and A + E ₂ + CPA resulted in morphological indications of stimulated GH and PRL cells and depressed gonadotrophs. This correlates well with an increase in PRL-dependent staining in glandular epithelium and fibromuscular tissue of the prostate gland. However, basal serum PRL and GH levels were not significantly affected. Treatment with A and A + E₂ stimulated, while additional treatment with CPA clearly suppressed adrenocorticotrophin/melanotrophin (ACTH/MSH) cells. These findings indicate that an endocrine imbalance in hypothalamic-pituitary-adrenal function may be involved in induction and prevention of prostatic hyperplasia in the dog.     

Synthesis of α-galacto-oligosaccharides by a cloned α-galactosidase from Bifidobacterium adolescentis

A genomic library of Bifidobacterium adolescentis was constructed in Escherichia coli and a gene encoding an -galactosidase was isolated. The identified open reading frame showed high similarity and identity with bacterial -galactosidases, which belong to Family 36 of the glycosyl hydrolases. For the purification of the enzyme from the medium a single chromatography step was sufficient. The yield of the recombinant enzyme was 100 times higher than from B. adolescentis itself. In addition to hydrolytic activity the -galactosidase showed transglycosylation activity and can be used for the production of -galacto-oligosaccharides.     

DNA polymorphisms in the ψβ1- and β-globin gene regions in asian macaques

DNA polymorphisms in the ₁--globin gene region in nine Asian macaques(Macaca fuscata, M. mulatta, M. nemestrina, M. cyclopis, M. fascicularis, M. arctoides, M. radiata, M. maura, andM. assamensis) were examined using several restriction endonucleases and the human ₁, IVS2, and IVS2 probes. TheBamHI site 3 to the -globin gene was polymorphic inM. fuscata andM. mulatta, while the HincII site and the EcoRI site in the ₁-globin gene region was highly polymorphic inM. fuscata andM. mulatta, respectively. These polymorphic sites also seem to be present in other Asian macaques. The present study of the polymorphism at theBamHI site 3 to the -globin gene in Asian macaques supports, at the nuclear DNA level, the idea that thefascicularis group includingM. fuscata, M. mulatta, M. cyclopis, andM. fascicularis is different from other Asian macaque groups.This study was supported in part by the Cooperation Research Program of the Primate Research Institute, Kyoto University.     

The structure of aPhaseolus vulgaris cDNA encoding the iron storage protein ferritin

A cDNA containing the entire coding region for the iron storage protein ferritin has been isolated from the French bean plant,Phaseolus vulgaris L. cv. Tendergreen. Ferritin protein was purified from young leaves and shoot meristem tissue and used to raise antisera in mice. A gt11 cDNA library was constructed from seed-derived poly(A)⁺ RNA, and screened with the mouse anti-ferritin serum. A 1.2 kb immunopositive phage DNA insert was isolated and sequenced. The derived amino acid sequence shows substantial similarity with other ferritin sequences. The 5 untranslated region contains two out-of-frame AUG codons, a region of extreme pyrimidine composition bias and potentially stable secondary structure.     

Carbon and nitrogen dynamics along the decay continuum: Plant litter to soil organic matter

Decay processes in an ecosystem can be thought of as a continuum beginning with the input of plant litter and leading to the formation of soil organic matter. As an example of this continuum, we review a 77-month study of the decay of red pine (Pinus resinosa Ait.) needle litter. We tracked the changes in C chemistry and the N pool in red pine (Pinus resinosa Ait.) needle litter during the 77-month period using standard chemical techniques and stable isotope, analyses of C and N.Mass loss is best described by a two-phase model: an initial phase of constant mass loss and a phase of very slow loss dominated by degradation of lignocellulose (acid soluble sugars plus acid insoluble C compounds). As the decaying litter enters the second phase, the ratio of lignin to lignin and cellulose (the lignocellulose index, LCI) approaches 0.7. Thereafter, the LCI increases only slightly throughout the decay continuum indicating that acid insoluble materials (lignin) dominate decay in the latter part of the continuum.Nitrogen dynamics are also best described by a two-phase model: a phase of N net immobilization followed by a phase of N net mineralization. Small changes in C and N isotopic composition were observed during litter decay. Larger changes were observed with depth in the soil profile.An understanding of factors that control lignin degradation is key to predicting the patterns of mass loss and N dynamics late in decay. The hypothesis that labile C is needed for lignin degradation must be evaluated and the sources of this C must be identified. Also, the hypothesis that the availability of inorganic N slows lignin decay must be evaluated in soil systems.     

The role of vanadium in green plants

In a series of experiments, it is demonstrated that the trace element vanadium (4·10^-7 g-at/l as NH₄VO₃) has a considerable positive influence on the synthesis of -aminolevulinic acid (-ALA) in the autotrophically growing green algaChlorella pyrenoidosa, the effect being visible by an enhanced output of the amino acid into the culture medium in presence of levulinic acid (LA). The level of intracellularly accumulated -ALA, however, is not changed in presence of the metal. The V-effect on exogenous found -ALA is suppressed, when LA is added to the nutrient medium at low pH (pH 5), although V-uptake into the algal cells is not disturbed by LA. As demonstrated in culture media with various nitrogen sources (urea, partially hydrolized urea, ammonium salts), the development of the pH during the cultivation time is important for the presentation of the V-effect on -ALA. It is suggested that vanadium acts as a catalyst in the conversion of 4,5-dioxovaleric acid to -ALA by transamination.Abbreviations -ALA -aminolevulinic acid - LA levulinic acid - DOVA 4,5-dioxovaleric acid     

A new protection/activation strategy for the synthesis of naturally occurring and non-naturalα-N-alkylamino acids

Summary A new method for the preparation of N-methylamino acids and some of their derivatives starting from hexafluoroacetone protected amino acids is described. The new concept results in saving of steps compared to conventional protection/activation techniques. Protection and deprotection proceed without racemization.     

Investigation of the Relationship between the Lectin Activity of Azospirilla and Their α-Glucosidase, β-Glucosidase,and β-Galactosidase

The activities of -glucosidase, -glucosidase, and -galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilenseSp7 and Azospirillum lipoferum59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol–acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied for the azospirilla studied. The cofunction of the A. brasilenseSp7 lectin and -galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum59b lectin and glucosidases was revealed. The lectin from A. lipoferum59b may possess saccharolytic activity.     

The Solution Structure of [d(CGC)r(amamam)d(TTTGCG)]2

The solution structure and hydration of a DNARNA hybrid chimeric duplex [d(CGC)r(a_ma_ma_m)d(TTTGCG)]₂ in which the RNA adenines were substituted by 2-O-methylated riboadenines was determined using two-dimensional NMR, simulated annealing, and restrained molecular dynamics. Only DNA residue 7T in the 2-OMe-RNA DNA junction adopted an O4-endo sugar conformation, while the other DNA residues including 3C in the DNA 2-OMe-RNA junction, adopted C1-exo or C2-endo conformations. The observed NOE intensity of 2-O-methyl group to H1 proton of 4a_m at the DNA 2-OMe-RNA junction is much weaker than those of 5a_m and 6a_m. The 2-O-methyl group of 4a_m was found to orient towards the minor groove in the trans domain while the 2-O- methyl groups of 5a_m and 6a_m were found to be in the gauche (+) domain. In contrast to the long-lived water molecules found close to the RNA adenine H2 and H1 protons and the methyl group of 7T in the RNA-DNA junction of [d(CGC)r(aaa)d(TTTGCG)]₂, there were no long-lived water molecules found in [d(CGC)r(a_ma_ma_m)d(TTTGCG)]₂. This is probably due to the hydrophobic enviroment created by the 2-O-methylated riboadenines in the minor groove or due to the wider minor groove width in the middle of the structure. In addition, the 2-O-methylation of riboadenines in pure chimeric duplex increses its melting temperature from 48.5°C to 51.9°C. The characteristic structural features and hydration patterns of this chimeric duplex provide a molecular basis for further therapeutic applications of DNARNA hybrid and chimeric duplexes with 2-modified RNA residues.     

Molecular cloning and characterization of a gibberellin-inducible, putative α-glucosidase gene from barley

A putative -glucosidase clone has been isolated from a cDNA library constructed from mRNA of barley aleurones treated with gibberellin A₃ (GA). The clone is 2752 bp in length and has an uninterrupted open reading frame encoding a polypeptide of 877 amino acids. A 680 amino acid region is 43% identical to human lysosomal -glucosidase and other glycosyl hydrolases. In isolated aleurones, the levels of the corresponding mRNA increase strongly after the application of GA, similar to the pattern exhibited by low-pI -amylase mRNA. High levels are also observed in the aleurone and scutellum after germination, while low levels are found in developing seeds. The genome contains a single form of this -glucosidase gene and two additional sequences that may be related genes or pseudogenes.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.