Occurrence of pppApp-synthesizing activity in actinomycetes and isolation of purine nucleotide pyrophosphotransferase

The occurrence of adenosine 5-triphosphate-3-diphosphate-synthesizing activity was detected in five strains of actinomycetes; Streptomyces morookaensis, Streptomyces aspergilloides, Streptomyces hachijoensis, Actinomyces violascens and Streptoverticillium septatum, out of 825 strains of actinomycetes, bacteria, fungi and imperfecti. Purine nucleotide pyrophosphotransferase were extracellularly excreted associating with the cell growth, and were purified partially or to apparent homogeniety from the culture filtrate. The enzymes are a monomeric protein with a molecular weight of 18000–26000 and synthesize adenosine, guanosine and inosine 5-phosphate (mono, di or tri)-3-diphosphate such as pApp, ppApp, pppApp, pGpp, ppGpp, pppGpp and pppIpp by transferring a pyrophosphoryl group from the 5-position of ATP, dATP and pppApp to the 3-position of purine nucleotides in the presence of a divalent cation and in alkaline state.Abbreviations pppApp adenosine 5-triphosphate 3-diphosphate - ppApp adenosine 5-diphosphate 3-diphosphate - pApp adenosine 5-monophosphate 3-diphosphate - pppGpp guanosine 5-triphosphate 3-diphosphate     

Formation of the imidazolides of dinucleotides under potentially prebiotic conditions

Summary Imidazolides of dinucleotides such as ImpApA can be formed from the corresponding dinucleotides in a two-stage process, which gives up to 15% yields under potentially prebiotic conditions. First a solution of the dinucleotide and sodium trimetaphosphate is dried out at constant temperature and humidity. This produces polyphosphates such as pnApA in excellent yield (80%). The products are dissolved in water, imidazole is added, and the solution is dried out again. This yields the 5-phosphorimidazolides.Abbreviations P₃! trimetaphosphate - A adenosine - U uridine - EDTA ethylenediaminetetraacetic acid - Ap adenosine 2(3)-phosphate - Ap! adenosine cyclic 2:3-phosphate - pA adenosine 5-phosphate - pA²p adenosine 2, 5-diphosphate - pA³p adenosine 3, 5-diphosphate - pAp! 5-phospho-adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - ImpA adenosine 5-phosphorimidazolide - A²pA adenylyl-[25]-adenosine - A³pA adenylyl-[35]-adenosine - A²pU adenylyl-[25]-uridine - A³pU adenylyl-[35]-uridine - pA²pA 5-phosphoadenylyl-[25]-adenosine - pA³pA 5-phospho-adenylyl-[35]-adenosine - pA²pU 5-phospho-adenylyl-[25]-uridine - pA³pU 5-phospho-adenylyl-[35]-uridine - pApN (N= A, U) 5-phosphate of a dinucleoside phosphate - p_nApN (N = A, U; n = 2, 3, 4.) 5-polyphosphate of a dinucleoside phosphate - ImpA²pA imidazolide of pA²pA - ImpA³pA imidazolide of pA³pA - ImpA²pU imidazolide of pA²pU - ImpA³pU imidazolide of pA³pU - ImpApN imidazolide of pApN     

Degradation of the diarylpropane lignin model compound 1-(3′,4′-diethoxyphenyl)-1,3-dihydroxy-2-(4′'-methoxyphenyl)-propane and derivatives by the basidiomycete Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 1-(3,4-diethoxyphenyl)-1,3(dihydroxy)-2-(4'-methoxyphenyl)-propane (XII) in low nitrogen stationary cultures, conditions under which the ligninolytic enzyme system is expressed. 3,4-Diethoxybenzyl alcohol (IV), 1,2(dihydroxy)-1-(4-methoxyphenyl)ethane (XX) and anisyl alcohol were isolated as metabolic products indicating an initial , bond cleavage of this dimer. Exogenously added XX was rapidly converted to anisyl alcohol, indicating that XX is an intermediate in the metabolism of XII. Fungal cleavage of the , bond of 1-(3-4-diethoxyphenyl)-1-(hydroxy)-2-(4'-methoxyphenyl)ethane (XI) also occurred, indicating that a hydroxymethyl group is not a prerequisite for this reaction. P. chrysosporium also metabolized 1-(4-ethoxy-3-methoxyphenyl)-2,2(dihydroxy)-2-(4'-methoxyphenyl)propane-1-ol (XIII). The major products of the degradation of this triol included 4-ethoxy-3-methoxybenzyl alcohol (III) and 2-hydroxy-1-(4-methoxyphenyl)-1-oxoethane (XXI). The nature of the products formed indicates that this triol is also cleaved directly at the , bond. The significant difference in the nature of the products formed from the diaryl propane (XII) and the triol (XIII), however, suggests that XIII is not an intermediate in the major pathway for the degradation of XII. Metabolites were identified after comparison with chemically synthesized standards by GLC-mass spectrometry.Abbreviations GLC Gas liquid chromatography - TMSi trimethylsilyl - TLC thin layer chromatography - MS mass spectrometry     

Sequence analysis of linked maize 22 kDa α-zein genes

We have determined the nucleotide sequence of a 7343 bp zein genomic clone (gZ22.8H3) from the maize inbred W64A. Computer-aided analysis of the DNA sequence revealed two contiguous 22 kDa -zein genes. The 5 gene (gZ22.8) encodes a complete polypeptide and contains putative regulatory sequences in both the 5 and 3 flanking regions that are typical of zein genes. In contrast, the 3 gene (gZ22.8) appears to be a pseudogene, because it contains numerous insertions and deletions that would prevent translation of the mRNA. Alignment of the 5 and 3 flanking sequences of both genes indicated that they resulted from a 3.3 kb DNA duplication event.     

Cyclic nucleotide phosphodiesterases of the renal cortex

Summary Cyclic nucleotide phosphodiesterase in the basal-lateral segment of plasma membranes from proximal tubule cells of the rabbit renal cortex was studied and compared to that in the brush border segment of the plasma membrane. Both adenosine 3,5-monophosphate and guanosine 3,5-monophosphate were hydrolyzed by the basal-lateral membrane, but activity varied differently with the two substrates in a complex concentration-dependent manner. Activity with adenosine 3,5-monophosphate was greater than, equal to, or less than with guanosine 3,5-monophosphate, at concentrations of 1000, 100, and 10 to 1 m, respectively. Basal-lateral membrane phosphodiesterase activities at 1 and 500 m substrate exhibited differential responses to pH, metals, heat, and a heat stable inhibitor. Stimulation by guanosine 3,5-monophosphate and inosine 3,5-monophosphate of adenosine 3,5-monophosphate hydrolysis was found in basal-lateral but not in brush border membranes. This stimulation was potentiated by ethyleneglycol-bis(-aminoethyl ether)N,N-tetraacetic acid and ethylenediaminetetraacetate, inhibited by Triton X-100, and totally blocked by Zn²⁺. The findings indicate that multiple forms of phosphodiesterase are present in the basal-lateral segment and these differ from the activities in the brush border region of the plasma membrane. The characteristics of (i) allosteric, guanosine 3,5-monophosphate-sensitivity of adensoine 3,5-monophosphate phosphodiesterase, and (ii) relatively high guanosine 3,5-monophosphate phosphodiesterase activity, in basal-lateral membranes, which are also enriched in adenylate and guanylate cyclase, suggest an important physiological role for these phosphodiesterases in the regulation of net production of cyclic nucleotides in the renal cortex.     

2′(3′),5′-ADP inhibits initiation-dependent protein synthesis in a cell-free system from Ehrlich ascites tumor cells

When tested in a poly(U)-dependent polyphenylalanine synthesizing system and in a postnuclear supernatant, both derived from Ehrlich ascites tumor cells, 2(3),5-ADP did not affect chain elongation of polypeptide synthesis. In a cell-free system which was dependent on initiation and programmed by natural mRNA, however, the amino acid incorporating activity was suppressed to about 10% of the control in the presence of 1 mM 2(3),5-ADP. The inhibitor was shown not to interfere with the attachment of poly(U) to the small ribosomal subunit and with the formation of mRNA-80S ribosome complexes in a complete protein synthesizing system. The subsequent attachment of a 40S ribosomal subunit to the mRNA-80S ribosome complex and the formation of polysomes, however, was depressed by the inhibitor. The experimental results suggest that 2(3),5-ADP inhibits initiation-dependent protein synthesis between monosome formation and the formation of the first peptide bond(s).     

Higher eukaryotic aminoacyl-tRNA synthetases in physiologic and pathologic states

Summary The aminoacyl-tRNA synthetases play a dual role in cell metabolism by synthesizing aminoacyl-tRNAs and an odd dinucleotide diadenosine-5, 5-P¹, P⁴-tetraphosphate which appears to be involved in DNA replication and the control of cell proliferation. This review is a synthesis of recent results on the structure, genetics, cell biology, physiology, role in neoplasia, and role in autoimmune myositis of the higher eukaryotic aminoacyl-tRNA synthetases.     

Non-coordinate synthesis of RNA polymerase beta beta'' subunits in a temperature-sensitive beta''-subunit mutant of Escherichia coli

Summary On exposure to high temperature of a temperature-sensitive RNA polymerase subunit (rpoC92) mutant of Escherichia coli, selective reduction was observed in the rate of synthesis of a group of proteins including RNA polymerase subunit. The finding that the synthesis of subunit but not subunit was specifically repressed in this mutant grown at non-permissive temperature indicates that the functionally intact RNA polymerase is required for the synthesis of subunits be coordinated. In addition, the assembly of newly synthesized RNA polymerase subunits was inefficient in this mutant at the steps where altered subunit was involved, and the unassembled enzyme subunits were rapidly and preferentially degraded. During recovery to non-restricted growth, the synthesis of both and subunits was transiently enhanced in parallel leading to recovery of the intracellular concentration of functional RNA polymerase.     

Stereoselective formation of a 2′(3′)-aminoacyl ester of a nucleotide

Summary Reaction ofDl-serine and adenosine-5-phosphorimidazolide in the presence of adenosine-5-(O-methylphosphate) and imidazole resulted in the stereoselective synthesis of the aminoacyl nucleotide ester 2(3)-O-seryl-adenosine-5-(O-methylphosphate). The enantiomeric excess ofd-serine incorporated into 2(3)-O-seryl-adenosine-5-(O-methylphosphate) was about 9%. Adenylyl-(5N)-serine and an unknown product also incorporated an excess ofd-serine; however, serylserine showed an excess ofl-serine. The relationship of these results to the origin of the biological pairing ofl-amino acids and nucleotides containingd-ribose is discussed.     

Species diversities analyzed by density and cover in an early volcanic succession

To evaluate alpha diversities, various variables such as density, cover, volume, and weight have been used. However, density is often a distinct variable from the remaining three. To clarify differences in diversity measured by those two kinds of variables, the data collected in fourteen 2×5 m permanently-marked plots on Mount Usu, Japan, which erupted during 1977 and 1978 in growing seasons from 1983 to 1989 was analyzed, using Shannon's species diversity (H) that is represented as a result of combination of species richness and evenness (J). H and J were evaluated by density (density H and J) and cover (cover H and J). Cover H and J were significantly lower than density H and J, indicating that cover H has different characteristics from density H. Those differences are due to differences in evenness, because species richness is the same. The rank orders of species density are different from those of cover. The predominance of a few perennial herbs greatly decreases cover evenness, while seedling establishment success influences density evenness. Therefore, I propose that, during the early stages of succession on harsh environments such as volcanoes, density diversity represents seedling establishment success rate while cover diversity expresses vegetative reproduction success rate.     

Sensitive 1H-31P correlations with 5′ methylene protons of DNA via homonuclear double-quantum coherence

A novel pulse sequence is presented for the correlation of 5 and 5 protons in DNA with phosphorus. Double-quantum coherence between the methylene protons is used to generate ¹H5-³¹P and ¹H5-³¹P cross peaks in an HMQC-type experiment. The resolution for these cross peaks is significantly improved over that of conventional HSQC experiments, as cross peaks between ¹H4 and ³¹P are largely suppressed and a 3D version of the experiment can be performed with little penalty in sensitivity. In addition, sensitivity is favoured by slower relaxation of the double-quantum coherence and a more favourable multiplet fine structure in the acquisition dimension.     

DNA variants within the 5′-flanking region of milk-protein-encoding genes II. The β-lactoglobulin-encoding gene

For the detection of polymorphisms within the 5-flanking region of the -lactoglobulin (-LG) -encoding gene a nucleotide sequence containing 795 bp of the promoter and 59 bp of exon I was cloned and sequenced. After comparing the sequence from the DNA of 11 diverse cows (different breeds and milk-protein yields), 14 singlebp substitutions were identified within the 5-flanking region and two in the 5-untranslated region (5-UTR) of exon I. Some of the variants are located in potential binding sites for trans-acting factors or in the 5-UTR. A PCR-based RFLP analysis was performed, and the genotypes of an additional 60 cows were identified at five variable 5-flanking sites. The results reveal three frequent combinations between the A and B alleles of the protein-coding region and the novel 5-flanking DNA variants. This finding may explain the differences of the protein-variant-dependent -LG synthesis (A>B) observed in vivo. A sequence comparison of the bovine and ovine promoters reveals an homology of 92.8% and shows a higher degree of conservation between positions -600 and -300.     

Alternative splicing of pre-mRNA encoding the <Emphasis Type="Italic">Musca domestica</Emphasis> latrophilin-like protein: Primary structures of four spliced forms of mRNA and their protein products

An internal DNA fragment (2000 bp) homologous to the conserved regions of genes encoding latrophilin-like proteins (LLPs) was obtained by the PCR technique using degenerate primers to these gene regions. The gene-specific primers were synthesized based on the results of sequencing of the isolated fragment, and all overlapping cDNA fragments of the llp gene encoding the Musca domestica LLP were obtained by the rapid amplification of cDNA 5- and 3-ends (5- and 3-RACE). Four alternatively spliced mRNAs were found while sequencing the obtained cDNA fragments. Two long mRNAs (6000 nt) differ in the structures of both the regions encoding signal peptides and 5-terminal untranslated regions. They encode large proteins (1800 aa), whose domain organization is similar to that of mammalian latrophilins. Each deduced protein contains a domain with seven transmembrane strands followed by an extended cytoplasmic C-terminal domain. Two other mRNA forms are derived from these long mRNAs; they encode proteins severely truncated at their C-termini (900 aa). They are composed of the domain with only three transmembrane regions and a short unique cytoplasmic C-terminal domain (23 aa). The limitations and drawbacks of the existing 3-RACE techniques found during study of the long alternatively spliced cDNAs are analyzed, and ways for overcoming these difficulties are proposed.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 175–185.Original Russian Text Copyright © 2005 by Andreev, Danilevich, Grishin.     

Cytochemical studies on the localization of 5′-nucleotidase in the acinar cells of the rat salivary glands

Summary The cytochemical localization of 5-nucleotidase (5-AMPase), and its validity, were investigated in parotid and submandibular acinar cells of a rat. Biochemical determinations showed that adequate treatment with glutaraldehyde could minimize the loss of enzymatic activity, and that 5-AMPase and non-specific alkaline phosphatase (-GPase) possessed different pH optima.The cytochemical distribution of the reaction products from the 5-AMPase activity was distinct from those of -GPase. 5-AMPase activity was localized on the surface membranes of acinar, ductal and myoepithelial cells of both salivary glands. -GPase activity was evenly distributed on the entire plasma membranes of myoepithelial cells and on the basal plasmalemma of acinar cells. The reaction products, which appeared on the luminal and lateral plasma membranes of the acinar cells, were presumed to reflect the presence of 5-AMPase, while those on the myoepithelial surface and basal plasma membranes of the acinar cells demonstrated both 5-AMPase and -GPase.The results indicate that 5-AMPase activity can be utilized as a reliable marker enzyme of plasma membranes in the salivary acinar cells.     

Inverse relationship between poly (ADP-ribose) polymerase activity and 2′,5′-oligoadenylates core level in estrogen-treated immature rat

Summary The activity of poly (ADP-ribose) polymerase (ADPRP) and the content of 2,5-oligodenylates core (2,5A_n; n = 2,3 and 4) were measured in homogenates of the uterus and of the liver of immature rats immediately before (time 0) or at different times after injection of estradiol-valerate. ADPRP activity increased gradualy, starting 6 hours after estrogen injection, for about 4 days. Instead, the content of 2,5 A_n decreased by about 50% within 6 hours, and thereafter more slowly for 4 days to about 20% of starting values. Estrogen increased ADPRP activity and decreased 2,5A_n concentration also in the kidney and in the cardiac muscle of the same animals, but not in the skeletal muscle, where neither of the two parameters was affected. Injection of vehicle only (sesame oil) had no effect on ADPRP activity nor on 2,5A_n content of immature rat tissues.     

Untersuchungen zum C′3-Polymorphismus (β 1c-Globulin)

Zusammenfassung Serumproben von 1322 Blutspendern aus Hessen, 40 Familien mit 89 Kindern, 20 Mutter-Kind-Kombinationen und 268 Sera einer Bantupopulation aus Portugiesisch Angola wurden mit einer modifizierten Technik der Hochspannungs-Dünnschichtelektrophorese auf Agarosegel hinsichtlich des C3-Polymorphismus untersucht. Die Genfrequenzen für Weiße (C3^S=0.779, C3^F=0.215) und Neger (C3^S=0.95, C3^F=0,048) stimmen gut mit den Werten anderer Autoren überein. Insgesamt ließen sich bei Weißen 9 Phänotypen sicher abgrenzen, bei Negern 3. Familienstudien bestätigten den für die Allele C3^S und C3^F angenommenen Vererbungsmodus (autosomal codominant) ausnahmslos. Die Frage der Lagerungsstabilität des C3 wurde abschließend untersucht.

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Investigations on C3-polymorphism ( _1c-Globulin)Gene frequencies and family studies in blood donors from Hessen and a Bantu population

Summary Serum samples of 1322 unrelated individuals from Hessen (Germany), 40 families with 89 children, 20 mother-child-combinations and 268 sera of a Bantu population from Angola were examined for C3 polymorphism using a modified technique of high voltage agarose gel electrophoresis. The gene frequencies for Caucasians (C3^S=0.779, C3^F=0.215) and negroes (C3^S=0.95, C^F=0.048) are in good accordance with those obtained by other authors. In total 9 different phenotypes were observed in Caucasians, 3 phenotypes in negroes. Family studies verify the supposed way of inheritance (autosomal codominant for C3^S and C3^F) without exception. Finally the problem of C3-inactivation by storage was investigated.

     

Two cDNAs encode two nearly identical Cu/Zn superoxide dismutase proteins in maize

Summary SOD-4, a cytosolic form of superoxide dismutase in maize, originally was defined as a single band of activity by zymogram analysis. The protein was purified to homogeneity as shown by a single band on native or denaturing polyacrylamide gels and a single spot on two dimensional gels. The N-terminal amino acid sequence for the first 20 residues was determined for the purified SOD-4 protein. All residues were clearly determined except for residue twelve, where both glutamic and aspartic acids were found. A maize gt11 cDNA library was constructed from scutellar poly(A)⁺RNA. Two cDNAs were isolated, restriction mapped, and their DNA sequences determined. The amino acid sequence deduced from both cDNAs matched perfectly the N-terminal sequence of the purified protein except for the residue at position 12. Significantly, at the twelfth codon, one cDNA was found to code for glutamic acid and the other cDNA had a codon for aspartic acid. Both cDNAs contained similar but not identical 5 and 3 untranslated sequences. Both cDNAs contained polyadenylation signals and tails. cDNA isolations, RNA, and genomic DNA blots confirm the existence and expression of two genes that produce indistinguishable SOD-4 proteins.     

Analysis of temperature dependence of cytoplasmic streaming using tonoplast-free cells of Characeae

Summary The temperature dependence of cytoplasmic streaming in intact and tonoplast-free cells ofNitellopsis obtusa was studied using a cryomicroscope. The streaming velocity decreases linearly with decrease in the temperature in well-buffered tonoplast-free cells but non-linearly in some intact cells. These results suggest that low temperature causes a disturbance in the homeostasis of calcium and protons, which inhibit cytoplasmic streaming in intact cells.Abbreviations ADP adenosine 5-diphosphate - APW artificial pond water - ATP adenosine 5-triphosphate - EGTA ethylene glycol-bis(-aminoethyl ether)N,N,N-tetraacetic acid - HEPES N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) - PIPES piperazine-N, N-bis(2-ethanesulfonic acid) - Tris tris(hydroxymethyl)aminoethane     

Genetic and biochemical studies on flavonoid 3′-hydroxylation in flowers of Petunia hybrida

Summary In flower extracts of defined genotypes of Petunia hybrida, an enzyme activity was demonstrated which catalyses the hydroxylation of naringenin and dihydrokaempferol in the 3-position. Similar to the flavonoid 3-hydroxylases of other plants, the enzyme activity was found to be localized in the microsomal fraction and the reaction required NADPH as cofactor. A strict correlation was found between 3-hydroxylase activity and the gene Ht1, which is known to be involved in the hydroxylation of flavonoids in the 3-position in Petunia. Thus, the introduction of the 3-hydroxyl group is clearly achieved by hydroxylation of C₁₅-intermediates, and the concomitant occurrence of the 3,4-hydroxylated flavonoids quercetin and cyanidin (paeonidin) in the presence of the functional allele Ht1 is due to the action of one specific hydroxylase catalysing the hydroxylation of common precursors for both flavonols and anthocyanins.     

Nucleotide sequence of the rrnB 16S ribosomal RNA gene from Mycoplasma capricolum

Summary The nucleotide sequences of the rrnB 16S ribosomal RNA gene and its 5-and 3-flanking regions from Mycoplasma capricolum have been determined. The coding sequence is 1521 base pairs long, being 21 base pairs shorter than that of the Scherichia coli 16S rRNA gene. The 16S rRNA sequence of M. capricolum reveals 74% and 76% identity with that of E. coli and Anacystis nidulans, respectively. The secondary structure model constructed from the M. capricolum 16S rRNA.gene sequence resembles that proposed for E. coli 16S rRNA. A large stem structure can be constructed between the 5- and 3-flanking sequences of the 16S rRNA gene. The flanking regions are extremely rich in AT.