CP-61,405, a novel polycyclic pyrrolether antibiotic produced byStreptomyces routienii Huang sp. nov.

Summary CP-61,405 (C₂₆H₂₉N₂O₇Na) is a novel polycyclic pyrrolether antibiotic produced by a new species,Streptomyces routienii Huang sp. nov. (ATCC 39446). Recovery, fractionation and purification were achieved using standard procedures. The crystalline form includes the CP-61,405 sodium salt, m.p. 334–335°C, [] _D ^25°C +315°C (c=1, chloroform). The structure is shown below. CP-61,405 was co-produced with the polycyclic ether antibiotics salinomycin and epi-17-deoxy-(0–8)-salinomycin. It exhibited activity in vitro against gram-positive and anaerobic bacteria, efficacy against poultry coccidia and stimulation in vitro of propionic acid production.     

Expression of cold-tolerant pyruvate,orthophosphate dikinase cDNA,and heterotetramer formation in transgenic maize plants

Maize is a typical C₄ plant of the NADP-malic enzyme type, and its high productivity is supported by the C₄ photosynthetic cycle, which concentrates atmospheric CO₂ in the leaves. The plant exhibits superior photosynthetic ability under high light and high temperature, but under cold conditions the photosynthetic rate is significantly reduced. Pyruvate orthophosphate dikinase (PPDK), a key enzyme of the C₄ pathway in maize, loses its activity below about 12 °C by dissociation of the tetramer and it is considered as one possible cause of the reduction in the photosynthetic rate of maize at low temperatures. To improve the cold stability of the enzyme, we introduced a cold-tolerant PPDK cDNA isolated from Flaveria brownii into maize by Agrobacterium-mediated transformation. We obtained higher levels of expression by using a double intron cassette and a chimeric cDNA made from F. bidentis and F. brownii with a maximum content of 1mg/g fresh weight. In leaves of transgenic maize, PPDK molecules produced from the transgene were detected in cold-tolerant homotetramers or in heterotetramers of intermediate cold susceptibility formed with the internal PPDK. Simultaneous introduction of an antisense gene for maize PPDK generated plants in which the ratio of heterolologous and endogenous PPDK was greatly improved. Arrhenius plot analysis of the enzyme extracted from one such plant revealed that the break point was shifted about 3 °C lower than that of the wild type.     

The biosynthetic origin of the pyridone ring of efrotomycin

Summary Nocardialactamdurans has been shown to catabolyse uracil via the reductive pathway. The end product of this pathway, -alanine, is incorporated into the pyridone ring of efrotomycin. Support for this proposal includes: (1) reversal of thymine inhibition of efrotomycin biosynthesis by dihydrouracil andN-carbamoyl--aline, two intermediates of the catabolic pathway; (2) incorporation of [5,6-³H]-uracil into efrotomycin with a relative molar specific activity of approximately 0.5, close to the theoretical maximum; and (3)¹³C coupling at C₄ and C₅ of efrotomycin after feeding resting cells with [4,5-¹³C]-uracil. Our results do not rule out the possibility of an alternative source of -alanine or the coexistence of uracil catabolism via oxidative reactions.     

Molecular and biochemical characterization of three aromatic polyketide synthase genes from Rubus idaeus

To play an essential role in C₄ photosynthesis, the maize C₄ phosphoenolpyruvate carboxylase gene (PPCZm1) acquired many new expression features, such as leaf specificity, mesophyll specificity, light inducibility and high activity, that distinguish the unique C₄ PPC from numerous non-C₄ PPC genes in maize. We present here the first investigation of the developmental, cell-specific, light and metabolic regulation of the homologous C₄ PPCZm1 promoter in stable transgenic maize plants. We demonstrate that the 1.7 kb of the 5-flanking region of the PPCZm1 gene is sufficient to direct the C₄-specific expression patterns of -glucuronidase (GUS) activity, as a reporter, in stable transformed maize plants. In light-grown shoots, GUS expression was strongest in all developing and mature mesophyll cells in the leaf, collar and sheath. GUS activity was also detected in mesophyll cells in the outer husks of ear shoots and in the outer glumes of staminate spikelets. We did not observe histological localization of GUS activity in light- or dark-grown callus, roots, silk, developing or mature kernels, the shoot apex, prop roots, or pollen. In addition, we used the stable expressing transformants to conduct and quantify physiological induction studies. Our results indicate that the expression of the C₄ PPCZm1-GUS fusion gene is mesophyll-specific and influenced by development, light, glucose, acetate and chloroplast biogenesis in transgenic maize plants. These studies suggest that the adoption of DNA regulatory elements for C₄-specific gene expression is a crucial step in C₄ gene evolution.     

Selective preparation of N-acetyl-D-glucosamine and N,N'-diacetylchitobiose from chitin using a crude enzyme preparation from Aeromonas sp

A bacterium, Aeromonas sp. GJ-18, having strong chitinolytic activity was isolated from coastal soil and used for crude enzyme preparations. This enzyme preparation contained N-acetyl-D-glucosaminidase and N,N-diacetylchitobiohydrolase. N-Acetyl-D-glucosaminidase was inactive above 50 °C, but N,N-diacetylchitobiohydrolase was stable at this temperature. Utilizing the temperature sensitivities of the chitin degradation enzymes in crude enzyme preparation, N-acetyl-D-glucosamine (GlcNAc) and N,N-diacetylchitobiose [(GlcNAc)₂] were selectively produced from chitin. At 45 °C, GlcNAc was produced as a major hydrolytic product (94% composition) with a yield of 74% in 5 d, meanwhile at 55 °C (GlcNAc)₂ was the major product (86%) with a yield of 35% within 5 d.Revisions requested 29 September 2004; Revisions received 1 November 2004     

The Synthesis and Cytotoxic Activity of D-Ribofuranosides and 2-Deoxy-D-Ribofuranosides of Substituted Bis(indolyl)furan, Bis(indolyl)pyrrole, and Indolo[2,3-a]carbazole Derivatives

1-(2,3,5-Tri-O-acetyl)--D-ribofuranosyl indole, the key compound in the synthesis of glycosides with the bis(indole) aglycone, was obtained for the first time by the indoline–indole method. There were synthesized 3-(1-methylindol-3-yl)-4-(1-glycosylindol-3-yl)furan(or pyrrole)-2,5-diones containing the residue of -D-ribofuranose or 2-deoxy--D-ribofuranose and analogous glycosides of indolo[2,3-a]furano(or pyrrolo)[3,4-]carbazol-5,7-diones, which are structurally relative to the antitumor antibiotic rebeccamycin. Their cytotoxicities toward a number of human tumor cell lines were studied in vitro, and the carbazole N-glycosides were shown to be more active than the bis(indole) glycosides. At the same time, the ribofuranosides were found to be less active than the corresponding ribopyranosides synthesized previously.     

Dihydroceramide hinders ceramide channel formation: Implications on apoptosis

Early in apoptosis, ceramide levels rise and the mitochondrial outer membrane becomes permeable to small proteins. The self-assembly of ceramide to form channels could be the means by which intermembrane space proteins are released to induce apoptosis. Dihydroceramide desaturase converts dihydroceramide to ceramide. This conversion may be removing an inhibitor as well as generating a pro-apoptotic agent. We report that both long and short chain dihydroceramides inhibit ceramide channel formation in mitochondria. One tenth as much dihydroceramide was sufficient to inhibit the permeabilization of the outer membrane by about 95% (C₂) and 51% (C₁₆). Similar quantities inhibited the release of carboxyfluorescein from liposomes indicating that other mitochondrial components are not necessary for the inhibition. The apoptogenic activity of ceramide may thus depend on the ceramide to dihydroceramide ratio resulting in a more abrupt transition from the normal to the apoptotic state when the de novo pathway is used in mitochondria.     

Mitochondrial cytochrome c release precedes transmembrane depolarisation and caspase-3 activation during ceramide-induced apoptosis of Jurkat T cells

Whilst the role of ceramide, a second messenger of the sphingolipid family, in the initiation of receptor-mediated apoptosis is controversial, a growing body of evidence is emerging for a role of ceramide in the amplification of apoptosis via mitochondrial perturbations that culminate in the activation of execution caspases. Treatment of Jurkat T cells with the cell-permeable analog, C₂-ceramide, resulted in the rapid onset of apoptosis as evidenced by Annexin V-FITC staining of externalised phosphatidylserine residues. Cells bearing this early apoptotic marker had a reduced mitochondrial transmembrane potential (m) that was preceded by the release of cytochrome c from mitochondria. Subsequent activation of caspase-3 provides the link between these ceramide-induced mitochondrial changes and execution caspases that ultimately result in the physical destruction of the cell. Collectively these results demonstrate that ceramide signalling results in caspase-mediated apoptosis via mitochondrial cytochrome c release and are further supportive of the role of ceramide in the amplification of apoptosis.     

A novel site for streptomycin resistance in the “530 loop” of chloroplast 16S ribosomal RNA

The chloroplast gene for 16S rRNA was cloned from two maternally inherited streptomycin-resistant mutants ofNicotiana differing in degree of resistance at the whole plant and isolated chloroplast level. A single-nucleotide change in the 16S rRNA gene was detected for each mutant: a C to T transition at nucleotide 860 (Escherichia coli coordinate C₉₁₂) which is an often mutated site, and a novel transition of C to T at nucleotide 472 (E. coli coordinate C₅₂₅). The novel mutation is located in the phylogenetically conserved 530 loop.     

Regulatory flexibility of methylotrophic yeasts in chemostat cultures: Simultaneous assimilation of glucose and methanol at a fixed dilution rate

The influence of the composition of methanol/glucose-mixtures as only sources of carbon and energy on growth and regulation of the synthesis of enzymes involved in methanol-dissimilation was studied under chemostat conditions at a fixed dilution rate with the methylotrophic yeasts Hansenula polymorpha and Kloeckera sp. 2201. Both carbon sources were found to be utilized completely independently of the composition of the C₁/C₆ mixture. Using mixtures of ¹⁴C-labelled methanol and glucose the growth yield for glucose was found to be constant for all C₁/C₆-mixtures tested and both yeasts. The growth yield for methanol, however, was reduced by up to 25% when the proportion of methanol in the inflowing medium was lower than 20% (w/w with respect to glucose) for H. polymorpha and 50% (w/w with respect to glucose) for Kloeckera sp. 2201 respectively. During growth with C₁/C₆-mixtures containing higher C₁-proportions of methanol regular growth yields for methanol were recorded which corresponded to the growth yields found with methanol as the only carbon source.The regulation of the synthesis of the enzymes of the dissimilatory pathway for methanol was found to be under multiple control. Although glucose was present in the medium methanol had a positive effect on the synthesis of these enzymes. Thus, in addition to derepression induction by methanol was also observed. This inductive effect was found to increase with increasing proportions of methanol in the mixture. Depending on the enzyme, 10–40% methanol in the mixture resulted in a maximal induction with enzyme specific activities equal to those found in cells grown with methanol as the only carbon source. No further enhancements in enzyme specific activities were observed during growth on mixtures containing more than 40% methanol.Abbreviations and terms C₁ Methanol - C₆ glucose - C₁/C₆ mixture compositions are given in % (w/w) - C₀ concentration of ¹⁴C in the inflowing medium (DPM ml^-1) - C(t) concentration of ¹⁴C incorporated in cells as a function of time t (DPM ml^-1) - d dilution rate (h^-1) - DPM disintegrations per minute - q _s q _C1 and q _C6 are specific rates of consumption of substrate, methanol and glucose respectively [g (g cell dry weight)^-1 h^-1] - q _O2 and q _CO2 are the specific rates of oxygen consumption and carbon dioxide release [mmol (g cell dry weight)^-1 h^-1] - RQ respiration quotient (q _CO2 q _O2 ^-1) - s _C1 and s _C6 are the residual concentrations of methanol and glucose in the culture liquid (g l^-1) - s _O/C1 and s _O/C6 are the concentrations of methanol and glucose in the inflowing medium (g l^-1) - Sp.A. enzyme specific activity - x cell dry weight concentration (g l^-1) - Y _X/C1 and Y _X/C6 are growth yields on methanol and glucose respectively (g cell dry weight (g substrate)^-1 - Y _C/C1 growth yield with methanol with respect to carbon (g carbon assimilated (g carbon supplied)^-1 - _m maximum specific growth rate (h^-1)     

Carbon isotope composition of intermediates of the starch-malate sequence and level of the crassulacean acid metabolism in leaves of Kalanchoe blossfeldiana Tom Thumb

Isotype analyses were performed on biochemical fractions isolated from leaves of Kalanchoe blossfeldiana Tom Thumb. during aging under long days or short days. Irrespective of the age or photoperiodic conditions, the intermediates of the starch-malate sequence (starch, phosphorylated compounds and organic acids) have a level of ¹³C higher than that of soluble sugars, cellulose and hemicellulose. In short days, the activity of the crassulacean acid metabolism pathway is predominant as compared to that of C₃ pathway: leaves accumulate organic acids, rich in ¹³C. In long days, the activity of the crassulacean acid metabolism pathway increases as the leaves age, remaining, however, relatively low as compared to that of C₃ pathway: leaves accumulate soluble sugars, poor in ¹³C. After photoperiodic change (long daysshort days), isotopic modifications of starch and organic acids suggest evidence for a lag phase in the establishment of the crassulacean acid metabolism pathway specific to short days. The relative proportions of carbon from a C₃-origin (RuBPC acitivity as strong discriminating step, isotope discrimination in vivo=20) or C₄-origin (PEPC activity as weak discriminating step, isotope discrimination in vivo=4) present in the biochemical fractions were calculated from their ¹³C values. Under long days, 30 to 70% versus 80 to 100% under short days, of the carbon of the intermediates linked to the starch-malate sequence, or CAM pathway (starch, phosphorylated compounds and organic acids), have a C₄-origin. Products connected to the C₃ pathway (free sugars, cellulose, hemicellulose) have 0 to 50% of their carbon, arising from reuptake of the C₄ from malate, under long days versus 30 to 70% under short days.Abbreviations CAM crassulacean acid metabolism - CAM pathway pathway with malate accumulation by -carboxylation of PEP, arising from glycolysis of starch (starch-malate sequence) - C₃-metabolism metabolism with primary carbon fixed by the Calvin and Benson pathway (C₃-origin) - C₄-metabolism metabolism with primary carbon fixed by the Hatch and Slack pathway (C₄-origin) - C₃-pathway pathway with RuBPC activity and the Calvin and Benson pathway, irrespective of the CO₂-source, atmospheric or reuptake of the C₄ from malate - ¹³C()=(R_sample-R_PDR)10³/R_PDB where PDB Pee Dee belemnite (belemnite from the Pee Dee formation, South Carolina) and R=¹³C/¹²C - D isotope discrimination - PEP phosphoenolpyruvate - PEPC (EC 4.1.1.31) PEP carboxylase - PGA phosphoglyceric acid - Py.di-PK (EC 2.7.9.1) pyruvate, Pi-dikinase - RuBP ribulose bisphosphate - RuBPC (EC 4.1.1.39) RuBP carboxylase - SD short days - LD long days     

Differences in Leaf δ13C Among Four Dominant Species in a Secondary Succession Sere on the Loess Plateau of China

Differences in leaf ¹³C among four dominant species as well as the species-specific response to the fluctuations of either soil moisture or monthly mean temperature were examined along a secondary succession sere with a time scale from 3 to 149 y on the Loess Plateau in north-western China. We used leaf ¹³C as a surrogate for water use efficiency (WUE) of the mentioned dominant species. Bothrichloa ischaemun as a dominant species in the final succession stage belongs to C₄ photosynthesis pathway, while the other three dominant species occurring in the first three succession stages belong to C₃ pathway. The overall trend of leaf ¹³C variation among the three C₃ species was Artemisia gmelinii (in the third stage) and Lespedeza davurica (in the second stage) > Artemisia scoparia (in the first stage). This suggests that species with higher WUE (more positive leaf ¹³C) would have substantial competitive advantages in the context of vegetation succession. Furthermore, species with highest WUE (i.e. C₄ pathway) have great potential to be dominant in the final succession stage in the habitats (such as the study area) undergoing strong water stress in growing season. The evolution of WUE among the dominant species occurring in different succession stages strongly depends on the time scale of given stage since abandonment. The longer the time scale is, the more significant the differences among them in terms of leaf ¹³C, hence WUE. Our results support the notions that leaf ¹³C may be more positive when water supply is less favourable.     

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd 1-free background (NirS−) of Pseudomonas stutzeri

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of Achromobacter cycloclastes nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd ₁ nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d ₁, indicating that both heterologous hosts synthesized a cytochrome cd ₁ without the d ₁-group.Abbreviations Cu-NIR Cu-containing nitrite reductase - DDC diethyldithiocarbamate - EPR electron paramagnetic resonance - IPTG isopropyl--D-galactoside - SDS sodium dodecyl sulfate - LB medium Luria-Bertani medium     

Enhancement of interferon-β production with sphingomyelin from fermented milk

A fermented milk, Kefir, contains an active substance which enhances IFN- secretion of a human osteosarcoma line MG-63 treated with a chemical inducer, poly I: poly C. The active substance in the fermented milk was identified to be sphingomyelin (SpM) by a combined use of a fast atom bombardment mass spectrometry (FAB-MS) and a fast atom bombardment tandem mass spectrometry (FAB-MS/MS). SpM from fermented milk (F-SpM) was a mixture of four molecular species of SpMs having C₂₁-, C₂₂-, C₂₃- and C₂₄-fatty acids. F-SpM enhanced the IFN secretion 14 times, SpMs from other sources also enhanced moderately (2–3 times). Sphingosine and lysosphingomyelin also enhanced the activity but ceramide and cerebroside did not.Abbreviations IFN- interferon- - SpM sphingomyelin - Lyso-SpM lysosphingomyelin - SpS sphingosine - FAB-MS fast atom bombardment mass spectrometry - FAB-MS/MS fast atom bombardment tandem mass spectrometry     

N2 fixation by red alder (Alnus rubra) and scotch broom (Cytisus scoparius) planted under precommerically thinned Douglas-fir (Pseudotsuga menziesii)

Summary From acetylene reduction assays over a 10-month period starting in April 1979, nodule activities averaged 18.78 (se 4.67) moles C₂H₄ g nodule dw^–1 h^–1 forAlnus rubra and 59.95 (se 12.14) moles C₂H₄ g nodule dw^–1 h^–1 forCytisus scorparius. Plant rates were 1.91 (se. 47) moles C₂H₄ plant^–1 h^–1 forA. rubra and 0.55 (se. 17) moles C₂H₄ plant^–1 h^–1 forC. Scoparius. Plant activity and total leaf N were strongly correlated with the dw of other plant parts, but nodule activity and percent leaf N were not. Plant and nodule activities were not associated with temperature, moisture stress, precipitation events or percent light for either species over the growing season nor for 54A. rubra sampled in mid-season 1979 on one replication. After 5 to 6 growing seasons, 14A. rubra on the same site ranged from 30 to 332 cm in height and showed strong correlation between nodule dw, leaf dw, plant size and total leaf N. Results from this study and others indicate logistic equations may be modified to predict the effect of adding a N₂ fixing plant to a population of non N₂ fixing trees.     

A new isolate of Hydrogenobacter,an obligately chemolithoautotrophic,thermophilic, halophilic and aerobic hydrogen-oxidizing bacterium from seaside saline hot spring

An obligately chemolithoautotrophic and aerobic hydrogen-oxidizing bacterium was isolated from a seaside saline hot spring in Izu Peninsula, Japan. The isolate was a Gram-negative, non-motile, non-spore-forming rod cell measuring 0.3 to 0.5 by 1.0 to 2.5 m. The optimal temperature for growth was around 70°C, and no growth was observed at 40°C or 80°C. Elemental sulfur or thiosulfate could be an alternative to molecular hydrogen as the sole energy source. The DNA base composition of the isolate was 46.0 mol% G+C. 2-Methylthio-3-VI,VII-tetrahydromultiprenyl⁷-1,4-naphthoquinone (methionaquinone) was the major component of the quinone system. C_18:0, C_18:1 and C_20:1 were the major components of the cellular fatty acids. These properties clearly indicate that the isolate belongs to genus Hydrogenobacter, but differed from H. thermophilus in some respects. Specifically, the isolate was a halophile which grew optimally at around 0.3–0.5 M NaCl, while H. thermophilus could not grow at such NaCl concentration levels. A new species name H. halophilus is proposed for this new halophilic isolate.     

Ceramide Induces Apoptosis to Immature Cerebellar Granule Cells in Culture

A recent study revealed that ceramide acts as a second messenger in the sphingomyelin pathway and thus plays an important regulatory role in programmed cell death (apoptosis) to cell the lines induced by tumor-necrosis factor (TNF)- and interleukin (IL)-1, although its effect remains controversial regarding primary neuronal culture. We investigated the effect of a cell-permeable ceramide analog (C₂-ceramide) on cultures of cerebellar granule cells, which is thought to have active sphingomyelin pathway during development. The presence of C₂-ceramide decreased the number of cerebellar granule cells (CGCs) in a concentration-dependent manner when added at DIV 1 (1 day in vitro). The ED₅₀ was 60 M. After DIV2, CGCs became less sensitive to C₂-ceramide and the ED₅₀ was 200 M at DIV 7. DNA staining with Hoechst 33258 showed the morphology of apoptotic nuclei in the degenerating neurons. Internucleosomal DNA degradation could also be observed by gel electrophoresis. Protein and RNA synthesis inhibitors prevented the death of neurons. C₂-dihydroceramide, which lacks the 4–5 trans double bond and failed to induce neuronal death. These results thus demonstrated that C₂-ceramide induces apoptosis to the CGCs at the early stage in vitro, however the CGCs were found to be less sensitive to C₂-ceramide at the later stage in vitro.