Enzymes involved in the metabolism of 3-hydroxy-3-methylglutaryl-coenzyme A in Catharanthus roseus

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) is an important intermediate in various metabolic pathways, e.g. sterol biosynthesis, ketogenesis and leucine catabolism. The reactions and enzymes involved in the metabolism of HMG-CoA are briefly reviewed. These enzymes have been studied in Catharanthus roseus, a model system for studies on the regulation of secondary metabolic pathways, particularly those leading to terpenoidindole alkaloids. By using HPLC, three HMG-CoA catabolizing enzyme activities have been detected in protein extracts from suspension cultured C. roseus cells: HMG-CoA lyase, 3-nucleotidase and (tentatively identified) 3-methylglutaconyl-CoA hydratase (HMG-CoA hydrolyase). The enzymes have been partially purified. HMG-CoA is formed from three molecules of acetyl-CoA, via reactions which are catalyzed by two (as in yeast and animal cells, via intermediacy of acetoacetyl-CoA) or by just one enzyme (as in e.g. radish). It is yet not clear which process occurs in C. roseus.Abbreviations AACT acetoacetyl-CoA thiolase - AACT/HMGS acetoacetyl-COA thiolase/HMG-CoA synthase - CoASH coenzyme A (reduced form) - HMG-CoA 3-hydroxy-3-methylglutaryl-CoA - MG-CoA 3-methylglutaconyl-CoA     

Variation in phlorotannin content within two species of brown macroalgae (Desmarestia anceps and D. menziesii) from the Western Antarctic Peninsula

Phlorotannins are metabolites found only in the Phaeophyceae (brown algae) and have a variety of metabolic roles, including both primary (e.g. cell wall construction) and secondary (e.g. herbivore defence and UV protection). This study aimed to establish the level of variation of phlorotannins in two species of dominant Antarctic macroalgae (Desmarestia anceps and D. menziesii). Thirteen samples were taken from specific locations throughout the thallus of multiple individuals at two depths at three locations near Anvers Island, Antarctica. Overall average concentrations were 0.117±0.003 g g^–1dwt in D. anceps and 0.052±0.002 g g^–1dwt in D. menziesii. Concentrations varied greatly at the 13 sampling locations in each individual, but not in any consistent manner. Significant variation occurred between locations in both species and between depths in D. anceps.     

Cell and tissue cultures ofCatharanthus roseus (L.) G. Don: a literature survey

The literature concerning the formation of secondary metabolites in cell and tissue cultures ofCatharanthus roseus has been reviewed. Several aspects involved in the formation of secondary metabolites are discussed; e.g. regulation of secondary metabolism, environmental factors influencing secondary metabolism, biosynthesis and enzymology of the products, analysis of product formation, immobilization of cultured cells and stability of cell lines. Some economical aspects of production processes are discussed.     

RNA pseudoknot prediction in energy-based models.

RNA molecules are sequences of nucleotides that serve as more than mere intermediaries between DNA and proteins, e.g., as catalytic molecules. Computational prediction of RNA secondary structure is among the few structure prediction problems that can be solved satisfactorily in polynomial time. Most work has been done to predict structures that do not contain pseudoknots. Allowing pseudoknots introduces modeling and computational problems. In this paper we consider the problem of predicting RNA secondary structures with pseudoknots based on free energy minimization. We first give a brief comparison of energy-based methods for predicting RNA secondary structures with pseudoknots. We then prove that the general problem of predicting RNA secondary structures containing pseudoknots is NP complete for a large class of reasonable models of pseudoknots.     

Influence of alkaline pH on the direct lethal action of miconazole againstCandida albicans

The imidazole group of miconazole is subject to protonation (pKa 6.5). Earlier we suggested that the direct lethal action (DLA) of miconazole againstCandida albicans requires nonprotonated drug molecules. DLA declined in intensity as pH was decreased from 6.0. At pH >6.5 most molecules of miconazole exist in the nonprotonated state, but drug also becomes less soluble. Viability studies were designed to assess DLA in relation to alkaline pH. DLA was clearly inhibited with increasing as well as decreasing pH (i.e., pH <6.0 and >7.0), suggesting that nonprotonated neutral drug molecules must be in solution or in extremely small aggregates to elicit DLA, and that the nonprotonated species itself is more soluble at pH 6.0–7.0 than under more alkaline conditions.     

Combinatorial properties of RNA secondary structures.

The secondary structure of an RNA molecule is of great importance and possesses influence, e.g., on the interaction of tRNA molecules with proteins or on the stabilization of mRNA molecules. The classification of secondary structures by means of their order proved useful with respect to numerous applications. In 1978, Waterman, who gave the first precise formal framework for the topic, suggested to determine the number a(n,p) of secondary structures of size n and given order p. Since then, no satisfactory result has been found. Based on an observation due to Viennot et al., we will derive generating functions for the secondary structures of order p from generating functions for binary tree structures with Horton-Strahler number p. These generating functions enable us to compute a precise asymptotic equivalent for a(n,p). Furthermore, we will determine the related number of structures when the number of unpaired bases shows up as an additional parameter. Our approach proves to be general enough to compute the average order of a secondary structure together with all the r-th moments and to enumerate substructures such as hairpins or bulges in dependence on the order of the secondary structures considered.     

Overproduction of microbial products--facts and ideas

Overproduction of microbial metabolites is related to developmental phases of microorganisms. Inducers, effectors, inhibitors and various signal molecules play a role in different types of overproduction. Primary and secondary metabolism are interconnected. Biosynthesis of enzymes catalyzing metabolic reactions in microbial cells is controlled by well-known positive and negative mechanisms, e.g. induction, repression, catabolite repression, mechanisms controlling enzyme activity include isosteric and allosteric interactions, e.g. competitive and non-competitive inhibition, allosteric effects, molecular conversion etc. Biosynthesis of secondary metabolites is catalyzed by unaltered enzymes of primary metabolism, by altered enzymes of primary metabolism and by specific enzymes of secondary metabolism. In addition to classical mutagenesis and selection of suitable microbial cells, methods of molecular genetics are used in the overproduction of microbial products.     

The effect of feedback regulation and in situ product removal on the conversion of sugar to cycloheximide by Streptomyces griseus

An addition of cycloheximide to cycloheximide-producing Streptomyces griseus cultures resulted in reductions in the production rate and in the conversion of sugar into cycloheximide. In situ cycloheximide adsorption was observed to enhance: total cycloheximide titers; productivities; and the conversion of sugar to cycloheximide. During the secondary metabolite-producing phase, sugar consumption was observed to be linearly dependent on cycloheximide productivity. From this analysis a true product yield and maintenance coefficient were estimated to be 0.08 g cycloheximide/g glucose and 0.028 g glucose/g cell-h, respectively. The sixfold difference between this true product yield and a theoretical value obtained from knowledge of the biosynthetic pathway is discussed. Since the maintenance sugar requirement for cycloheximide production is large, stimulation of biosynthesis through in situ adsorption significantly increases the overall efficiency of sugar conversion to this secondary metabolite.     

Effects of air-filled soil porosity and aeration on the initiation and growth of secondary roots of maize (Zea mays)

We studied the possibility whether the initiation of secondary roots is regulated by the air-filled porosity in soil, i.e. the availability of oxygen in the soil. Maize plants were grown in long PVC tubes (1 m long and 12 cm diameter) and were unwatered for different numbers of days so that variations of soil water content with depth were achieved on the same date with plants at the same age. The plants were harvested when their root systems were established in the whole soil column and watering had been withheld for 0, 15, 20, 25 days. A decrease of soil water content was significantly correlated with an increase of air-filled porosity in soil. The number of secondary lateral roots from segments of primary adventitious roots increased dramatically when soil water content decreased from field capacity to about 0.05 g water g^-1 dried soil. The total dried mass of roots at different soil depths was also positively correlated with soil air-filled porosity. It was observed that the elongation of the initiated secondary roots responded differently to the variations of soil air-filled porosity. The length of secondary roots increased initially when the soil was dried from field capacity to 0.18 g g^-1 dried soil (water potential at about−0.2 MPa, air-filled porosity 0.26 cm³ cm^-3), but was drastically reduced when the soil was dried further. Obviously elongation of secondary roots was inhibited when soil water potential began to deviate substantially from an optimum value. The present results suggested that the initiation of secondary roots was greatly promoted by the increase of air-filled soil porosity, i.e. availability of oxygen. This conclusion was further verified in a separate experiment where solution-cultured maize seedlings were subjected to different aeration treatments. An obvious increase in secondary root initiation was found in plants which were aerated with normal air (21% O₂) than in plants which were either not aerated or aerated with 5% O₂ air. ei]Section editor: B E Clothier     

Phylogeny and taxonomic revision of plastid-containing euglenophytes based on SSU rDNA sequence comparisons and synapomorphic signatures in the SSU rRNA secondary structure

Sequence comparisons and a revised classification of the Euglenophyceae were based on 92 new SSU rDNA sequences obtained from strains of Euglena, Astasia, Phacus, Trachelomonas, Colacium, Cryptoglena, Lepocinclis, Eutreptia, Eutreptiella and Tetreutreptia. Sequence data also provided molecular signatures for taxa from genus to class level in the SSU rRNA secondary structure, revealed by a novel approach (search for non-homoplasious synapomorphies) and used for taxonomic diagnoses. Photosynthetic euglenoids and secondary heterotrophs formed a clade, designated as Euglenophyceae (emend.) with two orders: Euglenales and Eutreptiales. The mostly marine Eutreptiales (Eutreptia, Eutreptiella; not Distigma) comprised taxa with two or four emergent flagella (the quadriflagellate Tetreutreptia was integrated within Eutreptiella). The Euglenales (freshwater genera with < or = one emergent flagellum) formed nine clades and two individual branches (single strains); however, only two clades were congruent with traditional genera: Trachelomonas (incl. Strombomonas) and Colacium. Euglena was polyphyletic and diverged into four independent clades (intermixed with Astasia, Khawkinea and Lepocinclis) and two individual branches (e.g. E. polymorpha). Phacus was also subdivided into Phacus s. str. and two combined lineages (mixed with Lepocinclis spp. or Cryptoglena). In consequence, Euglena (s. str.), Phacus and other genera were emended and one lineage (mixed Phacus/Lepocinclis-clade) was recognized as the previously neglected genus Monomorphina Mereschkowsky (1877). The sister clade of Phacus s. str. (mixed Euglena/Lepocinclis-clade) was identified as Lepocinclis Perty (emended).     

Parsimony analysis of the distribution pattern of Japanese primary freshwater fishes, and its application to the distribution of the bagrid catfishes

The geographic distribution of Japanese primary and some secondary freshwater fishes was analyzed using parsimony analysis of endemicity (PAE). Analysis of 73 taxa (species and intraspecific forms) on the four main islands of Japan (divided into 25 geographic areas), produced 44 most parsimonious cladograms. In all of the latter, a total of eight single and compound areas were recognized as endemic areas in nested relationships. The area cladograms showed Japan as comprising a middle-eastern Hokkaido area plus southern areas, the latter containing mainly a northeastern-Honshu endemic area and more heterogeneous southwestern areas, including four endemic areas (western Kyushu, southeastern Chugoku, middle Kinki and Tokai around Ise Bay) and several peripheral areas. Some patterns, e.g., the distinction in fauna across Fossa Magna, noted by previous studies, were supported by these results. Even though the analysis had some problems (e.g., not all geographic ranges of taxa could be included), it provided evidence for the detection of general distribution patterns, because the relationships or similarities among areas were clearly defined by shared taxa. To demonstrate the historical implications of the analysis, the allopatric distribution of, four bagrid catfishes was reconsidered in the area cladogram. The general pattern implied secondary extinction ofPseudobagrus nudiceps around lse Bay, which was in keeping with the fossil record.     

体外遗传学方法及应用

体外遗传学是利用核酸分子本身一定的表型（如结合、催化等）在试管中分离筛选特定核酸分子序列进行研究的方法．由于体外遗传学方法改变了自然界缓慢的进化过程,使人为的进化得以简单地实现,同时也使许多核酸功能区的识别和确定由被动变为主动,为进一步探索基因的调控规律及主动地调节生化反应过程提供了有效的手段,近年来体外遗传学方法及在分子生物学方面的应用得到了很大的发展．     

Cadherin-7 function in zebrafish development

Cadherin cell adhesion molecules play crucial roles in vertebrate development. Most studies have focused on examining the functions of classical type I cadherins (e.g., cadherin-2) in the development of vertebrates. Little information is available concerning the function of classical type II cadherins (e.g., cadherin-7) in vertebrate development. We have previously shown that cadherin-7 mRNA exhibits a dynamic expression pattern in the central nervous system and notochord in embryonic zebrafish. To gain insight into the role of cadherin-7 in the formation of these structures, we analyzed their formation in zebrafish embryos injected with cadherin-7-specific antisense morpholino oligonucleotides (MO). Notochord development was severely disrupted in MO-injected embryos, whereas gross defects in the development of the central nervous system were not detected in MO-injected embryos. Our results thus demonstrate that cadherin-7 plays an important role in the normal development of the zebrafish notochord.     

Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein

Using the methods reported by De Fossard et al. (11) the influence of various media constituents on the growth and the alkaloid and anthraquinone production in Cinchona ledgeriana callus cultures was studied. Growth and indole alkaloid production (e.g. cinchonamine) was improved by higher auxin levels. The best growth was observed in the light, although many media resulted in no growth at all in the light. Anthraquinone production was highest at lower auxin levels. Quinoline alkaloid levels (e.g. quinidine) were highest in media with low auxin concentrations. Low and medium cytokinin concentration benefited the quinoline alkaloid production.From the results it was concluded that the pathways leading to the various secondary products, anthraquinones, indole alkaloids and quinoline alkaloids are, at least partly, regulated independently.Abbreviations used IAA indol-acetic acid - IBA indol-butyric acid - NAA -naphtaleneacetic acid - NOA 2-naphtoxy-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - pCPA parachlorophenoxy-acetic acid - BA benzyladenine