Variation of Alkaloid Content in Erythroxylum coca Leaves from Leaf Bud to Leaf Drop

A sequential study describing the content (%) of alkaloids inleaves of Erythroxylum coca var. coca Lam. from leaf bud developmentto leaf drop has not previously been conducted. The length oftime the leaf resides on the plant and its concurrent metabolicactivity also has not been defined. In the present study, cocaine,methyl ecgonine, hygrine, tropinone, trans -cinnamoylcocaine,cis-cinnamoylcocaine, tropacocaine and cuscohygrine were monitoredto determine: (1) their content and patterns of accumulationfrom leaf bud to leaf drop; (2) the time leaves resided on theplant; and (3) whether leaves were metabolically active untilleaf drop. E. coca plants were grown in a controlled environmentfor 37 months. Leaves similar in chronological age were extractedand analysed for alkaloid content by gas chromatography (GC)and gas chromatography/mass spectrometry (GC/MS). Cocaine washighest in 7 d old rolled leaves (0·75%) and declinedto 0·39% at leaf drop. Cocaine, methyl ecgonine, hygrine,tropinone, trans -cinnamoylcocaine, cis-cinnamoylcocaine, cuscohygrineand tropacocaine content in 35 d old (mature) leaves was 0·61,0·59, 0·68, 0·08, 0·31, 0·55,0·52, and 0·05%. respectively. Cocaine, methylecgonine, hygrine, cis -cinnamoylcocaine, and cuscohygrine displayeda gradual decline from week 2 to week 36 of leaf duration. Tropinoneand tropacocaine were the least abundant of the alkaloids monitored.Cis-cinnamoylcocaine content exceeded cocaine at week 12, 16,and weeks 19 to 23 of leaf duration. Trans -cinnamoylcocainewas highest in rolled leaves (week 1) and in expanded leavesafter week 30. The monitored alkaloids appeared to be part ofthe metabolically active pool of the leaf. Leaves remained onthe E. coca plants for 36 weeks.Copyright 1994, 1999 AcademicPress Cocaine, methyl ecgonine, hygrine, tropinone, trans-cinnamoylcocaine, cis-cinnamoylcocaine, cusco-hygrine, tropacocaine, leaf bud, rolled leaves, expanded leaves, alkaloids, patterns, fluctuation, Erythroxylum coca var. coca, E, coca     

Selection of genetically diverse Trichoderma spp. isolates for suppression of Phytophthora capsici on bell pepper

Environmentally compatible control measures are needed for suppression of Phytophthora capsici on pepper. Twenty-three isolates of Trichoderma were screened for suppression of a mixture of 4 genetically distinct isolates of this pathogen on bell pepper (Capsicum anuum) in greenhouse pot assays. Of these 23 isolates, GL12, GL13, and Th23 provided significant suppression of P. capsici in at least 2 assays. These isolates were then compared with Trichoderma virens isolates GL3 and GL21 for suppression of this disease in the presence and absence of the harpin-based natural product Messenger. Isolates GL3 and Th23 provided significant disease suppression (P ≤ 0.05) in 3 of 4 assays, while GL12, GL13, and GL21 provided significant suppression in 2 of 4 assays. There was no apparent benefit from the application of Messenger. Phylogenetic analysis of these 5 isolates (based on the ITS1 region of the nuclear rDNA cluster and tef1), and an additional 9 isolates that suppressed P. capsici in at least 1 assay, separated isolates into 2 clades, with 1 clade containing GL3, GL12, GL13, and GL21. There were also 2 more distantly related isolates, one of which was Th23. We report here the identification of genetically distinct Trichoderma isolates for potential use in disease management strategies employing isolate combinations directed at suppression of P. capsici on pepper.     

Historical trends in frog populations in New Zealand based on public perceptions

Surveys were distributed to New Zealand land users in 1998 and 2008 to acquire information about New Zealand frogs with the aim of compiling and mapping their distribution and inferred population trends without costly and time-consuming field surveys. The overall frog population trend was reported as declining, with possible causes reported as an increase in agriculture, an increase in the distribution of predatory fish and disease. The resultant maps could be used for four main purposes: 1) to identify regions where Litoria populations are known to occur, which can be eliminated when considering suitable regions for translocation of Leiopelma; 2) to identify growing or stable populations of Litoria species, which may assist future disease surveys, population monitoring and to identify sources of genetic material that may serve as an Ark for declining Australian populations; 3) to highlight populations that are in decline to enable effective targeting of detailed disease studies; and 4) to approximate the stability of amphibian populations in the absence of more accurate, but costly, scientific monitoring.     

A novel harmony search-K means hybrid algorithm for clustering gene expression data

Recent progress in bioinformatics research has led to the accumulation of huge quantities of biological data at various data sources. The DNA microarray technology makes it possible to simultaneously analyze large number of genes across different samples. Clustering of microarray data can reveal the hidden gene expression patterns from large quantities of expression data that in turn offers tremendous possibilities in functional genomics, comparative genomics, disease diagnosis and drug development. The k- ¬means clustering algorithm is widely used for many practical applications. But the original k-¬means algorithm has several drawbacks. It is computationally expensive and generates locally optimal solutions based on the random choice of the initial centroids. Several methods have been proposed in the literature for improving the performance of the k-¬means algorithm. A meta-heuristic optimization algorithm named harmony search helps find out near-global optimal solutions by searching the entire solution space. Low clustering accuracy of the existing algorithms limits their use in many crucial applications of life sciences. In this paper we propose a novel Harmony Search-K means Hybrid (HSKH) algorithm for clustering the gene expression data. Experimental results show that the proposed algorithm produces clusters with better accuracy in comparison with the existing algorithms.     

Alterations in the production and concentration of selected alkaloids as a function of rising atmospheric carbon dioxide and air temperature: implications for ethno-pharmacology

The influence of recent and projected changes in atmospheric carbon dioxide concentration [CO₂] with and without concurrent increases in air temperature was determined with respect to growth characteristics and production of secondary compounds (alkaloids) in tobacco (Nicotiana tabacum L.) and jimson weed (Datura stramonium L.) over a ca. 50‐day period. Rising [CO₂] above that present at the beginning of the 20th century resulted in consistent, significant increases in leaf area, and above ground dry weight (both species), but decreased leaf area ratio (LAR) and specific leaf area (SLA) in jimson weed. Increased temperature resulted in earlier development and increased leaf area for both species, but increases in above ground final dry weight were observed only for jimson weed. The secondary compounds evaluated included the alkaloids, nicotine, atropine and scopolamine. These compounds are generally recognized as having impacts with respect to herbivory as well as human physiology. Rising [CO₂] reduced the concentration of nicotine in tobacco; but had no effect on atropine, and increased the concentration of scopolamine in jimson weed. However, because of the stimulatory effect of [CO₂] on growth, the amount of all three secondary compounds increased on a per plant basis in both species. Temperature per se had no effect on nicotine or scopolamine concentration, but significantly increased the concentration and amounts of atropine per plant. Overall, the underlying mechanism of CO₂ induced changes in secondary compounds remains unclear; however, these data suggest that the increase in [CO₂] and temperature associated with global climate change may have significant effects not only with respect to herbivory, but on the production of secondary compounds of pharmacological impact.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.     

Application of magnetic techniques in the field of drug discovery and biomedicine

Magnetic separation technology, using magnetic particles, is quick and easy method for sensitive and reliable capture of specific proteins, genetic material and other biomolecules. The technique offers an advantage in terms of subjecting the analyte to very little mechanical stress compared to other methods. Secondly, these methods are non-laborious, cheap and often highly scalable. Moreover, techniques employing magnetism are more amenable to automation and miniaturization. Now that the human genome is sequenced and about 30,000 genes are annotated, the next step is to identify the function of these individual genes, carrying out genotyping studies for allelic variation and SNP analysis, ultimately leading to identification of novel drug targets. In this post-genomic era, technologies based on magnetic separation are becoming an integral part of todays biology laboratory. This article briefly reviews the selected applications of magnetic separation techniques in the field of biotechnology, biomedicine and drug discovery.     

Phenotypic and Genetic Diversity of Aeromonas Species Isolated from Fresh Water Lakes in Malaysia

Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions—exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.     

Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.